The membrane outline

MEMBRANES

Alberts et al., selections from Chapters 1, 10 and 15

Why are membranes so relevant (to deserve the first lecture)?
Signalling is usually across the membrane
Many important molecules are in the membrane

Get a good source of membranes
red blood cell ghosts - only has plasma membrane, flat cell with 2 sides,
Gorter and Grendel showed that there is enough lipid to make 2 layers
Lipids orient on the basis of hydrophobicity TRANSPARENCY Fig. 1-9 , p. 10
Bilayer (Robertson) or micelles TRANSPARENCY Fig. 10.3 , p. 479
Fluid mosaic model TRANSPARENCY Fig. 10-1, p. 477
2 dense lines in EM with osmium
outsides of membrane: glycoprotein, glycolipid, S-S (disulfide) bonds

Lipid biochemistry: TRANSPARENCY panel 2-4, pp. 54-55
(lots of information)
fatty acids :C18-stearic, C16, palmitic, C18-1 oleic
also shown are triglycerides, phospholipids, glycolipids, steroids
Phospholipids TRANSPARENCY Fig. 10.10, p. 483
phosphatidylcholine (lecithin)
phosphatidylethanolamine
phosphatidylserine [amino acid]
sphingomyelin w/ ceramide (based on serine) not glycerol
others
TRANSPARENCY Fig. 10-2 , p. 479
Double bonds make more fluid, note - double bonds more likely in #2 position
Glycolipids TRANSPARENCY Fig. 10-12 , p. 484
One pentameric subunit of cholera toxin (not the best-known one which ADP-ribosylates the alpha subunit of the alpha-stimulatury subunit of the heterotrimeric G protein) binds to membrane using the GM1 ganglioside as a receptor
Lack of a specific enzyme to break down GM is the cause of the famous genetic lysosomal "storage disease" common in Ashkenase Jews called Tay-Sach's disease
in animal, usually based on serine as sphingomyelin

Chloroform - methanol extract - aqueous and organic phases SLIDE
Separation on TLC SLIDE and autoradiogram

There is a special case for inositol lipids
TRANSPARENCY Fig. 15-29, p. 745
phosphatidylinositol inositol alcohol like sugar
PI -> PIP -> PIP2 (PI 4,5-bisphosphate) ->[via PLC (phospholipase C)] ->
IP3 (inositol trisphosphate)+ DAG (diacyl glycerol)
TRANSPARENCY Fig. 15-30, p. 746
-> IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol)
IP3 involved in release of Ca2+ from non-mitochondrial intracellular membranes
DAG activates PKC (protein kinase C)
arachidonic acid is a special case with 4 double bonds
TRANSPARENCY Fig. 15-6, p. 725
arachidonic acid is oxidized to prostaglandins, etc.
One especially long and unsaturated fatty acid is DHA=docosahexaenoic acid=22-6

TRANSPARANCY Fig. 10-5, p. 480
artificial membranes - "black" (from bilayer)
can reconstitute with protein and study electrophysiologically

References:

E. Gorter and F. Grendel, On bimolecular layers of lipoids on the chromocytes of the blood, J. Exp. Med. 41, 439-443, 1925

J. D. Robertson, The membrane of the living cell, Scientific American 206, April 1962, 64-72

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The general signalling outline

Signal transduction - a general introduction

Alberts et al, selections from Chapters 3, 4, 11, 13, 15
(as with the membrane lecture, I will emphasize the text book)

Numbers with "#" sign refer to topis covered in more detail:
Membranes have channels
#1 For instance there are the Na+ channels of the action potential
TRANSPARENCY Fig. 11-22, p. 529
(bioelectric potentials will be covered early this semester)
Channels are gated with #1 voltage, #2 extra- & #3 intra- cellular ligands, and #4 mechanical force
TRANSPARENCY Fig. 11-18 p. 525
Such channels can be studied physiologically as individual molecules
by the patch clamp technique
TRANSPARENCY Fig. 4-55, p. 182 (see also Dwyer)
The 1991 Nobel prize was awarded jointly to: ERWIN NEHER and BERT SAKMANN for their discoveries concerning the function of single ion channels in cells (i.e. developing the patch clamp in 1976)

Extracellular ligand-gated channels are one type of receptor
TRANSPARENCY Fig.15-14 p. 732 is an outline of membrane receptor types
#2 (referring to above numbering) channel
#5 (adding to above numbers) G-protein-linked
#6 (adding to above numbers) enzyme linked

#7 In addition to cell-surface receptors, there are intracellular receptors
(the latter being the steroid hormone superfamily) [later]
TRANSPARENCY Fig. 15-2, p. 722

The ligand can diffuse or it can be membrane-bound
(the latter mechanism being used in situations like "sevenless" [later])
TRANSPARENCY Fig. 15-1 p. 722

NOW FOR A BIT MORE DETAIL
#1 action potentials are used for rapid signalling in nerve axons and muscle cells TRANSPARENCY Fig. 11-20, p. 528
#2 the nicotinic acetylcholine receptor is an example of a channel gated by an extracellular ligand TRANSPARENCY Fig. 11-32, p. 538
#3 the rod photoreceptor cell uses an intracellular ligand (cGMP) TRANSPARENCY Fig. 15-40, p. 754
#4 the hair cell used in hearing and balance is a very specialized cell with mechanically gated channels TRANSPARENCY Fig. p. 37
#5 the G protein linked receptor typically has 7 transmembrane spans TRANSPARENCY Fig. 15-17 p. 735
and is linked to the "heterotrimeric G protein" TRANSPARENCY Fig. 15-33 p. 749 (relates to membrane lecture) with a cascade of intracellular signalling
and intracellular signalling molecules like cAMP TRANSPARENCY Fig. 15-20, p. 737
(note there are also small G proteins)
#6 receptor tyrosine kinases are examples of enzyme receptors TRANSPARENCY Fig. 15-47, p. 760. phosphorylation VERY important
These cascades are long and usually wind up at the transcriptional level
Signals include things like insulin TRANSPARENCY Figs. 3-54, p. 128, Fig. 3-14, p. 104 and Fig. 13-38, p. 628
VERY IMPORTANT: protein domains and shuffling of genes for domains
#7 steroid hormones, thyroxine, retinoic acid cross in to the cell
TRANSPARENCY Fig. 15-11, p. 729
to activate a receptor involved in gene transcription TRANSPARENCY Fig. 15-12, p. 729

References:

T. Dwyer, A patch clamp primer, (Review Tutorial) J. Electrophysiol tech. 12, 15-29, 1985

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The signalling in the nervous system - electrophysiology outline

Electrophysiology

Alberts et al., pp 181-184, Chapter 11
(again, even though there are many classic papers, the text covers a lot so the readings are limited)

History

1791 Luigi Galvani (Italy) - nerve muscle electricity in frog
1840's Carlo Matteuci (Italy) and Emil du Bois Reymond (Germany)
1850 Herman von Helmholtz - speed of conduction (40 m/s)
Walther Hermann Nernst (Germany) (1864-1941) 1920 Nobel in Chemistry
1902 Julius Bernstein apply Nernst, K perm lost in a.p.
1939 K. C. Cole and H. J. Curtis (US) (Transparency) squid
1950's Sir Alan L. Hodgkin & Sir Andrew F. Huxley (Great Britain)
1963 Nobel "ionic mechanisms...excitation inhibition...nerve cell membrane"
Erwin Neher &Bert Sackmann (Germany) for patch clamp (transparency)
Nobel prize in 1992 "incredibly small electric currents that pass
through an ion channel"

Here are the Nobel prize speeches (Neher 1992) (Sakmann 1992) (and I am putting a copy of Neher's on reserve)

Electrical concepts

Circuits (equivalent circuits) Transparency
Battery, anode:+, anions:-, Cathode:-, cations:+
current = i, defined as + to -

Potential (potential difference): V or E
(1) Battery (source of electromotive force, EMF)
(2) Current flow through a resistor
battery and resistor in circuit
E = IR (Ohm's law), R in units of Ohms, W
G is conductance, 1/R, "mho" = Siemens (S)

Membrane capacitance

(another source of impedance)
typically it adds delays (draw) - cuts off high frequency symbol
High pass filter
low pass filter

Glass micropipette typically 1 pf - 10-12f
typically 20 MegOhm
t =RC=2ms (slow)
need negative capacity electrometer to compensate

Sodium - potassium "pump"
Uses 1/3 (2/3 if high electrical activity) of cell
Ouabain binds K+ site (glycoside)
Na+-K+-ATPase
Transparency (Fig. 11-11, p. 515)
"Electrogenic" - imbalance of 3 Na+ - 2 K+ cause current to flow, contribute a few mV
8 membrane spans
homologies with Ca++ pump in sarcoplasmic reticulum
homologies with bacterial K+-ATPase

Ion concentrations

Transparency Table 11-1, p. 508

Derivation of Nernst potential

See Transparency (Panel 11-2, p. 526)
In class, use another Transparency

Assume two compartments in communication
(ions like K+ or Na+ dissolved in each)
Free energy (of each system) = RT ln Ci + ziFF
chemical electrical
F is absolute potential, C is concentration, i is given ion, e.g. K+ or Na+
T is tempreature in degrees Kelvin
R = 8.31 Joules/moleoK
F = 9.65 x 104 Coulombs/mole
= 6.02 x 10 E23 ions/mole (Avagadro's number) x 1.6 x 10E-19 Coulombs/ion (elementary charge) ]
Assume equilibrium which means
(1) no flux
(2) electrical and chemical gradients equal and opposite
(3) energies of two compartments the same
Simple algebra and the fact that log10 = 2.3 x ln gives:
EK+ = 58 log [K+]out / [K+]in

Goldman equation

David Goldman, 1943
assume constant field
Transparency Contributions of Cole and urtis on Squid with AC bridge

Vm = 58 log PK[K+]out + PNa[Na+]out + PCl[Cl-]in
PK[K+]in + PNa[Na+]in + PCl[Cl-]out

Transparency reviews action potential vs. resting potential and permeabilities

Hodgkin-Huxley work

Transparency operational amplifier
voltage clamp
early and late conductances

INa = gNa m3 h (V-VNa)

IK = gK n4 (V-VK)

m and n are "activations" and h is "inactivation"

Sodium channels

how little tetrodotoxin (from puffer fish) does it take to block in lobster nerve
13 / (micro m)2 (Moore et al.)

also Keynes 1971 3.6 x 10E9 channels/cm2
1.5 x 10E-10 Siemens / channel

Reference (only Neher is on reserve right now)

J. W. Moore, T. Narahashi & T.I. Shaw, An upper limit to the number of sodium channels in the nerve membrane? J. Physiol. 1967, 188, 99-105

Neher, E., 1992 Ion channels for communication between and within cells. Science. 256: 498-502.

Sakmann, B., 1992 Elementary steps in synaptic transmission revealed by currents through single ion channels. Science. 256: 503-12.

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The signalling in the nervous system - channels outline

Channels

lots of primary literature here.

Here is a web site on channels I recently found

TRANSPARENCY
R. TAYLOR, 1994,

The first page gives history, some of which was covered before, a quite thorough treatment.
1780's Galvani
1834 - Faraday - "ion"
1887 - Arrhenius - salts dissociate
1890 - Nernst
1902 - Bernstein - - membrane's selective permeability to potassium breaks down
1902 - Overton - Sodium needed for action potential
1925 - Michaelis ion selective channels
1936 - Young Loligo
1939 - Cole and Curtis
1949-1952 - voltage clamp and Hodgkin - Huxley papers
1964 - Narahashi - TTX (tetrodotoxin)- sodium
1965 - Armstrong and Binstock - TEA - potassium
some isolations of channel with labeled toxins
Electrophorus electricus - eel mRNA => 1820 a.a. with 4 homologous domains
Noda, Numa et al.
1987 - Jan and Jan (and other groups) Drosophila potassium

Ball and chain inactivation:
TRANSPARENCY
T. Hoshi et al., 1990

O. P. Hamill & D. W. McBride, Jr., 1995

"mechanogated"

Numerous (e.g. 17,000 in palm and fingertips) and widespread (to bacteria and Paramecia, plants, fungi and animals, i.e. all kingdoms)

Classically, for touch, there are "phasic" e.g. Pacinian corpuscle and "tonic" e.g. Merkel cell based on how fast the adaptation and thus whether vibration or steady pressure is optimal

When Erwin Neher and Bert Sakman (Max Planck Inst) developed patch clamp (1097's, Nobel Prize 1991) - this helped a lot.

development of "pressure clamp" - TRANSPARENCY

oocytes of toad have channels, Amiloride blocks channels and fertilization

tension on channel may be important plus viscoelastic elements - intracellular and extracellular, TRANSPARENCY e.g. tip links in stereocilia in hair cells (later)
integrins cell surface link to extracellular like laminin
dystrophin found in studies of Duchenne muscular dystrophy - reverse genetics
link with actin etc.

O. P. Hamill & D. W. McBride, Jr., , 1994,

classic work is on VGNa where Electrophorus contributed
and AChR where Torpedo came in handy

cannot patch clamp bacteria - so make spheroplasts (6 micro m) for patch clamping

find channels called MscL and MscS for Mechanosensitive channel large (conductance - 3000 pS) and small (1000 pS)

use 37 a.a sequence near N terminus to get mscL gene

comparisons with alamethicin 20 a.a. pore forming peptide.

J. Liu et al., 1996,
also D. P. Corey & J. Garcia-Anoveros, , 1996,

advantages of C. elegans for genetics and neurobiology

Caenorhabditis elegans -unc-105 is a new member of channel superfamily
TRANSPARENCY
and let-2 is a collagen

DEGs (degenerins) - ion influx and swelling - mechanosensation

family tree (dendrogram) TRANSPARENCY (Fig. 1) with structures
MEC-4 and MEC-10 -- proteins which contribute to heteromultimers

related to epithelial sodium channels (ENaCs) which are sensitive to amiloride

Discuss hair cells - 30 - 300 stereocilia TRANSPARENCY (Fig. 2)
deflection toward tallest cause nonselective cation channels to open

some mec genes encode tubulins, some secreted proteins, one a collagen

no relation to bacterial

gap junctions

[background] (note there are also plasmodesmata in plants)
classic example is electrical connection between myocardial cells at intercalated disk
pass larger molecules (fluorescent dyes, sugars) as well as electrical current
basically, a hexagon of 6 transmembrane molecules on one cell are in register with the 6 molecules on the other, a patch (macula) of these is at site where membranes look very close in the EM but still pass dye (an electron dense tracer like lanthanum) extracellularly [as opposed to occluding junction where outer membrane dense lines (as seen in the EM) merge
connexons - protein can be very variable
MIP = major intrinsic protein (between lens cells)
big brain mutant Drosophila
conductance high - 120 pS
appropriate to cover this between "channels" & "chemical synapses"
pass lots of molecules
invertebrate and lower vertebrate nervous systems mostly, not in mammalian brain
a review paper on gap junctions
E. Edelson, Gap junctions: conduits for cell-cell communication, Mosaic 21, 1990, 48-56
TRANSPARENCY

J. Bergoffen et al., Connexin mutations in X-linked Charcot-Marie-Tooth disease, Science, 262, 1993, 2039-2042 (also "Connexin connection" - This week in Science, p. 1951)

disease is a degeneration of peripheral nerve
connexin-32 TRANSPARENCY (Fig. 3)
find 7 mutations in 8 families, one frameshift, the others nonconservative substitutions
gap junction channels - expression in myelin

P. M. T. Dean et al., Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine, Science 264, 1994, 92-95.

aquaporin-1 acts in descending thin loop of Henle and proximal tubule - constitutively high water permeability

collecting ducts regulated by antidiuretic hormone arginine vasopressin
There are mutations in receptor which cause Nephrogenic diabetes insipidus
but they had a patient who did not have this
TRANSPARENCY (Fig. 2) - protein and mutations

Cystic fibrosis

J. R. Riordan et al., Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA, Science 245, 1066-1072, 1989

J. L. Marx, The cystic fibrosis gene is found (news and comment) & The CF gene hits the news, Science 245, 923-925, 1989

most common genetic disorder in Caucasians (1/2000)
chromosome 7
many indications that a chloride channel was at fault
needed to do chromosome jumping
cDNA library from sweat glands (CF patients have very salty skin)
big gene (250 kb), 24 exons, TRANSPARENCY (Fig. 7)
cystic fibrosis transmembrane conductance regulator (CFTR)
1480 amino acids (168, 138 daltons), membrane protein probably channel
has sequences resembling consensus nucleotide (ATP)-binding folds (NBF's)R R domain with 69/241 a.a.'s being charged
consensus sequences for phosphorylation by PKA and PKC
sequence like White gene in Drosophila => transport
10/12 membrane spans have charge => channel
like GABA (gamma amino butyric acid) chloride channel
do not secrete chloride - water does not follow, thick mucus
phe at #508 is missing in 70% of cases near binding site for ATP

Drosophila

L. Salkoff & R. Wyman, Ion channels in Drosophila muscle, Trends in Neurosciences 6, 1983, 128-133

Use of Drosophila (see TRANSPARENCY [home drawn and from Salkoff and Wyman])
this work emphasizes fact that in muscle, a fast potassium channel can be
isolated by different developmental times

B. L. Tempel, Potassium channel genes and genetics in flies, mice and man, Seminars in the Neurosciences 2, 197-205, 1990

(this is a nice paper and an interesting one)

2 TRANSPARENCIES (home drawn and from Tempel)
Lots of different potassium channels with different functions
well-known is the delayed rectifier
then there is the transient K+ current or A-current
In pancreatic beta cells, blocked by intracellular ATP or ADP, extracellular glucose
cloning (walking), expression
S4 with charged arginines

L. Salkoff et al., An essential "set" of K+ channels conserved in flies, mice and humans (viewpoint), Trends in Neurosciences May, 1992, 15-5, 161-166.

List of K+ channels: voltage activated, Ca2+-sensitive, ATP-sensitive and others
all kinds of functions
human pancreatic beta cells, cardiac tissue, mammalian lymphocytes

Shaker gene in fly with alternative splicing - still there must be others
low stringency hybridization to find these - Shal, Shaw, and Shab
Shal, A-type K+ current
Shaw, and Shab - delayed rectifier-type K+ current
TRANSPARENCY (Figs 1 and 2)
fast inactivation "inactivation ball"

Evolution - gene duplication -
find lots in mice - TRANSPARENCY
mammals lack introns (no splicing except Shaw) - duplicated gene many times
ancestral K+ channel had already diverged into 4 types by Cambrian radiation

Drosophila Shaw has only 4 (not 6) S4 charges - less low voltage sensitivity
Co-express in Xenopus oocyte - and compare properties with real ones
-probably no heteromultimers

M. C. Trudeau, HERG, a human inward rectifier in the voltage-gated potassium channel family, Science 269, 92-95, 1995

ether-a-go-go (eag) outward rectifying K+ channel in family with Elk (eag-like) and Erg (eag-related)

inward rectifiers carry currents at voltages negative to K+ equilibrium
in heart, at positive voltages they close contributing to action potential plateau

heterologous expression of HERG in frog oocytes

M-EAG is outward, typical data TRANSPARANCY (Figs 1 and 2)
note outward current is up
some modeling based on S4 properties most recent work "has focused on a family of channels sharing a hydrophobicity plot that predicts two transmembrane domains"

KAT-1 in Arabidopsis is inward rectifier with S4 to sense very negative V in plants

HERG is locus for a long QT syndrome (LQT-2)
review electrocardiogram

L. M. Manuzzu et al., Direct physical measure of conformational rearrangement underlying potassium channel gating, Science 271, 213-216, 1996

Shaker site-specific fluorescent labeling

Fig. 6 (Jan and Jan 1997) is a comparison of the voltage gated K+ channel and the inwardly-rectifying potassium channel. Note, the latter has a very different structure, only 2 membrane spans but does have the pore-lining loop

Fig. 8 (Jan and Jan 1997) is a description of how this channel is activated by the muscarinic cholinergic recrptor (through the G-protein cascade).

R. Hedrich & P. Dietrich, Plant K+ channels: similarity and diversity, Bot. Acta. 109, 1-8, 1996
This paper refers to "green" (from plants) and "red" (from animals)
this work is electrophysiological, and application of the patch clamp has contributed greatly
Of course, nutritionally, for plants and herbivores, potassium is very relevant (macronutrient)
several rapid volume change responses
for instance, here is a TRANSPARENCY from this department's introductory biology text to show the involvement of K+ in guard cell
responses, responsible for opening and closing of stomata
an overall outline:
Inward rectifying voltage dependent K+ channels for K+ uptake
Outward rectifying voltage dependent K+ channels for K+ release
Also there is a high affinity K+ transporter (HKT1) about which little is known though it is thought that protons are cotransported. Such a
system would be used to get K+ in from low concentration in soil.
This paper concentrates on uptake channels like those cloned from:
Arabidosis thaliana (KAT1)
Solanum tuberosum (KST1)
channel conductance is 5-30 pS
for a 10 x change in K+ gradient, voltage changes 56-58 mV in accord with the Nernst potential (see earlier this semester, discussion of
Paramecium)
not many insect, scorpion, snake, frog or dinoflagellate toxins which affect animal channels affect plant channels
But external Cs+ and TEA+ do block, but weakly
In contrast with animal channels, KST1 and KAT1 have ATP and cyclic nucleotide cassettes and several channels are ATP dependent
Like shaker in S1-S6 and H5 or P (pore forming) - TRANSPARENCY with 21 conserved amino acids except that plant channel has extra 14
amino acids
No N-terminal ball and chain and no inactivation
there are AKT1 = Arabidosis K+ transporters which have ankyrin binding domains


Paramecium

Y. Naitoh & R. Eckert, Ionic mechanisms controlling behavioral responses of Paramecium to mechanical stimulation, Science 164, 1969, 963-965

R. Eckert, Bioelectric control of ciliary activity, Science 176, 1972, 473-481

C. Kung et al., Genetic dissection of behavior in Paramecium, Science 188, 1975, 898-904.

Jennings (1906) avoidance reaction (literature onsimple behaviors of lower organisms goes back a long way)
TRANSPARENCY (home drawn and from papers)

Eckert - anterior stimulation - depolarization - spike - Calcium
-posterior stimulation hyperpolarization - Potassium
(These are good figures for understanding the Nernst equation)

Genetics - good (as well as size)
TRANSPARENCY
Autogamy - makes homozygous - good for isolating recessives

Get mutants - "pawn" (pw) cannot swim backwards - no spike- eliminate calcium current
fast-2 (f) - no depolarization
paranoiac (Pa) no downstroke of the action potential
and others - (see below)

R. R. Preston et al., Calmodulin mutants and Ca2+-dependent channels in Paramecium, Annual Review of Physiology, 53, 1991, 309-319

TRANSPARENCY Calmodulin (Alberts et al., Fig. 15.34, p. 750)

Intracellular Calcium is usually low, Ca2+/CaM complex if micromolar
expose hydrophobic patches for interaction with >20 enzymes
Paramecium tetraurelia - one CaM gene; true for most, yeasts to eel
ORF 444 bases, no introns
Calcium transient increases concentration from <10-7 to > 10-5
then delayed K+ channels repolarizes calcium action potential
(this is eliminated in pantophobiac, CaM structural gene
change gene name from pntA to cam1)
ser -> phe at 101 TRANSPARENCY (Fig. 1)
shows cam 1, 2, 3 mutants all near C-tterminus

also there is a calcium-dependent (not voltage-dependent!) sodium current
lacking in fast
enhanced in paranoiac
cam 11, 12, 13 changes (sometimes two changes!) near N-terminus

Maybe two sites interact differently
Other mtants affect Ca2+-dependent K+ channels without affecting CaM
tea for TEA-insensitive and rst for restless

Chloride:

T. J. Jentesch, Chloride channels: a molecular perspective, Current opinion in neurobiology, 1996, 6, 303-310

There are 3 types of chloride channel:
(1) postsynaptic receptors (like for GABA = gama amino butyric acid) and glycine
(2) CFTR (traffci ATPase superfamily, though cystic fibrosis channel is passive)
(3) CLC
TRANSPARENCY
(1) note, for neurotransmitter, there are 4 membrane spans, called M1-M4
(2) the CFTR, with its 12 TM spans with R and NBF (nucleotide binding fold) between 6 & 7 and NBF near C terminus was covered earlier, R domain is for phosphorylation
(3) CLC different, perhaps still some uncertainty

(1) inhibitory, dampen membrane excitability (anion, 110/5-15 gradient, out to in)
(3) TRANSPARENCY dendrogram - show expression and defects (like myotonia CLC-1, Kidney stones CLC-5)

Channels

K. Mikoshiba, The InsP3 receptor and intracellular Ca2+ signaling, Current opinion in Neurobiology, 1997, 7, 339-345
TRANSPARENCY quick-freeze deep-etch technique actually shows channel arrangement
cartoon shows cut-away of 2 of the 4 pore subunits each with 6 transmembrane domains
note that, relative to Shaker, inside lumen is topologically like outside of cell

C.C.H.Peterson, Store operated calcium entry, Seminars in the Neurosciences, 1996, 8, 293-300
TRANSPARENCY Fig. 1 shows a diagram which overlaps considerably with what will be shown later (Friel paper, Invertebrate Phototransduction lecture)
Note that there must be a pump (here called SERCA) to concentrate calcium into ER TRANSPARENCY here is a detail of trp channel
note suggestion that ankyrin repeats may see to trp - InsP3R proximity

In this lecture so far, I have short-changed calcium channels, and now I will only summarize what will be repeated in the synaptic transmission lecture:

In presynaptic terminal:Ca2+ in through Q or N type voltage gated channel
(N stands for "neither, as opposed to T=transient or L=long lasting, the N channel is blocked by omega toxin from Conus [snail genus])

In Muscle: t-tubules get excitation to near sarcoplasmic reticulum
dyhydropyridine (blocking drug) receptor in t-tubule
homology to sodium channel - voltage sensitive

In Muscle: ryanodine receptor in sarcoplasmic reticulum same family as IP3 receptor
coupled with t-tubule

G. A. Cottrell, The first peptide-gated ion channel (Review), The Journal of Experimental Biology 200, 1997, 2377-2386
TRANSPARENCY Fig. 3 shows FMRF amide and related agonists and antagonists
TRANSPARENCY Fig. 1 - shows unusual structure with only 2 transmembrane domains which is like the epithelial Na+ channels and the degenerins

W. N. Zagotta and S. A. Siegelbaum, Structure and function of cyclic nucleotide-gated channels, Annual Review of Neuroscience, 1996, 19, 235-263
TRANSPARENCY compares Shaker and CNG for domains
- there is a cGMP binding domain beyond (toward C-terminus) S1-S5, P, S6
Figure also shows the predicted membrane topography and the 4 subunit structure
TRANSPARENCY amino acid structure and comparisons of pores

D. Ren et al., A prokaryotic voltage-gated sodium channel, Science 294, 2372-2375, 2001.
also
W. A. Catterall, A one-domain voltage-gated sodium channel in bacteria (perspective), Science 294, 2306-232308., 2001
Took until 2001 to get this. Bacillus halodurans - salt-loving
274 aa -> homotetramer
for eukaryote, ion selectivity probably determined by a few a.a.'s in 2nd half of pore loop
some discussion of critical amino acids and how bacterial channel differs from eukaryotic
tetrodotoxin does not block but some calcium channels do block
loop too small for S4 to move, so gating is probably different
inactivation is 100x slower
the ball in chain now has the jargon N-type inactivation,
by contrast, C-type inactivation requires all the loops and that is likely here.

References

M. Barinaga, Playing tetherball in the nervous system, Science 250, 506-507,1990

J. Bergoffen et al., Connexin mutations in X-linked Charcot-Marie-Tooth disease, Science, 262, 1993, 2039-2042

also D. P. Corey & J. Garcia-Anoveros, Mechanosensation and the DEG/ENaC Ion channels (Perspectives), Science 273, 19 July, 1996, 323-324

G. A. Cottrell, The first peptide-gated ion channel (Review), The Journal of Experimental Biology 200, 1997, 2377-2386

P. M. T. Dean et al., Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine, Science 264, 1994, 92-95.

R. Eckert, Bioelectric control of ciliary activity, Science 176, 1972, 473-481

E. Edelson, Gap junctions: conduits for cell-cell communication, Mosaic 21, 1990, 48-56

O. P. Hamill & D. W. McBride, Jr., The cloning of a mechano-gated membrane ion channel (Research News) Trends in Neurosciences 17-11, Nov., 1994, 439-443

O. P. Hamill & D. W. McBride, Jr., Mechanoreceptive membrane channels, American Scientist 83, Jan-Feb 1995, 30-37

R. Hedrich & P. Dietrich, Plant K+ channels: similarity and diversity, Bot. Acta. 109, 1-8, 1996

T. Hoshi et al., Biophysical and molecular mechanisms of Shaker potassium channel inactivation, Science 250, 533-538, 1990

Jan, L. Y., and Y. N. Jan, 1997 Voltage-gated and inwardly rectifying potassium channels. J Physiol (Lond). 505: 267-82.

T. J. Jentesch, Chloride channels: a molecular perspective, Current opinion in neurobiology, 1996, 6, 303-310

C. Kung et al., Genetic dissection of behavior in Paramecium, Science 188, 1975, 898-904.

J. Liu et al., Interaction between a putative mechanosensory membrane channel and a collagen, Science, 273, 1996, 361-364.

J. L. Marx, The cystic fibrosis gene is found (news and comment) & The CF gene hits the news, Science 245, 923-925, 1989

L. M. Manuzzu et al., Direct physical measure of conformational rearrangement underlying potassium channel gating, Science 271, 213-216, 1996

K. Mikoshiba, The InsP3 receptor and intracellular Ca2+ signaling, Current opinion in Neurobiology, 1997, 7, 339-345

Y. Naitoh & R. Eckert, Ionic mechanisms controlling behavioral responses of Paramecium to mechanical stimulation, Science 164, 1969, 963-965

C.C.H.Peterson, Store operated calcium entry, Seminars in the Neurosciences, 1996, 8, 293-300

D. Ren et al., A prokaryotic voltage-gated sodium channel, Science 294, 2372-2375, 2001.
also
W. A. Catterall, A one-domain voltage-gated sodium channel in bacteria (perspective), Science 294, 2306-232308., 2001

J. R. Riordan et al., Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA, Science 245, 1066-1072, 1989

L. Salkoff et al., An essential "set" of K+ channels conserved in flies, mice and humans (viewpoint), Trends in Neurosciences May, 1992, 15-5, 161-166.

L. Salkoff & R. Wyman, Ion channels in Drosophila muscle, Trends in Neurosciences 6, 1983, 128-133

R. TAYLOR, Evolutions: The voltage-gated sodium channel, The Journal of NIH Research 6, November, 1994, 100-102 & 111-112.

B. L. Tempel, Potassium channel genes and genetics in flies, mice and man, Seminars in the Neurosciences 2, 197-205, 1990

M. C. Trudeau, HERG, a human inward rectifier in the voltage-gated potassium channel family, Science 269, 92-95, 1995

W. N. Zagotta and S. A. Siegelbaum, Structure and function of cyclic nucleotide-gated channels, Annual Review of Neuroscience, 1996, 19, 235-263

This page was last updated on Jan. 24, 2002

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The synapses and transmitters lecture

Synaptic transmission (today's lecture features channels)
channels (ionotropic) ACh, glutamate, GABA, Glycine
vs. metabotropic receptors- peptides and others

Review of major points (that mostly everybody knows), thus, since a lot is text book material, I only present a few references, the rest is from Alberts et al.

Chemical synaptic transmitter substances:
Monamines (acetylcholine, catecholamines, serotonin)
Peptides
Amino Acids
Nucleotides
Gasses

Presynaptic membrane, cleft, Postsynaptic membrane
Biochemistry - synaptosomes
Postsynaptic receptors, multiplicity defined by pharmachology
agonist, antagonist
Breakdown, reuptake

TRANSPARENCY Fig. 11-30, p. 537 Alberts et al. (SEM of neuromuscular junction)
on n.m.j. (100 - 1000 square microns as opposed to synapse - 1 square micron)
TRANSPARENCY Fig. 11-35, p. 541 Alberts et al. motor neuron - final common path
TRANSPARENCY Fig. 13-43, p. 632 Alberts et al., synaptic vesicle formation
TRANSPARENCY Fig. 11-34, p. 540 Alberts et al., ion channels of neuromuscular junction
(Here is where that information I mentioned in the "channels" outline about calcium channels comes in, so, for convenience, I repeat it here:)
In presynaptic terminal:Ca2+ in through Q or N type voltage gated channel
(N stands for "neither, as opposed to T=transient or L=long lasting, the N channel is blocked by omega toxin from Conus [snail genus])

In Muscle: t-tubules get excitation to near sarcoplasmic reticulum
dyhydropyridine (blocking drug) receptor in t-tubule
homology to sodium channel - voltage sensitive

In Muscle: ryanodine receptor in sarcoplasmic reticulum same family as IP3 receptor
coupled with t-tubule

D. W. Tingley, Evolutions: Synaptic transmission, Journal of NIH Research 7, Nov. 1995, 103-104 & 98-101. TRANSPARENCY

From Tingley, 1995 and other sources:
reticular theory - cells are not separated from each other
vs. Schwann's 1839 cell theory
1891 Waldeyer coined "neuron"
1897 Held described "boutons"
Muller had students - DuBois-Reymond (chemical communication)
and Helmholtz - electrical
early 1900's - 1932 Nobel for "functions of neurons" - Sherrington (England) Integrative Action of the Nervous System 1906 coined "synapse," studied spinal reflex
1904 - Elliot adrenalin from adrenal mimics sympathetic nerves
1905 - Langley - chemical transmission in fish
1921 Loewi vagus
1936 Dale - acetylcholine
share 1936 Nobel "chemical transmission of nerve impulses"
1953 Palay sees synapse in EM
1959 Furshpan and Potter electrical transmission in crayfish
1963 Nobel Prize - Eccles (with Hodgkin and Huxley) - Resistance decreases - channels open EPSP (PK & PNa up) IPSP(Cl- conductance)
1964 DeRobertis - omega figures
1965 Katz and Miledi - low calcium -> "quanta"
1970 Nobel Sir Bernard Katz (England) Ulf von Euler (Sweden),Julius Axelrod (US) "humoral transmitters...nerve terminals....storage release inactivation"
Science publishes Nobel speeches - Julius Axelrod, Noradrenalin: Fate and control of its biosynthesis, Science 173, 598-606, 1971
1974 Heuser et al quick freezing
early 1970's - Katz et al, use noise analysis to understand AChR
1976 Neher and Sackmann - record from individual channels
alpha-bungarotoxin binds nicotinic
1978 Karlin alpha, beta, gamma and delta
1980's cloning from Torpedo
Calcium channels (T, N, and L) - calcium entry critical
glutamate and GABA
work with NMDA and non-NMDA receptors
metabotropic receptors
proteins involved in vesicle
nitric oxide, etc

Aceylcholine "metabolism"

TRANSPARENCY
Dietary choline -reuptake or uptake-> intraneural choline
-Choline-O-acetyltransferase-> H3-CO-O-CH2-N+-(CH3)3
Acetyl Co-A
release stimulated by Ca2+, blocked by botulism
over release by black widow spider venom
nicotinic receptors - nicotine stimulates, curare blocks.
labeled by 125I alpha-bungarotoxin - banded krait - snake
receptors low in mysthenia gravis - autoimmunity to nicotinic receptors
autonomic ganglia, muscle
muscarinic receptors - muscarine stimulates, atropine (belladonna) blocks
parasympathetic neuro-effector junctions (incl. smooth muscle)
Acetylcholinesterase blocked by malathion and neostigmine

TRANSPARENCY from Axelrod's 1971 Nobel Prize speech
" Metabolism" of epinephrine (amine from amino acid)
(phenylalanine->) l-tyrosine -> (tyrosine hydroxylase, rate limiting)
-> l-DOPA (dihydroxyphenylalanine)
given to Parkinson's patients, precursor of quinones which polymerize to melanin
-> (DOPA decarboxylase = l-aromatic amino acid decarboxylase, low substrate specificity used also in 5HT=5-hydroxy tryptamine =serotonin synthesis)
-> dopamine (not l or d)
low in nigro-striatal dopamine system (from substantia nigra to striatum) in Parkinsons
-> (dopamine beta hydroxylase)
-> l-NE -> (PNMT [N-methyl transferase]) E
MAO breaks down intracellularly and inhibitors are antidepressants
COMT breaks down extracellularly
but by far, reuptake is the way that NE is inactivated

The Nicotinic Acetylcholine receptor (information from here and there)

TRANSPARENCY Fig. 11-32, p. 538 Alberts et al.
Torpedo - electric ray
up to 75 V (not that much) but 20 Amps
there are fish with electric sense, not just thost that stun prey
lots of generator potentials added up (Electrophorus - lots of spikes 600 V 1 A)
Structure likely spans the membrane 4 times (though there is extra alpha-helix)
expression - need all 4 types of subunit
autoimmunity in myasthenia gravis
mechanisms of muscular relaxatants used in surgery (like succinylcholine)
when nerve-muscle junction is made, diffuse receptors cluster
in Torpedo 10,000/micro meter2, i.e. 1 per 100 x 100 A
concentrated at n.m.j. crest, 20,000 - 30,000
whereas AChE is evenly distributed
6.5 A in diameter, probably water filled pore
0.2 - 3.0 x 106 ACh molecules from one a.p. into n.m.j. cleft
2.5 x 105 channels transiently open
400 nA n.m.j. end plate current
1 ms open time
10,000 Na+'s flow through each channel in this time
channel conductances of 25 pS

TRANSPARENCY - Fig. 11-31, p. 538 channel opening and closing
occupied and closed - analogy to inactivated vs closed

TRANSPARENCY - REVIEW channel structure Fig. 11-33, p. 539 Alberts et al

Amino acid transmitter systems

Glutamate central excitatory - like inputs to hippocampus
a lot of diversity
AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
reversal potential is at 0 mV so it is likely opening for K+ & Na+ channels
NMDA (N-methyl-D-aspartate) blocked by APV (2-amino-5-phosphonovalerate)
reversal is more positive, => open Na+ & Ca2+
Calcium influx - cytotoxicity in injury or stroke
(There are other cation selective subclasses defined pharmacologically)

GABA (gamma amino butyric acid)
GAD glutamic acid decarboxylase
GABAA is Cl- channel blocked by bicuculline & picrotoxin
Benzodiazepines (like Valium) bind- reduce anxiety
Barbiturates also enhance Cl- conductance

Glycine blocked by strichnine
5 subunits - 2 alphas & 3 betas
4 (?) membrane passes with M2 lining pore

A very simple experiment, (Miller 1998) (Rosenmund, Stern-Bach and Stevens 1998) the glutamate channel is suggested to be a tetramer.

Vesicle fusion

TRANSPARENCY Fig. 13-58, p. 645, Alberts et al. - proteins in vesicle fusion
TRANSPARENCY Fig. from D. W. Tingley, Synaptic-vesicle release: New pieces of a puzzling process (in focus) Dec. 1995 J. NIH Res. 7, 46-49
Here (Fig. 5.10) is the information in Purves et al., the text book I am currently using to teach my neuro course
Docking, Priming (requires ATP), Fusion/exocytosis, Endocytosis
Ca2+ in through Q or N type voltage gated channel
(N stands for "neither, as opposed to T=transient or L=long lasting, the N channel is blocked by omega toxin from Conus [snail genus] toxin
Membrane proteins and cytoplasmic proteins
also the names change to protect the innocent
Vesicle proteins:
Synaptobrevin / VAMP (vesicle-associated membrane protein) = v-SNARE
Botulinum and Tetanus toxin binding sites
synaptotagmin - binds calcium
Rab3 (like ras, small GTP binding protein) (lots of rab's, specific for transport)
synapsins get phosphorylated (by CaM Kinase and PKA) interact with actin
target membrane proteins:
Syntaxin = t-SNARE
Neurexin - black widow spider venom (alpha Latrotoxin)
Cytoplasmic:
NSF - N-ethylmaleimide sensitive factor (ATPase activity when complex dispersed)
SNAP - soluable NSF associated protein
rabphillin

Metabotropic receptors -> signalling
like for beta adrenergic receptor, CAPM increased, action potentiated by caffeine which is a cAMP phophodiesterase inhibitor

George Wald Nobel Prize 1967 rhodopsin
later they found that it was 7-TD receptor
Sutherland Nobel prize 1971 Signal transduction cAMP father of signal transduction

References


J. Axelrod, Noradrenalin: Fate and control of its biosynthesis, Science 173, 598-606, 1971

Rosenmund, C., Y. Stern-Bach, and C. F. Stevens, 1998 The tetrameric structure of a glutamate receptor channel [see comments]. Science. 280: 1596-9.

D. W. Tingley, Evolutions: Synaptic transmission, Journal of NIH Research 7, Nov. 1995, 103-104 & 98-101.

D. W. Tingley, Synaptic-vesicle release: New pieces of a puzzling process (in focus) Dec. 1995 J. NIH Res. 7, 46-49

This page was last updated on Dec. 19, 2001

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The G protein coupled signalling outline

G Protein signalling

A review of "textbook version" of G-protein signalling

1994 - The Nobel Prize in Physiology and Medicine was awarded jointly to: ALFRED G. GILMAN and MARTIN RODBELL for their discovery of G-proteins and the role of these proteins in signal transduction in cells. (Discovering G-Proteins)

TRANSPARENCY Fig. 15-23 p. 739 Alberts et al.
receptor -> G protein (and GTP, GDP)-> adenylyl cyclase -> cAMP
mammalian adenylate cyclase:
2 portions with 6 membrane spans
catalytic domains between and at C-terminus (cytosolic side)
TRANSPARENCY Fig. 15-24 p. 740 Alberts et al.
PKA (A kinase has 2 regulatory (inhibitory) subunits and 2 catalytic
each regulatory subunit binds 2 cAMP's

Dictyostelium discoideum - Alberts et al. 831-833, also 838, 846-847

classic paper: J. T. Bonner, Hormones in social amoebae and mammals, Scientific American, June, 1969, pp 78-91

TRANSPARENCY slime mold
Fig. 16-62, p. 832 shows the chemotaxis of a cell
Fig. 16-63, p. 832 shows the involvement of sudden actin development
Fig. 16-60, p. 831 shows the slug
Fig. 16-61 p. 831 shows the fruiting body

The discovery that the receptor was related to the G protein linked receptor (e.g. the beta adrenergic receptor)

TRANSPARENCY from:

P. S. Klein et al. A chemoattractant receptor controls development in Dictyostelium discoideum, Science 241 615-616

main points
the 7 transmembrane protein (inferred from hydrophobicity plot) is the cAMP (pheromone) receptor and signals through the G protein to an adenylyl cyclase which makes cAMP for export. The receptor can be phosphorylated. A phosphodiesterase is out there to break down cAMP. The receptor looks like the metabotropic adrenergic receptor. A PDE outside the cell can brak down the cAMP.

sometimes cAMP has pathway to affect gene transcription:
S. Collins et al. From ligand to gene expression: new insights into the regulation of G-protein-coupled receptors. Trends in Biochemical Sciences 17 37-39, 1992
cAMP -> PKA -> CREB (phosphorylation) to control mRNA for receptor
CREB = cAMP response element binding protein
(but that will not be emphasized here)

Some general information on G proteins

TRANSPARENCY from:
E. M. Ross, Signal sorting and amplification through G protein coupled receptors (review) Neuron 3, 141-152, 1989

GTP-gamma-S useful in that it is a non-hydrolysable analogue of GTP and thus
causes a persistent dissociation of subunits and activation
Vibrio cholera toxin (heterodimer) cytosolic subunit affects Gs
Add an ADP-ribose unit ("ADP ribosylation") to arginine-174 of alpha
NAD+ (next TRANSPARENCY) is the donor of ADP-ribose
blocks deactivation of Gs by blocking GTP hydrolysis, cAMP up 100x
Bordetella pertussis toxin usually affects Gi
prevents release of ADP, renders Go and Gi insensitive to stimulation
both toxins affect transducins

at least 4 different alphas's alternative splicing, probably one gene
alpha0, alphai-1, alphai-2, alphai-3
different genes on different chromosomes
transducins different because segregated into different cells

Guanine nucleotide binding site conserved:
oncogene ras
bacterial elongation factor Tu (EFTu) - binds amino acyl tRNA to ribosome
ras is 170 amino acids long. it requires GAP (GTPase activating protein)
"extra 133 a. a. of heterotrimeric G protein alpha subunit helps to activate GTPase

B. Zheng et al., RGS-PX1, a GAP gor Gas and sorting nexin in vesicular trafficking, Science 294, 1939-1942, 2001
also
M. von Zastrow and K Mostov, A new thread in an intricate web (perspective), Science, 294, 1845, 1847, 2001
TRANSPARENCY (from von Zastrow and Mostov)
RGS(regulators of G protein signaling) also contribute to GAP function.
For stimulatory alpha subunit, it is on endosome
alpha must leave membrane, depalmitoylation and repalmitoylation
Note this figure also shows turnover of receptor like beta adrenergic receptor
for non-stimulatory G-proteins, alpha is stuck to membrane by myristoyl group, not detached, thus no cycling away from membrane.

2 beta subunits: 36k & 35k

maybe more gammas

additional reference:

E. J. Neer & D. E. Clapham, Roles of G protein subunits in transmembrane signalling. Nature 333 129-134, 1988

Metabotropic receptors use "7TD" receptor and G protein signalling
for instance, Muscarinic Cholinergic
most famous is beta-adrenergic receptor

activity is modulated
beta adrenergic receptor kinase BARK phosphorylates
then a protein like the visual system molecule arrestin (Barrestin) binds

There are metatobotropic receptors for
Dopamine, Serotonin, Histamine, Octopamine, Amino acids, Peptides

Background and review

M. J. Berridge, The molecular basis of communication within the cell, Scientific American 253 October 1985 pp. 142-152

TRANSPARENCY

stimulation pathway - cholera toxin blocks GTP->GDP so keep making cAMP
inhibitory pathway - pertussis toxin blocks GTP binding
Forskolin stimulate AC directly
Theophylline (caffeine) block cAMP phosphodiesterase

Pathway involving PIP2 -> IP3 and DAG
Phorbol esters stimulate C-kinase directly
Ca2+ iontophores mimic IP3 in cell
Note that he calls PLC PDE

Later structural work on G proteins

D. E. Coleman & S. R. Sprang, How G proteins work: a continuing story, Trends in Biochemical Science 21 41-44, 1996.

D. G. Lambright et al., Structural determinants for activation of the alpha subunit of a heterotrimeric G protein. Nature 369 621-628, 1994.

TRANSPARENCY to show difference in alpha if GTP vs GDP is bound

H. E. Hamm & A. Gilchrist, Heterotrimeric G proteins, Current Opinion in Cell Bilolgy, 8, 189-196, 1996.
(this is a very interesting article format in that it has an annotated bibliography)

TRANSPARENCY to show the trimer

A. Wittinghofer, Deciphering the alphabet of G proteins: the structure of the alpha, beta, gamma heterotrimer (Minireview). Structure 15 357-361, 1996

TRANSPARENCY from Wittinghofer cartoon shows major points

The receptor: N terminus outside cell, C-inside (heptahelical)
> 1000 receptors (from olfactory diversity)
2nd and 3rd loops and C terminus for interaction with alpha subunit of G protein
causes GDP to dissociate so acts like GEF= guanine nucleotide-exchange factor
at that point, anything (incl. GDP) could come in but GTP does since it is plentiful

G - proteins - at least 20 isoforms.
alpha-20 subunits. Size - alpha subunit of transducinG is 350 aa; 39-52 kDa
N & C terminals of alpha interact with receptor
Arg174 is cholera-sensitive site - Arg174 side chain interacts with PO4's
(Cys at C terminus of Gi is for ADP ribosylation by pertussis toxin)
not in p21Ras - may have to do with need for GAP
GTPase activating protein
myristoylation and palmitoylation contribute to membrane localization.
2 domains -
GTP - GDP binding like p21ras
alpha helical
beta: 5 subunits - all highly conserved, 36 kDa
has WD"propeller" domains found in some enzymes, 7 blades with 4 beta sheets each, with conserved WD sequence motif
gamma- 12 subunits, divergent, posttranslational modifications too, 6-9 kDa
isoprenylated - membrane association

earlier, there had been structural studies of Ras and EF-Tu (elongation factor)
Ras - conformation changes (GTP-GDP) just a few places
EF-Tu - majoral structural change
recently, there has been conformational studies of Gt
GDP - GTP alters Switch I (173-183), Switch II (195-215) and Switch III (227-238)
III is unique. I & II like Ras and EF-Tu in gamma-phosphate of GTP proximity
and in conserved Thr177 and Gly199

I. R. Vetter & A. Wittinghoffer, The guanosine nucleotide-binding switch in three dimensions, Science, 294, 1299- 1304, 2001.
A lot can be achieved by comparative structural analysis
TRANSPARENCY Switch 1 and 2 are more invariant in GTP bound form
the following two are just presented to show the awesome mechanistic detail now available
TRANSPARENCY GEF = guanine-nucleotide exchange factor
you have to actively kick out the GDP, then anything easily binds and GTP is abundant
the G-protein linked receptor is the GEF for the alpha in heterotrimeric like transducin
TRANSPARENCY GAP = GTPase activating protein

Here's an interesting tidbit:

D. E. Clapman, Mutations in G-protein-linked receptors: Novel insights on disease. Cell 75 1237-1239, 1993.

Ca2+ in blood is regulated by parathyroid hormone and thyrocalcitonin in push-pull system as well as vitamin D which is also a hormone to facilitate dietary absorption
bovine parathyroid Ca2+-sensing receptor 1085 a.a. (large)
like metabotropic glutamate receptor
elevates intracellular calcium via PLC to inhibit parathyroid hormone secretion (PTH)
PTH= 84 a.a. to enhance calcium
familial hypocalciuric hypercalcemia, autosomal dominant at 3q2, lethal if homozygous unless parathyroid glands removed

References:

M. J. Berridge, The molecular basis of communication within the cell, Scientific American 253 October 1985 pp. 142-152

J. T. Bonner, Hormones in social amoebae and mammals, Scientific American, June, 1969, pp 78-91

D. E. Clapman, Mutations in G-protein-linked receptors: Novel insights on disease. Cell 75 1237-1239, 1993.

D. E. Coleman & S. R. Sprang, How G proteins work: a continuing story, Trends in Biochemical Science 21 41-44, 1996.

S. Collins et al. From ligand to gene expression: new insights into the regulation of G-protein-coupled receptors. Trends in Biochemical Sciences 17 37-39, 1992

H. E. Hamm & A. Gilchrist, Heterotrimeric G proteins, Current Opinion in Cell Bilolgy, 8, 189-196, 1996.

P. S. Klein et al. A chemoattractant receptor controls development in Dictyostelium discoideum, Science 241 615-616

D. G. Lambright et al., Structural determinants for activation of the alpha subunit of a heterotrimeric G protein. Nature 369 621-628, 1994.

E. M. Ross, Signal sorting and amplification through G protein coupled receptors (review) Neuron 3, 141-152, 1989

I. R. Vetter & A. Wittinghoffer, The guanosine nucleotide-binding switch in three dimensions, Science, 294, 1299- 1304, 2001.

A. Wittinghofer, Deciphering the alphabet of G proteins: the structure of the alpha, beta, gamma heterotrimer (Minireview). Structure 15 357-361, 1996

M. von Zastrow and K Mostov, A new thread in an intricate web (perspective), Science, 294, 1845, 1847, 2001

B. Zheng et al., RGS-PX1, a GAP gor Gas and sorting nexin in vesicular trafficking, Science 294, 1939-1942, 2001

This page was last updated on January 11, 2002

Return to Stark home page
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The nitric oxide outline

Nitric oxide (and carbon monoxide) [gas "transmitters"]

In 1998, the Nobel Prize in Physiology and Medicine was awarded jointly to: ROBERT F. FURCHGOTT, LOUIS J. IGNARRO and FERID MURAD for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system.

TRANSPARENCY (from Snyder, 1992)
NO (nitric oxide, not nitrous oxide = laughing gas)
arginine is converted by NOS to NO and citrulline
diffuses to next cell, no vesicles or release mechanism
EDRF=endothelial derived relaxation factor, mediates vasodilation -corpus cavernosum
mediates penile erection via the parasympathetic nervous system
(Viagra blocks an enzyme that interferes with NO's effect.)
stimulates G-cyclase
lipophilic, crosses membranes
very unstable, so no breakdown mechanism needed
no breakdown, so synthesis should be carefully controlled
TRANSPARENCY (from Barinaga)
CO - derived from heme by heme oxygenase to stimulate guanylyl cyclase

Nitric oxide (NO) - Nitric oxide synthase (NOS) domains:
TRANSPARENCY (Fig. 1, Brenman & Bredt, 1997)
[1] N-terminal 1-498 amino acids of iNOS is oxygenase
binds heme (iron protoporphyrin IX, tetrahydrobiopterin and substrate (L-Arg)
binding the latter 2 cause enzyme to dimerize
[2] CaM binding - amino acids 499-530, thus Ca2+ regulates
[3] amino acids 531-1144
binds FMN (flavin adenine mononucleotide), FAD (FA dinucleotide), NADPH


There are (at least) 3 kinds (the first and third are constitutive):
(1) eNOS (endothelial) [recall that NO was originally called EDRF=endothelial derived relaxation factor
(2) iNOS (inducible by cytokines) for defensive cytotoxin production by macrophages
and
(3) nNOS neuronal (where a muscle form, nNOSm [mu] has an exon spliced between CaM & FMN
nNOS has PDZ domain near N-terminal

muscle:
nNOS is linked to dystrophin complex (specifically to PDZ domain on syntrophin)
developmentally, involved in myoblast fusion

neurons:
PDZ links to NMDA glutamate receptors via PSD-95 (and PSD-93)
TRANSPARENCY (Fig. 2, Brenman & Bredt, 1997)
P = postsynaptic density (PSD-25) [rat]
D = disc-large (dlg) [Drosophila]
Z = zonula occludentes-1 [mouse]
PSD=postsynaptic density, has 3 PDZ domains
NMDA implicated in neurotoxicity and LTP=long term potentiation
PDZ domains bind to PDZ domains or short amino acid consensus sequences

recent structural studies - unusual in that heme is exposed,
(originally, researchers expected that it would be like cytochrome P-450 with heme in pocket)
TRANSPARENCY Fig. 1 Crane et al.

References:

J. E. Brenman & D. S. Bredt, Synaptic signalling by nitric oxide, Current Opinion in Neurobiology 1997, 7, 374-378.

D. E. Koshland, Jr., The molecule of the year (Editorial) Science 258, 1861, 1992. Also: NO news is good news (Molecule of the year) pp. 1862-1863.

S. H. Snyder, Nitric oxide: First in a new class of neurotransmitters? (Perspectives) Science 257, 1992, 494 - 496.

M. Barinaga, Carbon monoxide: Killer to brain messenger in one step (Research News Neuroscience) Science 259, 309, 1993

I. Wickelgren, Biologists catch their first detailed look at NO enzyme (Research news, structural biology) Science 278, 389, 1997

B. R. Crane et al., The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science 278, 425-431, 1997

This page last revised on December 19, 2001

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The Chemoreception in Eukaryotes lecture

Eukaryotic microbe system

Alberts et al. information on:
Dictyostelium discoideum - especially 831-833, also 838, 846-847
Saccharomyces cerevisiae - 441-442, 833-834, 521-522, 721-722


classic paper: J. T. Bonner, Hormones in social amoebae and mammals, Scientific American, June, 1969, pp 78-91

TRANSPARENCY slime mold
Fig. 16-62, p. 832 shows the chemotaxix of a cell
Fig. 16-63, p. 832 shows the involvement of sudden actin development
Fig. 16-60, p. 831 shows the slug
Fig. 16-61 p. 831 shows the fruiting body

The discovery that the receptor was related to the G protein linked receptor (e.g. the beta adrenergic receptor)

TRANSPARENCY from:

P. S. Klein et al. A chemoattractant receptor controls development in Dictyostelium discoideum, Science 241 615-616

and cf.

J. L. Marx, Receptors highlighted at NIH symposium (research news) Science 238 615-616

main points
the 7 transmembrane protein (inferred from hydrophobicity plot) is the cAMP (pheromone) receptor and signals through the G protein to an adenylyl cyclase which makes cAMP for export. The receptor can be phosphorylated. A phosphodiesterase is out there to break down cAMP. The receptor looks like the metabotropic adrenergic receptor. A PDE outside the cell can brak down the cAMP.

Recent research
reserve paper:
J. Van Houten, Chemosensory transduction in eukaryotic microorganisms: trends for neuroscience? Trends in Neurosciences 17 62-71, 1994.

slime mold
TRANSPARENCY
detect bacteria ("prey") by folic acid, but cAMP signalling better understood
cAMP secreted in waves, phosphorylation and PDE help avoid self detection
in addition to aggregation, cAMP also used in prestalk and prespore
several receptor genes (cAR1-4) and 6 G protein genes
note that the calcium that comes as a result of PLC comes from intracellular stores and from extracellular sources
note here that PLC is delta type

yeast
TRANSPARENCY
haploid mating types a and a
peptides a-factor (13 a.a.) and alpha-factor (12 a.a.)
interestingly a-factor is secreted by multidrug-resistance transporter (&CFTR)
receptors are 7-transmembrane, but share no homology (convergent evolution?)
can be replaced by beta adrenergic
G protein, but beta and gamma more important than in the typical cascade
->-> (cascade)kinase arrests in G1 of cell cycle

paper also discusses sea-urchin spermatozoa
egg releases pheromones speract or resact, depending on species
guanylate cyclase is receptor and generates second messenger
as is: 1receptor for atrial natriuretic peptide (relax blood vessels and makes kidney
secrete sodium)
and E. coli enterotoxin receptor

paper also discusses several examples of chemoreception in ciliates
mating pheromones in Euploites
and, in Paramecia, folic acid and cAMP and glutamate

some important terminology introduced in end of paper
ERK = extracellular-signal-regulated kinase
= MAPK = mitogen activated protein kinase
phosphorylated by MEK = MAPK kinase (MAPKK) or ERK kinase (ERKK)

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The chemoreception in prokaryotes outline

Bacterial chemotaxis

enteric bacteria are motile: E. coli and Salmonella
importantly, bacteria are so small that they cannot compare concentrations along gradient
therefore must make temporal comparisons
go toward attractants and away from repellents (acids and alcohols)
4-8 flagella

Historical references:
J. Adler, Chemoreceptors in bacteria, Science 166 1588-1597, 1969
TRANSPARENCY Fig 1, Adler, showing attraction to aspartate
like Fig. 15-60, p. 773
J. Adler, The sensing of chemicals by bacteria, Scientific American 234 , #4, April 1976, 40-47

Early, there was question of whether, e.g., galactose metabolism (cf lac operon) was involved
Plenty of evidence that there are actually (multiple) receptors
1. some metabolized molecules do not attract
2a. even if mutations block metabolism, attraction can still be there
2b. chemicals like D-fucose (not used by bacteria) are attractants
3. attraction even if there is still another source of nutrition
4. competion within related compounds but not with others.
5. some mutants block taxis without blocking metabolism
Seemed to be separate receptors for galactose, glucose, ribose, aspartate, serine (5)
Now thinking is that there are 4 (see below)
specific and general mutants which lose chemotaxis
the behavior involves runs and twiddles - vary depending on direction along gradient
TRANSPARENCY Alberts et al., Fig. 15-63, p. 775 - what behavior looks like
counterclockwise - run
clockwise - tumble Text, Fig. 15-62, p. 774 TRANSPARENCY

Newer references:
Alberts et al., pp. 773-778
J.B.Stock, G.S.Lukat & A.M.Stock, Bcterial chemotaxis and the molecular logic of intracellular transduction networks, Ann. Rev. Biophys. Biophys. Chem. 20, 109-136, 1991.
Text, Fig. 15-61, p. 774 TRANSPARENCY - nature of falgellum and motor
flagellin is protein, uses H+ gradient for energy
30 genes to assemble
receptor, Fig. 7, Stock et al., TRANSPARENCY
there are 4 receptors: Tar (for galactose), Tsr, Trg & Tap
How receptors are oriented TRANSPARENCY Fig. 15-65, p. 776
Receptor is methylated at 4 sites for adaptation
TRANSPARENCY Fig. 15-67, p. 777
about 600 amino acids - methylation of glutamates
signalling pathway Stock et al, Fig. 1 and Table 1, TRANSPARENCY
receptors and motors are in membrane but not signalling molecules
Motor switches FliG, FliM and FliN
CheY - 129 amino acids, phosphorylated at Asp57, gets phosphorylated from His48 in CheA
CheY's phosphoaspartyl group is hydrolyzed
CheY is analogous to ras
Chez makes big (20) homopolymer and acts like GAP (GTPase activating protein)
CheA puts phosphate from ATP to histidine
CheAshould have MW of 73,000, but it is 250,000, so multimer
Recall that CheA is soluble, most histidine kinases are membrane
CheA is like src, a tyrosine kinase

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The sensory introduction lecture

SENSORY

Stimulus - NS - Response

Sensory coding
tuning at the receptor level - specialist, generalist
processing - lateral inhibition, feature detection
projection to CNS - question of "localization of function"
History (of general sensory mechanisms)-
Ernst Weber (1934)- DI/I = k
Gustav Fechner (1860) psychophysics - exponential
Modalities - (e.g. vision and audition, but there can be multiple submodalities in one modality, like, for somesthesis (touch), pressure is very different than nociception
Johannes Muller - 1830's law of specific nerve energies - like if you could transplant so that optic nerve connected up to brain through auditory nerve, then light would be perceived as if it were sound, so it is based on central connectivity rather than receptors that qualities are perceived, and, for submodalities of somesthesis, even the ascending tracts in the spinal cord are separated
Muller was an expert witness at a trial where a witness claimed to see his assailant by light of the phosphene from getting hit over the head
Stimulus - notion of "adequate" stimulus - Sherrington -the stimulus for which the least amount of energy works, like you see if you press on your eyeball, but it takes much less light energy, so rod is for light.
Receptors - Transduction -
receptor potential
generator potential
amplification
low threshold = high sensitivity
the most famous work on high sensitivity: S. Hecht, S. Schlaar & H. Pirenne, 1942, Energy, quanta and vision, J. Gen. Physiol. 25, 819-840 - a person can see 6 - 14 quanta over an area of 500 rods => each rod can see 1 photon
acuity (vs. sensitivity)
adaptation
phasic - rapidly adapting - Pacinian corpuscle
tonic - slowly adapting - muscle receptors
Receptive field the sensory field converging on the central neuron studied
each receptor would diverge to many neurons in the brain by lateral connections, and, by the same token, each cell high up in the nervous system would get input from many receptors
excitation and inhibition
Lateral inhibition -
H. K. Hartline (US) (1967 Nobel)
"primary physiological and chemical visual processes in the eye"
Feature abstraction -> pattern recognition

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The vertebrate visual transduction outline

Vision

(lots of information on transparencies and some figures from Alberts et al.)

Retina, rods and cones
TRANSPARENCY Fig. 22.6 p. 1144 retina
Rod, periphery, dim black and white, sensitivity
Very sensitive - 1 photon
Cone, ..., fovea, color, acuity
Stacks of disks.TRANSPARENCY Fig.22-7, p.1145 & Fig.15-39, p.753
G-protein coupled receptor, with its 7 membrane spans, would have N-terminus on outside (and glycosylation site is near there)
shows N-terminal of opsin in lumen
Lumen is topologically like the outside of the cell TRANSPARENCY (home made)

Visual pigment
Halobacterium halobium TRANSPARENCY Fig. 10-30, p. 495
Bacteriorhodopsin TRANSPARENCY Fig. 10.31, p. 496

G. Wald, Molecular basis of Visual excitation (Nobel prize speech) Science 162, 230-239, 1968
TRANSPARENCY (Figs 3, 7 & 8)
Vitamin A cis->trans conversion (from Wald)
vitamin A is the chromophore
Metarhodopsins. "bleaching" into opsin and trans vitamin A
TRANSPARENCY conversion steps isolated by liquid nitrogen, etc.
old terminology (prelumirhodopsin now bathorhodopsin)
note, now it is known that MII is R*
TRANSPARENCY some carotenoids - two vitamin A's end to end
TRANSPARENCY Rhodopsin mutants- ADRP-
(autosomal dominant retinitis pigmentosa) (Dryja)
T. P. Dryja et al., Mutation spectrum of the rhodopsin gene among patients with autosomal retinitis pigmentosa, PNAS 88, 9370-9374, 1991

Color vision
turtles, fish, birds have good color vision, needed to re-evolve for mammals
evolutionary bottleneck hypothesis - selection pressure on color vision relaxed in early mammalian evolution when animals were nocturnal
Young -Helmholtz trichromatic theory
3 kinds - evolutionarily related 2 (red and green) on X
color blind people might be "dichromats" as opposed to trichromats
NW Monkeys - usually 2 but females may have variants on 2 X's
and retina would be mosaic because of Mary Lyon X-inactivation hypothesis and Barr body
J.D.Mollon "Tho' she kneel'd in that place where they grew..." The uses and origins of primate colour vision. J. Exp. Biol. 146, 21-38, 1989
Color blindness - on X in males - superfamily
TRANSPARENCY Fig. from Nathans et al.
J. Nathans et al., Molecular genetics of human color vision: the genes encoding blue, green and red pigments, Science, 232, 193-202, 1986
differences between yellow and green absorbing
does not show 2 palmitylated cysteines on C terminus
or phosphorylation sites
TRANSPARENCY Fig from Nathans et al. on color blindness genes
J. Nathans et al., Molecular genetics of inherited variation in human color vision, Science, 232, 1986, 203-232
odd in that numbers of gene copies is screwed up
more recent work confirms and extends - there can be many copies
M. Neitz and J. Neitz, Numbers and ratios of visual pigment genes for normal red-green color vision, Science 267, 1013-1016

Transduction and the dark current
mitochondria packed in inner segment pump sodium out, and it leaks into the inner segment, but less so when signal transduction cascade closes channels in the light in in the dark into the TRANSPARENCY (home made, like 15-40 shown early)
lots of information, wiring of retina, rod association with retinal pigment epithelium, involved in turnover of vitamin A, membrane shedding in the phagosomal-lysosomal system, and accumulation of lipofuscin, the aging pigment, as the indigestible residue of the phagolysosomal system in these post-mitotic r.p.e. cells which must last a lifetime)
Rod works backwards, physiologically, hyperpolarizes in response to light
TRANSPARENCY like Fig. 4-54, p. 182 - Baylor rod current recording - like a whole organelle patch clamp

Generations of Transduction models (very much updated from 1998)

TRANSPARENCY Here (Polans, Baehr and Palczewski 1996) is one diagram of the phototrandduction cascade (Rhodopsin gets excited by a quantum to R*; R* activates transducin. The alpha subunits of this heterotrimeric protein effect the removal of inhibitory gamma subunits from the PDE alpha-beta enzyme. The cGMP hydrolyzed would have opened channels, so, with light, channels close. Since Ca2+ comes in through the channel, it can affect adaptation (3 ways). (1) Ca2+ binds recoverin (Rec) which binds and inhibits rhodopsin kinase (RK) which phosphorylates rhodopsin making it ready for arrestin binding. [Arrestin is also known as S-antigen (S=soluable) or 48 k (kD) protein.] (2) Also Ca2+ binds GCAP (GC activating protein) inhibiting its activation of GC (gualyate cyclase) which makes cGMP. (3) Ca2+ binds to CaM (calmodulin) which binds to the beta subunit of the CNG channel. In summary, lowering calcium (after photon absorption) increases cGMP synthesis, increases channel sensitivity to cGMP and decreases the lifetime of R* function, helping to counteract excitation

It is useful to reiterate the cascade with different diagrams, emphasizing where things take place

TRANSPARENCY Fig 4 (Molday 1998) is another diagram

TRANSPARENCY Fig 1 (Lem 1998) is another diagram.

TRANSPARENCY Fig. 2 (Jindrova 1998) is yet another diagram

TRANSPARENCY The molecules peripherin/rds and rom-1 are thought to be involved in maintaining the structure of the rod disk (Fig. 11, same TRANSPARENCY as Fig. 4 above (Molday 1998))

TRANSPARENCY Now, peripherin/rds is mapped with respect to mutations which cause retinal degeneration (Fig. 13 (Molday 1998))

TRANSPARENCYThe ABCR/RIM protein of Stargardt's degeneration is shown (Fig. 15 (Molday 1998)). There are 12 transmembrane segments and 2 ATP binding casettes

TRANSPARENCY Fig. 17 (Molday 1998) shows the location and ATP dependence of ABCR/RIM. For orientation purposes, Peripherin/rds - rom-1, GC and rhodopsin are shown. "Peripherin" is the name based on EM immunocytochemistry, it is localized to the periphery of the disks. "rds" is the genetic name, "retinal degeneration slow."

Fig. 1 (Polans et al. 1996) is a diagram of the domains of GC. An intracellular kinase domain is where GCAP interacts. The intracellular catalytic domain is where GTP is converted to cGMP. What the extracellular domain does is not clear.

TRANSPARENCY This model of the phototransduction cascade emphasizes diseases (Fig. 18 (Molday 1998, same TRANSPARENCY as above). Abbreviations:
ADRP - autosomal dominant retinitis pigmentosa
ARRP - (recessive)
CSNB - congenital stationary night blindness
CRD - cone rod dystrophy
MD - macylar dystrophy
XLRP - X-linked retinitis pigmentosa
XLRS - X-linked retinoschisis

Fig 1 (Molday 1996) shows alpha and beta subunits of bovine rod CNG's. For rods (and in olfactory receptors) the channel probably is a hetero-oligimer. Many features are familiar, S1 - S6 with S4 having a voltage-sensor motif and a pore between S5 and S6. There are cGMP binding sites. It should be intuitively obvious to the most casual observer that "C" (alpha helix) stands for bacterial catabolite gene activator protein. By the same token, GARP = glutamic-acid-rich protein.

Fig. 7 (Molday 1998) repeats the previous ingormation but includes linear representatiuons for the protein domains and the now familiar representation of the tetrameric channel.

From (Lem 1998). Table 1 is a list of diseases resulting from mutations in G-protein-coupled receptors, Table 2 for G-protein subunits and Table 3 for other molecules of the cascade.

Several general and summary notes:
There are 10x lower numbers of molecules further into cascade R > G > PDE
S = S antigen = 48 kD protein = arrestin

TRANSPARENCY, PDE - rd mutant mouse is in beta subunit (Bowes)
C. Bowes et al., Isolation of a candidate cDNA for the gene causing retinal degeneration in the rd mouse, PNAS 86, 9722-9726, 1989
C. Bowes et al., Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase, Nature, 347, 677-680, 1990

Review of calcium involvement
first thought of as second messenger in 1970's until cGMP won out
then a calcium binding protein recoverin was discovered
after some confusion,
now known to inhibit rhodopsin kinase at high calcium
These calcium binding proteins have EF-hands with negatively
charged a.a.'s
GC's (there are 2 in retina) are big proteins with one membrane pass
GCAP (guanylate cyclase activating proteins [there are 2])
activate target in absence of calcium
contribute to adaptation
Calcium is more obviously important in invertebrate phototransduction

References:

C. Bowes et al., Isolation of a candidate cDNA for the gene causing retinal degeneration in the rd mouse, PNAS 86, 9722-9726, 1989

C. Bowes et al., Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase, Nature, 347, 677-680, 1990T. P. Dryja et al., Mutation spectrum of the rhodopsin gene among patients with autosomal retinitis pigmentosa, PNAS 88, 9370-9374, 1991

Jindrova, H., 1998 Vertebrate phototransduction: activation, recovery, and adaptation. Physiol Res. 47: 155-68.

Lem, J., 1998 Diseases of G-protein-coupled signal transduction pathways: The mammalian visual system as a model. Sem. Neurosci. 9: 232-239.

Molday, R. S., 1996 Calmodulin regulation of cyclic-nucleotide-gated channels. Curr Opin Neurobiol. 6: 445-52.

Molday, R. S., 1998 Photoreceptor membrane proteins, phototransduction, and retinal degenerative diseases. The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 39: 2491-513.

J.D.Mollon "Tho' she kneel'd in that place where they grew..." The uses and origins of primate colour vision. J. Exp. Biol. 146, 21-38, 1989

J. Nathans et al., Molecular genetics of human color vision: the genes encoding blue, green and red pigments, Science, 232, 193-202, 1986

M. Neitz and J. Neitz, Numbers and ratios of visual pigment genes for normal red-green color vision, Science 267, 1013-1016

Polans, A., W. Baehr, and K. Palczewski, 1996 Turned on by Ca2+! The physiology and pathology of Ca(2+)-binding proteins in the retina. Trends Neurosci. 19: 547-54.

G. Wald, Molecular basis of Visual excitation (Nobel prize speech) Science 162, 230-239, 1968

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The retinitis pigmentosa and autosomal macular degeneration lecture

Retinitis pigmentosa - 50,000-100,000 Americans
Night blindness, loss of midperipheral vision (where rods are in high concentration)
autosomal and x-linked, dominant and recessive
there is a staggering list Table (Bird)
Candidate gene analyses, SSCP = single stranded conformational polymorphism,
rely on screening many unrelated people
Figure from Huang et al.
Some genes which are obvious candidates are in the phototransduction cascade
Figure from Farber
"Membrane" proteins usually inferred from Kyte-Doolittle hydrophobicity plots
rhodopsin missense mutations cause 20-20% of ADRP
there is a rhodopsin null mutation which causes ARRP
Figure from Bird covers the vast number of opsin mutants found, many studies

M. E. McLaughlin et al., Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nature Genetics 4, 130-134, 1993
gene has 22 exons
Figure from Farber - note that it is really odd that there are 2 defects in rd mice
screen 7 exons so far in 99 patients
Gln298X nonsense, Arg531X (nonsense), Pro496(1-pb del), His557Tyr

S. H. Huang et al., Autosomal recessive retinitis pigmentosa caused by mutations in the alpha subunit of rod cGMP phosphodiesterase. Nature Genetics 11, 468-471, 1995
22 exons on human chromosome 5q31.2-34
ARRP Tyr583Ter nonsense,Ser344Arg nonsense, Trp561Ter nonsense

T. P. Dryja et al., Mutations in the gene encoding the a subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa. Proc. Natl. Acad. Sci. USA 92, 10177-10181, 1995
10 exons - SSCP
find nonpathological changes - 3 silent, 2 missense, 2 1-base intron changes
and one variation in length of a poly-A repeat in an intron
5 pathological changes - 3 nulls and 2 missense

also there are other animal models which suggest candidate genes like the rds mouse
Figures from Molday
R. S. Molday, Peripherin/rds and rom-1: Molecular properties and role in photoreceptor cell degeneration. Chapter 11 in Progress in Retinal and Eye Research 13, 271-299, 1994
rds on short arm of human chromosome #6, has 2 introns
346 a.a. polypeptide with 4 membrane spans
rom-1 on human chromosome 11q13
351 a.a.
Kajiwara et al., Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science 264, 1604-1608, 1994.
Peripherin: Pro219del, Pro216Leu and Ser212Gly -> ADRP
Leu185Pro in peripherin like autosomal dominant but transmit only 1/4 of offspring
Peripherin on human chromosome 6p
ROM1 on human chromosome 11q find Gly80(1-bp ins) and Leu114(1-bp ins)
cause premature stop at 131 - early - probably null
If both genes mutant heterozygous, patient has RP
if only one, people are asymptomatic, do not know about homozygous for either

Age related macular degeneration (ARMD)

R. Allikmets et al., Mutation of the Stargardt's disease gene (ABCR) in age-related macular degeneration. Science 277, 1805-1807, 1997
also
E. Pennisi, Gene found for the fading eyesight of old age (Research news, Human genetics) Science 277, 1765-1766, 1997
"Macula" is a term for the central part of the retina, like fovea
Macular degeneration (11 million Americans) comes in 2 types:
(1) wet (20%) (exudative) can be an emergency treatable with laser
one model, Sorsby's fundus dystropht - TIMP3 - tissue inhibitor of metalloproteinase-3
(2) dry (80%) with drusen of debris under retina
a gene on 1p21 with 51 exons codes STDG1 also ABCR
=ATP-binding casette transporter-retina
TRANSPARENCY Fig 1 from paper -different mutations for Stargardt's vs AMD
looks like CFTR
it is also the protein called "rim protein" RmP (in rods)
There is a lot of controversy over this point, and critics claim that in such a large gene, there can be neutral mutations, so it is necessary to show that the genetic alteration cosegregates with the disorder.
But the work goes on (Shroyer et al., 2002). This is a huge gene (50 exons). There is a missense mutation V767D in an individual who also has W1408R and R1640W, and this appears to be null and causes RP.
This protein is of likely importance as a transporter of retinal based on the fact that retinal stimulates ATP hydrolysis (Sun et al., 1999)

References

R. Allikmets et al., Mutation of the Stargardt's disease gene (ABCR) in age-related macular degeneration. Science 277, 1805-1807, 1997

A. C. Bird, Retinal photoreceptor dystrophies LI Edward Jackson Memorial Lecture. Am. J. Ophthalmology 119, 543-562, 1995

T. P. Dryja et al., Mutations in the gene encoding the a subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa. Proc. Natl. Acad. Sci. USA 92, 10177-10181, 1995

D. B. Farber, From mice to men: The cGMP phosphodiesterase gene in vision and disease (The Proctor Lecture). Investigative Ophthalmology and Visual Science 36, 263-275, 1995

S. H. Huang et al., Autosomal recessive retinitis pigmentosa caused by mutations in the alpha subunit of rod cGMP phosphodiesterase. Nature Genetics 11, 468-471, 1995

Kajiwara et al., Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science 264, 1604-1608, 1994.

M. E. McLaughlin et al., Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nature Genetics 4, 130-134, 1993

R. S. Molday, Peripherin/rds and rom-1: Molecular properties and role in photoreceptor cell degeneration. Chapter 11 in Progress in Retinal and Eye Research 13, 271-299, 1994

E. Pennisi, Gene found for the fading euesight of old age (Research news, Human genetics) Science 277, 1765-1766, 1997

N. F. Shroyer et al., Null misense ABCR (ABCA4) mutations in a family with Stargardt disease and retinitis pigmentosa, Invest. Ophthalmol. Vis. Sci., 42, 2757-2761

Sun, H., RS Molday and J. Nathans, Retinal stimulates ATP hydrolysis by purified and reconstructed ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease. J. Biol. Chem., 274, 8269-8281, 1999

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The Invertebrate visual transduction outline

Invertebrate visual transduction

There is a long history of studying invertebrate model preparations. Because of the large cells, the horshshoe crab Limulus was used to demonstrate how lateral inhibition enhances contrast detection. Nobel prize paper -- H. K. Hartline, Visual receptors and retinal interaction, Science 164, 270- 278, 1969

(empahsis on Drosophila)

unlike vertebrate, photoreceptors depolarize in response to light, not cGMP PDE, but PLC
Review phosphoinositide cascade: PIP2 -> IP3 & DAG
S. Kim, Analysis of norpA encoded multiple subtypes of phospholipase C in Drosophila melanogaster, 1997, Ph.D. Dissertation, SUNY Buffalo
TRANSPARENCY PI -> PIP -> PIP2 -> IP3 + DG
IP3 -> IP2 ->IP
DG -> PA [ATP donates phosphate](phosphatidic acid) -> CDP-DG (citidine diphosphate diacylglycerol)
TRRANSPARENCY - a comparison of how a neurotransmitter would activate PLC beta (already covered) vs. how a growth factor (with its tyrosine kinase) would act through PLC gamma
TRANSPARENCY the domain structures of PLC's:
beta-1 ia 1215 amino acids, has X and Y (conserved) domains
gamma has SH (src homology) domains between X and Y, 1289 amino acids
delta1 is 756 amino acids

PICTURE (from the research portion of my web site) ommatidia (about 800/compound eye) with R1-6, R7 and R8, plus 3 simple eyes (ocelli)
photoreceptive organelle is called rhabdomere
"genetic dissection"field started in late '60's with screens for mutants by (mostly) Benzer and Pak
genetic diisection or retina : work by Harris Stark and Walker, 1976, J. Physiol., 256, 415-439:
PICTURE (from the research portion of my web site) R1-6 is sensitive to blue and UV, R7 to UV and R8 to blue-green
ninaE gene for R1-6 rhodposin, Rh1
(there are other opsins):
Rh2 (ocelli)
Rh3 & Rh4 (UV rhodopsins in R7) (work by Zuker -- Feiler et al., J. Neurosci., 12, 3862-3868, 1992)
Rh5 & Rh6 in R8 (work by Britt -- Chou et al., Neuron 17, 1101-1115, 1997, also Paulsen -- Huber et al., FEBS Lett. 406, 1997, 6-10)
TRANSPARENCY shows rhodopsin homologous to vertebrates, Figure is from:
W. L. Pak, Use of Drosophila mutants in vision research, Mol. Cells 6 117-124, 1996
"nina" stands for "neither inactivation nor afterpotential" (refers to ERG = electroretinogram)
ninaA is a photoreceptor - specific cyclophilin
pepitidyl-proline cis-trans isomerase
also chaperone

norpA = "no receptor potential"
shown to code for PLC
recent paper:
R.R.McKay, D.-M.-Chen, K.Miller, S.Kim, W.S.Stark,R.D.Shortridge, Phospholipase C rescues visual defect in norpA mutants of Drosophila melanogaster. J. Biol. Chem. 27013271-13276, 1995
attach norpA coding sequence to ninaE promoter,
put in P-element, insert gene into norpA mutant
recovery in:
western
activity
LM & EM immunocytochemistry
and ERG (electroretinogram)

TRANSPARENCY (that I drew) and TRANSPARENCY - Signal transduction cascade from
Zuker, C. S. The biology of vision in Drosophila. PNAS 93 571-576, 1996
talk about:
regeneration of PIP2
rdgA (retinal degeneration) in diacylglycerol kinase
rdgB is PI transfer protein
inaC (inactivation - no afterpotential) is eye-specific PKC
and others
TRP and TRPL (transient receptor potential [like]) channels
TRANSPARENCY shows comparison of vertebrate and invertebrate phototransduction cascades - from K. Scott & C. Zuker, Trends in Biochemical Sciences, 22, 350-354, 1997

To understand channels, TRANSPARENCY transduction scheme:
B. Minke & Z. Selinger, Inositol lipid pathway in fly photoreceptors: Excitation, calcium mobilization and retinal degeneration. In Prog. Retinal Res. vol 11, eds. N. N. Osborne & G. J. Chader, Oxford, Pergamon Press
microvilli vs submicrovillar cisternae

D. D. Friel, TRP: Its role in phototransduction and store-operated Ca2+ entry (minireview). Cell 85 617-619, 1996
1275 a.a. 6 membrane spans (=> 4/channel)
TRANSPARENCY store-operated channel = SOC

PDZ domains (already covered with respect to NMDA receptor - NOS interaction
INAD (ina = inactivation, no afterpotential) protein binds channel
The molecules of transduction are compartmentalized together in a transduceosome
TRANSPARENCY (from Kim thesis) Rhodopsin, trp, Cam, norpA, G-protein, inaC, PLC all nearby, and transduction is thus very fast
R. van Huizen et al., Two distantly positioned PDZ domains mediate multivalent INAD - phospholipase C interactions essential for G-protein-coupled signalling, EMBO Journal, 1998, in press
TRANSPARENCY (van Huizen) INAD has 5 PDZ domains, 2 binding PLC

References

D. D. Friel, TRP: Its role in phototransduction and store-operated Ca2+ entry (minireview). Cell 85 617-619, 1996

Harris, W.A., Stark, W.S. and Walker, J.A. Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster. Journal of Physiology, 1976, 256, 415-439.

H. K. Hartline, Visual receptors and retinal interaction, Science 164, 270- 278, 1969

S. Kim, Analysis of norpA encoded multiple subtypes of phospholipase C in Drosophila melanogaster, 1997, Ph.D. Dissertation, SUNY Buffalo
(this one is not on reserve)

McKay, R. R, Chen, D.-M., Miller, K., Kim, S., Stark, W. S., Shortridge,R. D. Phospholipase C rescues defect in norpA mutant of Drosophila melanogaster. Journal of Biological Chemistry, 1995, 270, 13271-13276. PubMed

B. Minke & Z. Selinger, Inositol lipid pathway in fly photoreceptors: Excitation, calcium mobilization and retinal degeneration. In Prog. Retinal Res. vol 11, eds. N. N. Osborne & G. J. Chader, Oxford, Pergamon Press

W. L. Pak, Use of Drosophila mutants in vision research, Mol. Cells 6 117-124, 1996

K. Scott & C. Zuker, Trends in Biochemical Sciences, 22, 350-354, 1997

van Huizen, R., Miller, K., Chen, D.-M., Li, Y., Lai, Z.-C., Raab, R.W., Stark, W. S., Shortridge, R. D., Li, M. Two distantly positioned PDZ domains mediate multivalent INAD-phospholipase C interactions essential for the G protein-coupled signalling. EMBO Journal, 1998, 17, 2285-2297. PubMed

Zuker, C. S. The biology of vision in Drosophila. PNAS 93 571-576, 1996

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The Chemical senses lecture

Vertebrate chemoreception

Smell (olfaction)

traditional knowledge

Smell- Olfaction chemicals (air, but definition hard for aquatic animals) - Complex
unusual primaries like camphoraceous, musky, ... pungent and putrid
many primaries, receptors difficult to reach, in nasal epithelium
the idea is that there would be receptor molecules that bind odorants which fit:
J.E.Amoore et al. The stereochemical theory of odor, Scientific American, Feb. 1964
interestingly, though it seems to be a forgotten field, Amoore studied human anosmias -- there are specific genetic smell-blindnesses
receptors are ciliary and their cilia have "9 + 2" microtubules- histology
these cells are unusual in that they turn over
7 transmembrane domain receptor
channel is like that of photoreceptor
vomeronasal organ snake tongue olfaction, hamster sex pheromone

A small reference to recent research:
L. Stowers et al., Loss of sex discrimination and male-male aggression in mice deficient for TRP2, Science, 295, 1493-1500, 2002
mice engineered to lack this "transient receptor potential" (see invertebrate phototransduction lecture) channel lack expression in the vomeronasal organ and lack pheromonal detection for male discrimination of females as well as for male-male aggression.

The news of cloning of olfactory receptors:
M. Barinaga, How the nose knows: Olfactory receptor cloned, Science 252, 209-210, 1991

new work

Diego Restrepo et al., Second messenger signalling in olfactory transduction. J. Neurobiol. 30, 37-48, 1996
TRANSPARENCY of olfactory neuron and transduction mechanism
like Fig 22-18 on p. 1156 which covers how basal (stem) cell -> neuron
(not many neuron types "turn over," needs to make new connections)
Fig.: note that the gCa(L) is voltage-dependent and gK(Ca) is Ca dependent
there is a sodium-calcium antiport
cAMP -> Ca2+ influx -> Cl- channel
IP3 ->Ca2+ channel in plasmalemma which opens nonspecific cation and K+ channel

H. Breer et al., Molecular genetics of mammalian olfaction, Behavior Genetics 26, 209-219, 1996 on reserve
(this is an easy-to-read paper)
Olfactory marker protein (OMP) 19 kDa 162 a.a. but big mRNA with 1600 untranslated
upstream binds Olf-1 (olfactory factor)
expressed in mature neurons
OBP odorant binding protein19kDa homodimer in mucus
like lipocalins: serum retinol binding protein and hamster urinary "aphrodisin"
Golf is sort of like a Gs; there is a specific adenylate cyclase (type III)
nonspecific channel most like that of photoreceptor
(similar mechanism - gating from internal cyclic nucleotide)
the IP3-gated channels have properties like endoplasmic reticulum IP3 receptor-channel
receptor - minimal in that loops are small and thus like opsin,
TRANSPARENCY lots of variability in transmembrane spans #4 & #5
900-950 bp intronless gene, come in clusters
20 in 400 kb on 17p, also on human chromosome #19
by contrast, in immune system, somatic DNA is rearranged to give diversity, but not in olfaction
TRANSPARENCY - expression
each cell expresses one or several, and arranged in spatial clusters
control of expression will be the next big question. Olf-1 is helix-loop-helix factor

TRANSPARENCY Fig. 2 (Molday 1996) elaborates on the olfactory transduction pathway through Golf to AC to cAMP which opens CNG channel causing an influx of Ca2+. Calcium ions further activate a Cl- channel reinforcing the depolarization. But Ca2+ also feeds back by binding calmodulin to activate cAMP PDE

Taste (gustation)

I corresponded with Dr. Lindemann who has an interesting site about taste.

Traditional knowledge

Taste [on tongue] Several types of papilla including the circumvallate papillae on the back of the tongue, shown in this picture from our histology course
Within each papilla are numerous clusters of cells called taste buds shown in this histology picture
Papillae -> taste buds. 1 & 2 support, 3 sensory, <- 4 basal
Unique feature: turnover of receptor cells
Gustation - chemicals - Many "flavors" are smell
dissolved in water

According th Lindemann, the following dogma is not quite true:
sweet - tip of tongue - cAMP close K+ channel - depolarize
salt - front sides of tongue - amiloride blocked Na+ channel
sour - back sides of tongue - pH sensitive K+ channel
bitter - back middle of tongue - K+ Channel or PLC
note that there could be different molecular receptors on one cellular receptor:
some people are taste "blind" for PTC=phenylthiocarbamide
TT and Tt genotypes are tasters

New work

D. W. Tingley, Transduction in the retina is also a matter of taste (research brief), The Journal of NIH Research 7 (October 1995) 44-48
(review of recent work by Margolskee et al. and the discussion which arose)
TRANSPARENCY - bitter in circumvallate papillae
Use bitter tasting chemical denatonium but others like quinine may be different
earlier found gusducin, now transducin, the two are 80% homologous at amino acid level
transducin activates taste specific phosphodiesterase,
but, unusually, channels are probably blocked by cyclic nucleotides
questions still to answer
what are the receptor molecules?
do transducin and gusducin work in the same cells?
how do IP3 and cyclic nucleotide pathways relate?
what are differences between different taste primaries?
how do different species relate? (frog not respond to sweet)
G.T.Wong et al., Transduction of bitter and sweet taste by gusducin, Nature 381, 796-800,
knockout mice for alpha subunit shows same one used in bitter and sweet

Fig. 1 a (Kinnamon and Margolskee 1996) shows taste stimuli, at the apical surface, can interact through channels or via receptors and second messengers. Ultimately, channels on the basolateral surface are affected, with the calcium channel being involved in transmitter release.

Fig. 1 b details these mechanisms for channels:
(1) salt is via a sodium channel blocked by amiloride
(2) acid is by:
(i) a proton blocked potassium channel
(ii) an amiloride blocked sodium channel which is carrying protons
(iii) a proton gated cation channel
(3) bitter is by quinine or divalent ion blocked potassium channel

Fig. 2 (Kinnamon and Margolskee 1996) elaborates the mechanisms involving receptors signalling through second messengers (as well as additional channel-mediated tastes)
(1) amino acids:
(i) the metabotropic glutamate receptor is for umami
(ii) the arginine indirect cation mechanism is for catfish
(2) sweet:
(i) amiloride blocked cation channel
(ii) AC mechanism
(iii) PLC mechanism
(for ii and iii, phosphorylation of potassium channel, channel closes, cell depolarizes)
(3) bitter:
(i) PLC
(ii) PDE mechanisms

Recent advances:

C. Holden, A taste for MSG (random samples) Science 287 799, 2000,
umami is like brain glutamate receptor except less sensitive

M. Barinaga, Family of bitter taste receptors found, Science 287, 2133-2135, 2000.
Start with tasters vs non-tasters ofPROP, locate mutation, look for G protein coupled receptors.
Find family of about 50, overlap in cells that use gusducin

RJDavenport, New genes may be key to sweet tooth, Science 292, 620-621, 2001
Till now, sweet receptor was elusive. It's a G protein-coupled receptor.
Using tasters and non-tasters (genetic difference in mice), a chromosomal region was identified.
Then looking at the corresponding area in the human genome, the gene expressed in taste cells was found.

References:

J.E.Amoore et al. The stereochemical theory of odor, Scientific American, Feb. 1964
This page was last updated on September 27, 2000

M. Barinaga, Family of bitter taste receptors found, Science 287, 2133-2135, 2000.

M. Barinaga, How the nose knows: Olfactory receptor cloned, Science 252, 209-210, 1991

H. Breer et al., Molecular genetics of mammalian olfaction, Behavior Genetics 26, 209-219, 1996

RJDavenport, New genes may be key to sweet tooth, Science 292, 620-621, 2001

C. Holden, A taste for MSG (random samples) Science 287 799, 2000

Kinnamon, S. C., and R. F. Margolskee, 1996 Mechanisms of taste transduction. Curr Opin Neurobiol. 6: 506-13.

Molday, R. S., 1996 Calmodulin regulation of cyclic-nucleotide-gated channels. Curr Opin Neurobiol. 6: 445-52.

Diego Restrepo et al., Second messenger signalling in olfactory transduction. J. Neurobiol. 30, 37-48, 1996

L. Stowers et al., Loss of sex discrimination and male-male aggression in mice deficient for TRP2, Science, 295, 1493-1500, 2002

D. W. Tingley, Transduction in the retina is also a matter of taste (research brief), The Journal of NIH Research 7 (October 1995) 44-48

G.T.Wong et al., Transduction of bitter and sweet taste by gusducin, Nature 381, 796-800

This page was last updated March 20, 2002

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The development in the Drosophila eye lecture

Development

I'm rather fond of this material. Partly because my work introduced the sevenless gene. At the time, the sevenless mutation was used to show that R7 is a UV receptor.

Know from earlier courses:
induction important "optic vesicle"
TRANSPARENCY from introductory textbook (like Fig. 22-2, p. 1141 Alberts)
signifies that retina is outgrowth of CNS
know that induction from retina makes ectoderm turn into lens placode

Drosophila is a model for understanding development, generally
Lewis, Weichaus, Nusslein-Volhard - 1995 Nobel Prize
Order of action: maternal genes, zygotic genes, homeotic genes
Imaginal discs TRANSPARENCY
(introductory book version of Fig. 21-71 p. 1097 Alberts)
homeotic mutants TRANSPARENCY
(introductory textbook version of Fig. 21-67, p. 1093 Alberts - antennapedia)
homeotic gene has homeobox (183 bp of DNA),
DNA binding protein has homeodomain (helix turn helix)

R. Mestel, Secrets in a fly's eye, Discover (July, 1996) 106-114
useful to introduce the ommatidial structure first
TRANSPARENCY - diagram from Cagan (now at Wash. U) and Zipursky
How do > 750 ommatidia with some 19 cells develop?
(receptors (R1-6, R7 & R8, cone cells, bristles, pigment cells)
History
Benzer (late 1960's) started screen for receptor mutants (like sev)
(vision is not essential [for mating] in Drosophila melanogaster)
Ready - 1976 - showed usefulness of imaginal disk in development
TRANSPARENCY - development in the eye imaginal disk
morphogenetic furrow
American plans of development- who your neighbors are is important
vs European (exemplified inC. elegans where lineage is important
- 1986 - showed in sev that R7 precursor becomes cone cell
Rubin - early 1980's - step up molecular developmental approach
Later, Zipursky, Banerjee, Simon, many others
Walter Gehring (Basel) fly eyeless like small eye mouse and aniridia in human, PAX-6 gene
eye development in fly, human, mouse, flatworm, squid may have common control even though different eyes had been previously thought of as being an example of convergent evolution

tyrosine kinase signalling
small G protein
sevenless signalling pathway
TRANSPARENCY Fig. 15-52, p. 764 - sequential addition of receptor cells in Drosophila eye: R8, R2 & R5, R3 & R4, R1& R8, R7
TRANSPARENCY Fig. 15-53, p. 765 Alberts et al. early R7 signalling steps
Boss = bride of sevenless is 7 transmembrane domain ligand
C-terminal intracellular, N-terminal extracellular
sevenless is receptor tyrosine kinsae -
2 transmembrane subunits, 2 extracellular subunits
expressed everywhere except R2 R5 and R8
It is a topic of intense present interest how this signals across membrane
Drk = downstream of receptor tyrosine kinase
which is a small SH adaptor protein, SH = src homology
src = oncogene of Roux sarcoma virus
Sos = son of sevenless
which is a GNRP (guanine nucleotide releasing protein)
to exchange GTP for GDP on ras
ras = rat sarcoma [viral ras oncogene of normal protooncogene]
ras is actually linked to membrane by fatty acid
GAP (sextra in Drosophila) does opposite (GTPase activating protein)

TRANSPARENCY (my drawing)
other steps -> signalling to nucleus
MAPK = mitogen activated protein kinase
alias ERK = extracellular signal regulated kinase


TRANSPARENCY Fig. 1 from Yamamoto, another diagram of the cellular architecture of the Drosophila compound eye
TRANSPARENCY from Cagan and Zipursky showing sequential recruitments of receptors plus cone cells and pigment cells
TRANSPARENCY a table from Yamamoto listing genes, phenotypes, gene products and developmental roles for sev, boss, ro, svp, drk, Sos, ras, sina, raf, Dsor, rl, and others
TRASPARENCY diagram of the sev signalling pathway from Yamamoto
TRANSPARENCY from Moses showing expression patterns of several genes (rough, glass, seven-up, sina) which are transcription factors (cf. Yamamoto table)

ras oncogenes may be involved in 30% of human cancers. mutations usually block GTPase or GAP stimulation
the 21 refers to 21 kDa
TRANSPARENCY from Hall
Raf=MAPKK first found from v-raf retroviral oncogene
there is a c-raf proto-oncogene
ras is localized to plasma membrane by fatty acid
suggestion that ras's function is to bring raf to the membrane
TRANSPARENCY from Marshall showing interactions with SH2 and SH3 domains in GAP and Switch I and Switch II in p21ras which is reminescent of structural work on alpha subunit of G protein (e.g. Lambright, et al., covered earlier this semester, Nature 369, 1994, 621-628)

References:

R.L.Cagan and S.L.Zipursky, Cell choice and patterning in the Drosophila retina, in Determinations of Neuronal Identity (eds. M. Shankland and E.R.Macagno) NY, Academic Press, 1992. (not on reserve)

A. Hall, A biochemical function for ras - at last, Science, 264, 1413-1414, 1994

M.S.Marshall, The effector interactions of p21ras (Review), Trends in Biochemical Sciences, 1993, 18, 250-255.

R. Mestel, Secrets in a fly's eye, Discover (July, 1996) 106-114

K. Moses, The role of transcription factors in the developing Drosophila eye, Trends in Genetics, 7#8, 250-255, 1991 (not on reserve)

D. Stokoe et al., Activation of Raf as a result of recruitment to the plasma membrane, Science, 264, 1463-1467, 1994

D. Yamamoto, Signaling mechanisms in induction of the R7 photoreceptor in the developing Drosophila retina (Review Article) BioEssays 16 #4, 1994, 237-244


This page was last updated on Dec. 19, 2001

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The hedgehog outline

Hedgehog


TRANSPARENCY
M. Murone et al., Hedgehog signal transduction: From flies to Vertebrates, Exp. Cell. Res. 253, 25-33, 1999
Drosophila segment polarity gene
Shh (one of several in mammals) controls patterning and is involved in left-right
if gene is missing, cyclopia and many structural defects
cleavage to active 19 kDa N-terminal segment by catalysis by 26-28 kDa C-terminal peptide
N-terminal is palmitoylated at N-terminus and has cholesterol at C-terminus
Patched (Ptc)(12 transmembrane) negative regulator
Smoothened (Smo) positive regulator but it does not bind Shh
Smo is like Frizzled (Wnt/Wingless receptor)
Hh binds to Ptc, Ptc stops repressing Smo
Cubitus interruptus Ci/Gli is a downstream zinc finger transcription factor
target genes: ptc, wb and dpp (decapentaplegic)
Ci-155 active or N-terminal cleaved Ci-75 repressor

PWIngham, Transducing hedgehog: the story so far, EMBO Journal 17, 3505-3511, 1998, on line
Another figure to show the same thing

Steven Dickman, The left-handed gene, Discover 17-8, 70-75, August, 1996.
development from embryonic limb bud
1968 John Sanders showed that transplanted tissue has signal to make digits and polartiy comes from region ZPA (zone of polarizing activity)
Christiane Nusslein-Volhard & Eric Wieschaus 1980-> work on Drosophila
hedgehog - embryo lacking hh is porcupine-like
signal that maintains polarity in segments
Cliff Tabin - Sonic hedgehog is ZPA signal in chicken (because that lab likes loud music)
there's olts of vertebrate hedgehogs like tidiwinkle (who was a hedgehog)
More Sonic on left than right. Change distribution by transplantation, heart not necessarily on left

Mark Peifer, The two faces of hedgehog, Science 266, 1492-1493, 1994
Christiane Nusslein-Volhard & Eric Wieschaus late 1970's did their work which results in the following run-down of types of developmental molecules:
signal - wingless like vertebrate Wnt
receptors - DER Drosophila EGF receptor
transcription factors - Paired like vertebrate Pax
Hedgehog is segment polarity signal

S. S. Blair, Hedgehog digs up an old friend (News and views - Developmental Biology), Nature 373 656-657, 1995
diffusable morphogen
reception via cAMP -> PKA
clones lacking PKA have phenotype like clones expressing hh
expression in posterior cells controls gene expression to the anterior
vertebrate who's who of inducers: notochord, ZPA, floor plate express hh
receptors poorly understood:
Patched (Ptc) in Drosophila transmembrane protein is a candidate
TRANSPARENCY
hh posterior -> anterior Dpp (TGF-beta family) (compartments and eye)
Wingless (Wnt family)

R. L. Johnson & C. Tabin, The long and the short of hedgehog signalling (Minireview), Cell 81 313-316, 1995
morphogens organize cell fates relative to discrete inducing tissue
hh found in 1980 by Nusslein-Volhard and Wieschaus
over short range induce cells to secrete dpp which can act over long range
SHH D - V patterning of neural tube
A - P axis of limb bud
non-cell autonomous consistent with secreted
Processing (1) signal sequence cleavage
(2) self-cleaving into N-terminal (local active) & C-terminal (distance proteolysis)
maybe lower dose for distant effects of second messenger
polarizing activity P limb bud transplanted to anterior limb bud->mirror image extra
SHH mimics ZPA activity (both long and short range

M. Dominguez et al., Sending and receiving the Hedgehog signal: Control by the Drosophila Gli protein Cubitus interruptus, Science 272 1621-1625, 1996
E. Pennisi, Gene linked to commonist cancer (Research News) Science 272 1583-1584, 1996
[Ed. by P. Ssuromi, A patched path to cancer (This Week in Science) Science 272 1561, 1996]
basal cell carcinoma most common cancer:
sporatic in middle age
basal cell nevus syndrome mutant in human homologue of patched gene
Drosophila patched is transmembrane protein which down-regulates
growth factor genes
TRANSPARENCY
GLI (oncogene in rare brain tumor in human) = Ci
cubitus interruptus is a transcription factor
protein from Wnt1 (mice) = Drosophila wg stimulates Hedgehog production
cause mammary tumors when overactive

W. Roush, Hedgehog's patterning call is patched through, smoothly (Developmental biology), Science 274, 1996, 1304-1305
Patched works by interrupting the Smo (smoothened) signal
with Shh, the Smo signal gets through

G. Martin, Pass the butter... (Perspectives), Science 274, 203-204, 1996
hedgehog acts in short and long rang signalling
NH2-terminal (Hh-N) has signalling activity
COOH-terminal (Hh-C) has determinants for autoprocessing
processing is to cleave Hh-N from Hh-C and to attach lipid which, oddly, is cholesterol
It is thought that the cholesterol form, by sticking to membranes would work for short range signalling while a non-modified form would go further

D.I.Lewin, A single gene may tell the embryo left from right (News), Journal of NIH Research, 9 (January), 32- 33, 1997
dorsal-ventral and anterior-posterior better studied than left right in bilaterally "symetric" animals
refers to C. Tabin's work on how there is an asymmetry of shh
maternal mRNA from vegetal 1 (Vg1), in TGF-beta superfamily of diffusable growth factors, processed differently in left and right
also an asymmetry in Xenopus nodal-related 1 (Xnr-1), another maternal mRNA
injection of modified Vg1 containing processing region of bone morphogenic protein 2 (BVg1 mRNA) reverses symmetry
thus symmetry is from differing processings of Vg1 protein
thus there is something like TGF-beta working
chicken nodal-related (cNR), like the mouse nodal required for primitive streak in early embryo, are in TGF-beta family
iv=situs inversus viscerum - regulates Nodal, also:
inv=inversion of embryonic turning (inv)- if mutant mice are reversed for Nodal expression
lefty codes TGF-beta homologue expressed on the left, on right in inv mutant
thus inv important in regulating right-left

References:

M. Murone et al., Hedgehog signal transduction: From flies to Vertebrates, Exp. Cell. Res. 253, 25-33, 1999

PWIngham, Transducing hedgehog: the story so far, EMBO Journal 17, 3505-3511, 1998, on line


Steven Dickman, The left-handed gene, Discover 17-8, 70-75, August, 1996

Mark Peifer, The two faces of hedgehog, Science 266, 1492-1493, 1994

S. S. Blair, Hedgehog digs up an old friend (News and views - Developmental Biology), Nature 373 656-657, 1995

R. L. Johnson & C. Tabin, The long and the short of hedgehog signalling (Minireview), Cell 81 313-316, 1995

M. Dominguez et al., Sending and receiving the Hedgehog signal: Control by the Drosophila Gli protein Cubitus interruptus, Science 272 1621-1625, 1996

W. Roush, Hedgehog's patterning call is patched through, smoothly (Developmental biology), Science 274, 1996, 1304-1305

G. Martin, Pass the butter... (Perspectives), Science 274, 203-204, 1996

D.I.Lewin, A single gene may tell the embryo left from right (News), Journal of NIH Research, 9 (January), 32- 33, 1997

J. D. Axelrod et al. Interaction between Wingless and Notch signalling pathways mediated by disheveled, Science 371 1826-1832, 1996

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The notch lecture

Notch (named after notch in wing)

S, Artavanis-Tsakonas et al., Notch signalling, Science 268 225-232, 1995
Here's the scheme:
Multicellular development: sequential action of genes
maternal-effect: (1) anterior, (2) posterior, (3) terminal, (4) dorso-ventral
zygotic: (1) gap, (2) pair rule, (3)segment polarity
then homeotic assigns segments
Specification
(1) lineage
(2) regulation by cell interaction
(i) "lateral" specification if cells were initially equivalent
(ii) ""Inductive" if nonequivalent
Notch in Drosophila and homologues in vertebrates
also lin-12 and glp-1 in C. elegans
TRANSPARENCY 300 kD w/ 36 EGF (epidermal growth factor) repeats
3 cysteine rich Notch/Lin-12 repeats
6 Ankyrin repeats
1 PEST (signal sequence for ubiquitinization)
ectoderm -> neuroblasts (delaminate)
dermmblasts
pro-neural express achaete-scute transcription factors
without Notch, all ectodermal cells continue to express
So Notch was called "neurogenic" but that does injustice to its multitude effects
Participates in both lateral and inductive signalling
in C. elegans, participates in anchor cell, uterine precursor & vulval precursor
vertebrate throughout development and in certain neoplasms
ligands shown in figure
some question about scabrous product involved in R8 spacing
also possibility of wingless product related to Wnt-1 proto-oncoprotein
pathway (TRANSPARENCY)
deltex encodes 737 a.a. cytoplasmic protein binds to ankyrin repeats
Suppressor of Hairless - product like several transcription factors
does detex multimerize Notch to interfere with Su(H)?
Hairless (novel basic protein) vs its supressor - adult sensory
Enhancer of split "complex" - 7 bHLH and another protein
mastermind shows up in modifier screens

J. D. Axelrod et al. Interaction between Wingless and Notch signalling pathways mediated by disheveled, Science 371 1826-1832, 1996
S.S.Blair, Notch and Wingless signals collide Science 371 1822-1823, 1996
ed. by P. Szuromi Inhibited by cross talk (This week in Science) Science 371 1785, 1996
The last paper was cautious in implicating wingless, here it is shown
TRANSPARENCY
dishevelled (dsh) protein of unknown protein
zeste-white 3 (zw3) = shaggy serine-threonine kinases
armadillo (arm) homologue of b-catenin (intracellular attachment protein)
(hooks membrane things to actin)
wingless signal is not known
Dsh inhibits Notch
Sgg-Zw3 phosphorylates and inactivates Arm

H. Kramer, RIPping notch apart: a new role for endocytosis in signal transduction? Science's STKE
DSL = Delta Serrate (Drosophila) Lag-2 (C. elegans
RIP = regulated intramembrane proteolysis
CSL = CBF1, Su(H), Lag-1 [CSL is a family of DNA binding proteins]
S1 cleavage makes notch receptor, S2 cuts off all but 12 a.a's on outside, S3 releases intracellular transcriptionally active domain (ICD)
S3 cut made by enzyme like gamma-secretase (Presenilin) thaty cuts APP (amyloid precursor protein) to make beta-amyloid that contributes to extracellular plaques

References

S, Artavanis-Tsakonas et al., Notch signalling, Science 268 225-232, 1995

J. D. Axelrod et al. Interaction between Wingless and Notch signalling pathways mediated by disheveled, Science 371 1826-1832, 1996

S.S.Blair, Notch and Wingless signals collide Science 371 1822-1823, 1996

ed. by P. Szuromi Inhibited by cross talk (This week in Science) Science 371 1785, 1996

H. Kramer, RIPping notch apart: a new role for endocytosis in signal transduction? Science's STKE


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The JAK/STAT outline

JAK/STAT pathway
(I also put in this outline a paper suggesting a useful organizational scheme)

J. E. Darnell, Jr., STATs and gene regulation, Science 277, 1997, 1630-1635

there is a page in Alberts et al (p. 767) on tyrosine-kinase-associated receptors & non-receptor tyrosine kinases

JAK = Janus kinase (or, fondly, "just another kinase")
Cytokine (small proteins which mediate proliferation or differentiation); receptor ligands:
interferon alpha beta and gamma, interleukins 2-7, 10-13 & 15, erythropoietin, growth hormone, prolactin, thrombopoietin

STAT = signal transducers and activators of transcription
gets phosphorylated on tyrosine and then dimerizes by phosphotyrosines (pY) on SH2's
SH = src homology
TRANSPARENCY (Fig.1, Darnell,1997)-have domains (SH2 & 3, TAD=transactivation domain)
protein is 750 - 850 amino acids in length
7 mammalian STAT genes -> at least 12 proteins from multiple mRNA splicing
specific ligands activate specific STATs in specific cell types
knockouts have shown that 5 of the 7 known STATs have specific roles in adult cells
TRANSPARENCY Table 1
STATs bind specific nucleotide sequences
marelle (an embryonic lethal) is Drosophila null mutant, and Dictyostelium has STAT => ancient

JAK STAT pathway TRANSPARENCY (Fig. 2, Darnell, 1997)

A. H. Brivanlou and J.E.Darnell Jr., Signal transduction and the control of gene transcription, Science 295, 813-818, 2002

There are about 200-300 general transcription factors, coactivators, etc. that combine with RNA polymerase in mammals.
There are more (2000-3000 that bind specific DNA sequences and activate transcription.
TRANSPARENCY (Fig. 1) organization
Constitutive (in all cells, like CCAAT binding protein) vs regulatory
Regulatory: "Developmental (go into nucleus directly like Hox cluster of homeobox genes sequentially expressed anterior to posterior) vs signal dependent.(1, 2, 3)
1. steroid receptor, about 50, all but glucocorticoid start in nucleus
2. activation by internal (cell autonomous signals) (new and presently esoteric)
3. cell surface receptors (a- resident nuclear factors, b- latent cytoplasmic factors)
a. RTK and G protein-coupled receptors lead to serine phorphorylation cascade
b. TRANSPARENCY (Fig 2) Many in animals, none in fungi or plants (phosphorylation, proteolysis)
TRANSPARENCY (Fig. 3) receptor activation delivers active transcription factor to the nucleus.
SMAD (like for TGFbeta), STAT (above, this outline), Hh (a recent outline)
TRANSPARENCY (Fig. 3 continued)

References

A. H. Brivanlou and J.E.Darnell Jr., Signal transduction and the control of gene transcription, Science 295, 813-818, 2002This page was last updated on March 21, 2002

J. E. Darnell, Jr., STATs and gene regulation, Science 277, 1997, 1630-1635

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The retinoic acid and steroid hormones lecture

Steroids and Retinoids

D.W.Tingley, Evolutions: Steroid-hormone receptor signalling (Bench Notes), Journal of NIH Research 8, April 1996, , pp. 81-84 & 87-88.
History TRANSPARENCY
Egyptians already knew that breast feeding delayed next pregnancy
Greek - silphion extract is contraceptive and aborttifacient (may bind estrogen receptors)
Eustachi - 1552 - drew adrenal glands
Addison - 1850 disease with deficiency of adrenal cortex hormones
Adrenalin isolated in 1901
Starling refers to adrenalin and secretin as hormones
1930's researchers isolated some steroids
1960's - explanation of estradiol-binding protein being in cytoplasm and nucleus
1972-1974 Ashburner shows that ecdysone induces puffs on Drosophila polytene chromosomes
Recent stuff drawn on main diagram
Steroid comes in. In cytoplasm-displaces Hsp proteins, functions as dimer in nucleus
Zinc finger aspect involved in DNA binding,
example is gene whose product inhibits Nuclear Factor (NF-kappaB)
TRANSPARENCY Figs 9-14 and 9-15 on p. 411 of Alberts et al.-
shows the structure of Zn-fingers in terms of amino acid sequence
and the binding of such a protein to DNA

TRANSPARENCY a transplantation experiment on limb bud from:
M. Hoffman, The embryo takes its vitamins (research news) Science 250, 372-373, 1990.
see also erratum on p. 1320 with the correct double bonds in retinoic acid
(this relates to the material on hedgehog)
retinoic acid is thought to be a morphogen
TRANSPARENCY showing transport, storage and binding proteins for retinoids from:
R. Blomhoff, et al., Transport and storage of vitamin A, Science 250, 399-403, 1990.
Note CM=chylomicrons involved in absorption of fats
There is conversion into and out of retinyl esters
in general, there is a "bucket-brigade" of binding proteins
TTR = transthyretin, a 55 kDa protein stuck on so that the carrier is too big for glomerular filtration

D.J.Mangelsdorf, The retinoid receptors (Chapter 8) in The Retinoids: Biology, Chemistry and Medicine 2nd edition Ed. by M.A. Sporn et al., Raven Press Ltd., New York, 1994.
in Xenopus embryo, all-trans retinoic acid reduces anterior structures - morphogenTRANSPARENCY formation or retinoic acid
TRANSPARENCY shows how hormone receptors are "nuclear receptors" (with and without ligand)
In this field, many receptors are called "orphan receptors" to denote that the ligand has not been found (yet)
bind to hormone response element
TRANSPARENCY shows all-trans- and 9-cis-retinoic acid
RXR is turned on preferentially by 9-cis, while RAR is equally sensitive to 9-cis and all-trans
TRANSPARENCY homology of RAR with vitamin D, glucocorticoid and thyroid hormone receptors
and RXR with the same and Drosophila ultraspiracle (usp)
TRANSPARENCY - shows conversions and receptor-response element interactions
note that there are cellular retinoid and retinoic acie binding proteins in cytoplasm
AGGTCA typically as direct repeats with 1,2,3,4 or 5 nucleotide separations
TRANSPARENCY shows dimerization and separations
TRANSPARENCY domain structure

S. M. Pemrick et al., The retinoid receptors (Review) Leukemia 8, 1994, 1797-1806
possibly receptors without hormones inhibit transcription
nuclear receptors bind as dimers - have dimerization interfaces in DNA and ligand binding domains
TRANSPARENCY depicts RXR bound to 9-cis RA and RAR bound to all-trans-RA
also shows binding to response elements with separation of 5 nucleotides
Figure 2 RAR & RXR alpha beta and gamma - these are about 450 amino acids (varies between types)
Domain structure, RAR: A-F, RXR: A-E
C is DNA binding domain and is highly conserved
E is ligand binding domain which is also highly conserved
TRANSPARENCY shows how direct regulation of one gene by retinoids can indirectly regulate another

W.A.Segraves, Something old, some things new: The steroid receptor superfamily in Drosophila (minireview), Cell 67, 225-225, 1991
Think about steroidal hormone signalling in, say, humans:
lots, adrenal cortex, gonads, (not to mention close relatives like thyroid)
in insects, ecdysone is the only one really known
TRANSPARENCY homology in members of steroid superfamily

A. Chawla et al., Nuclear receptors and lipid physiology: Opening the X-files, Science, 294, 1866-1870, 2001
48 members of this transcription factor family in human genome
receptors for steroids, thyroid hormones, vitamins A & D
Then there are orphan receptors where ligands etc. are (or were) not known
AF-1 a transcriptional activation function (ligand independent), AF-2 ligand dependent
Steroid receptors bind to DNA as homodimers
Others (orphans) function as heterodimers with RXR receptors

References

R. Blomhoff, et al., Transport and storage of vitamin A, Science 250, 399-403, 1990.

M. Hoffman, The embryo takes its vitamins (research news) Science 250, 372-373, 1990.
see also erratum on p. 1320 with the correct double bonds in retinoic acid

D.J.Mangelsdorf, The retinoid receptors (Chapter 8) in The Retinoids: Biology, Chemistry and Medicine 2nd edition Ed. by M.A. Sporn et al., Raven Press Ltd., New York, 1994.

S. M. Pemrick et al., The retinoid receptors (Review) Leukemia 8, 1994, 1797-1806

W.A.Segraves, Something old, some things new: The steroid receptor superfamily in Drosophila (minireview), Cell 67, 225-225, 1991

D.W.Tingley, Evolutions: Steroid-hormone receptor signalling (Bench Notes), Journal of NIH Research 8, April 1996, , pp. 81-84 & 87-88.

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The growth factors lecture

Growth factors

Alberts et al., several places including pp 893-894, 1125-1126

Their role in growth, maintenance, and survival makes them of potential interest in clinical applications. For instance, in my field (vision) there has been an interest in transplantations (and similar interventions) in restoring visual cells after their loss (in animal models and ultimately in patients). It became apparent that control (sham) treatments sometimes prevented loss and the idea was that disruption might release growth factors. Matthew LaVail and his lab at UCSF have specialized in this. The Nishi paper below suggests that some factors were of interest for treatment of ALS (amyotropic lateral sclerosis) which brings us to the topic of some famous baseball personalities, Lou Gehrig and Cal Ripken Jr.

This lecture is a collection of stories, some unrelated, but, this late in the semester, having overlap with material covered earlier.

From earlier this semester, there is a diversity in how growth factor signalling works:
TRANSPARENCY from general signalling shows RTK's
TRANSPARENCY relates to sevenless (ras MAPK) [figure from E. Culotta and D. E. Koshland, Jr. in "Molecule of the year" (a runner up) 1993, Science 262, 1958-1961
TRANSPARENCY S. Kim, referenced in Invertebrate vision shows signalling via PLC-gamma


Also, there is some Nobel Prize work:
R. Levi-Montalcini, The nerve growth factor 35 years later, Science 237, 1154-1162, 1987
see:
J.L.Marx, The 1986 Nobel Prize for physiology and medicine, Science 234, 543-544, 1986 "discoveries of growth factors"
dimer of 13 kDa protein found in snake venom and mouse salivary gland (strangely)
made by target, nerve binds receptors and is then transorted up to cell

But there is recent evidence that this retrograde transport is not necessary for effect, F.D.Miller and D.R.Kaplan TRK makes the retrograde, (Perspectives) Science 295, 1471-1473, and B. L. MacInnis and R. B. Campenot, Retrograde support of neuronal survival without retrograde transport of nerve growth factor, Science 295, 1536-1539

Nerve growth factor is a trophic factor which maintains sympathetic nervous system and some sensory neurons
TRANSPARENCY Fig. 21-109, p. 1125 - with NGF, there is neurite outgrowth in sympathetic ganglion.
Anti NGF antibody during development eliminates sympathetic nervous system
bind to transmembrane receptors, trk = tyrosine kinase receptors related to protooncogene
Effect in sympathetic- nerves using NE (or DA)
In CNS - affect cells using ACh including thise lost in Alzheimer's and Huntington's


TRANSPARENCY Table 17-2, p. 894 lists factors, related family members, specificity and representative actions
Overall scheme:
Cytokines
Neurotrophins like NGF
Neuropoietic factors (likely you are not familiar)
Hematopoietic factors (like interleukins)
Growth factors like EGF, FGF, TGF, IGF
EGF is 53 amino acids
Notch & lin-12 have EGF repeats (outside the cell and tyrosine kinase intracellularly)
NGF from targets like glands.

Historically, found in blood clots which sounds pretty esoteric, but there is a need for healing after injury. Hence one of the best known is PDGF (platelet-derived growth factor)
TRANSPARENCY Fig. 15-48, p. 761 - ligand dimer helps to dimerize receptor
(receptor dimerization covered ealrier - Fig. 15-47, p. 760)

K.D.Kimura et al., daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans, Science 277, 8942-946, 1997
also
W.Roush, Worm longevity gene cloned (Research news), Science 277, 897-898, 1997
in non-insulin-dependent diabetes, receptor is implicated
leprechaunism from receptor loss, growth arrest at birth
a lot can be accomplished because of the C. elegans genome project, but daf-2 was in unsequenced 10% of genome
dauer phase (dauer=durable) analogous to diapause in insects
pheromone signal indicates overpopulation
daf mutants - dauer-formation defect
TRANSPARENCY shows homology between insulin receptor and DAF-2
ligand binding, transmembrane, tyrosine kinase and insulin receptor substrate-1 domains shown
like insulin receptor and IGF-I receptor
ligand binding domain is 500 amino acids and cysteine rich, 36% & 35% identy respectively
tyrosine kinase domain is 275 amino acids, 70% and 50% identity
a human diabetic insulin-resistant patient has Pro1178->Leu, just like one daf-2 mutant
another human mutant is just like daf-2(e1391), morbidly obese 14-year-old
interesting that genes are related - nematodes and humans diverged 700-800 million years ago
IRS-1 is phosphorylated to recruit SH2 domain proteins
TRANSPARENCY - signal pathway
DAF-7 is TGF-beta-like signal
AGE-1 is PI 3-kinase which is involved in PIP2 generation (coded by daf-23)

M. Inoue et al., Activating mechanism of CNTF and related cytokines, Molecular Neurobiology, 12, 195-209, 1996
CNTF = ciliary neurotrophic factor is like hematopoietic cytokines
survival of ciliary ganglion neurons
in family with leukemia inhibitory factor (LIF), oncostatin M (OSM) cardiotropin-1 (CT-1), interleukins 6 & 11 (IL-6 & IL-11)
"lesion factor" (not secreted, released from damaged cells)
TRANSPARENCY (Fig. 2) 4 alpha helix compares CNTF receptor (left) and growth hormone (GH) receptor (right)
Also used for granulocyte colony-stimulating factor (G-CSF) prolactin (PRL) and erythropoietin (EPO)
The 4-helix would be so useful and stable that it would arise by convergent evolution
TRANSPARENCY (Fig. 3) CNTF binds to CNTF-R-alpha linked to membrane by GPI (glycosyl-phosphatidyl-inositol) anchor
break to TRANSPARENCY Fig, 12-51, p. 591 - GPI
coupled with transmembrane proteins gp 130 and LIF-R
Without CNTF-R, gp 130 and LIF-R used for LIF, OSM and CT-1
Without CNTF-R, gp130 and OSM-R is OSM receptor
IL-6 uses gp130 (2) and IL-6-R-alpha
& IL-11 uses IL-11R and gp130
these have overlapping biological activities
TRANSPARENCY (Fig. 5) domains of gp 130 and LIF-R
note that STAT's go to specific response elements, HRRE=hematopoietin RE
ras pathway used too

R. Nishi, Neurotrophic factors: two are better than one (perspective) Science 265, 1052-1053, 1994
TRANSPARENCY BDNF, FGF-5, CNTF, acidic FGF, basic FGF from different locations all involved in the maintenance of motorneuron
relevance is to prevention of neurodegenerations, in this case Amyotrophic lateral sclerosis (Lou Gehrig's disease)
pmn mutant mouse (motor neuron disease model) CNTF helps rescue
wobbler mutant mouse helped by BDNF and CNTF

P.C.Maisonpierre et al., Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis, Science 277, 55-60, 1997
also
D. Hanahan, Signaling vascular morphogenesis and maintenance (Perspectives, Cell biology) Science 277, 48-50, 1997
Vasculogenesis is when blood vessels are first made
Angiogenesis is when new vessels are formed
the latter is positively important in wound healing, the female reproductive system
negatively important in tumor progression and diabetic retinopathy
TRANSPARENCY the involvement of ligands (VEGF and Ang's 1 & 2, the latter about 75 kD) and receptors VEGF-R's 1 & 2, and Tie's 1 & 2
TRANSPARENCY now show in signal diagram that Ang2 is inhibitory via Tie2

C.-H. Heldin et al., TGF-beta signalling from cell membrane to nucleus through SMAD proteins (review article) Nature 390, 465-471, 1997.
TGF-beta = transforming growth factor.
family of cytokines includes Mullerian inhibiting substance and bone morphogenetic proteins (BMPs)
TRANSPARENCY (Fig. 3)
receptor is tetramer, 2 of 2 types, extracellular cysteine rich intracellular serine threonine kinase, upon ligand binding, type 2 receptor phosphorylates type 1 receptor.
Drosophila Decapentaplegic (dpp) is like BMP, receptors are Punt, Thick veins and Saxophone, and an enhancer screen turned up Mad (mothers against dpp, these Drosophila researchers are a barrel of laughs)
C. elegans has daf-1 and daf-4 that are serine/threonine kinases, and there is a homologue to mad that they called sma (I don't know why)
Anyhow, SMAD (vertebrate homologues) is mad and sma combined, presumably a negotiated settlement between C. elegans and Drosophila researchers.
Lots of different SMADs, hence the expression "different pathway-restricted SMADs"
Smad 2 and 3 get phosphorylated and form multimer with Smad4 goes to nucleus where it may recruit another protein to activate transcription. Also there are inhibitory Smads that inhibit phosphorylation.


References:

C.-H. Heldin et al., TGF-beta signalling from cell membrane to nucleus through SMAD proteins (review article) Nature 390, 465-471, 1997.

M. Inoue et al., Activating mechanism of CNTF and related cytokines, Molecular Neurobiology, 12, 195-209, 1996

K.D.Kimura et al., daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans, Science 277, 8942-946, 1997
also
W.Roush, Worm longevity gene cloned (Research news), Science 277, 897-898, 1997

R. Levi-Montalcini, The nerve growth factor 35 years later, Science 237, 1154-1162, 1987
see:
J.L.Marx, The 1986 Nobel Prize for physiology and medicine, Science 234, 543-544, 1986 "discoveries of growth factors"

P.C.Maisonpierre et al., Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis, Science 277, 55-60, 1997
also
D. Hanahan, Signaling vascular morphogenesis and maintenance (Perspectives, Cell biology) Science 277, 48-50, 1997

R. Nishi, Neurotrophic factors: two are better than one (perspective) Science 265, 1052-1053, 1994


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The apoptosis lecture

Apoptosis

Alberts et al., 1076, 1173-1175, 1238
Two types od cell death:
1. necrosis - from injury, cells burst, there is inflammation
2. programmed cell death = apoptosis
nuclei condense & fragment,
TRANSPARENCY (Fig. 22-35, p. 1174
cell is phagocytosed (by macrophages for instance), no inflammation
TRANSPARENCY (Fig. 21-46, p. 1146)
examples: lymphocytes eliminated, tadpole tail lost, get rid of webs between digits
in C. elegans, ced-3 and -4 (cell death abnormal [can't these guys spell?])
-131 cells which should die survive if mutant
ced-9, if normal, represses death
protein Ced-3 is a protease,
Ced 9 is like Bcl-2, product of bcl-2 proto-oncogene
(as of 1997, see Fox paper, below) the mammalian homologue of C. elegans CED-4
DNA gets chopped into 50-300 bp lengths, hence "ladder" seen in gel
(but note, this would only be seen transiently during the death)
A. Kumar, Defective TNF-alpha-induced apoptosis in STAT1-null cells due to low constitutive levels of caspases, Science 278, 1997 1630-1632
TRANSPARENCY Figure 2 shows ladder
K. McCall and H. Steller, Requirement for DCP-1 caspase during Drosophila oogenesis, Science 279, 230-234, 1994
TRANSPARENCY Fig. 1, shows TUNEL (Tdt-mediated deoxyuridine triphosphate nick end labeling, of course) which stains cells which are dying of apoptosis
(like above, this would only be seen transiently during the death)

Some say the world will end in fire,
Some say in ice.
From what I've tasted of desire
I hold with those who favor fire.
But if it had to perish twice,
I think I know enough of hate
To say that for destruction ice
Is also great
And would suffice
R. Frost

M. Barinaga, Cell suicide: by ICE, not fire (Research news) Science 263, 1994, 754-756.
TRANSPARENCY a frequently shown figure of how homo- or hetero-dimers of BCL-2 and BAX determines life or death respectively
"Apoptosis" Greek word describing plant leaf shedding
C. elegans
ced-3 and ced-4 death genes
ced-3 like gene for ICE = interleukin-1 beta-converting enzyme
CED-3 is also like CPP32 (caspase) (see Fox paper below)
ICE activates interleukin 1 beta by cutting an inactive precursor protein
(ICE has 2 subunite chopped out of a 45 kDa precursor and works as tetramer of 2 of each, Umansky)
ced-9 anti-death genes = (23% identical with) bcl-2
bax blocks bcl-2

M. Barinaga, Forging a path to cell death (Research news), Science 273, 1996, 735-737
(also a quick path from membrane to nucleus, a common theme in this last part of the course)
TRANSPARENCY a figure to show how Fas-ligand and TNF signal through receptors to activate or inhibit apoptosis through proteases
note mention of nuclear factor kappa B (NF-KB), a transcription factor which regulates transcriptions of certain proteins
TNF, Tumor Necrosis Factor and recepors TNFR1 & TNFR2
TRADD = TNF receptor associated death domain protein
FADD = Fas associated death domain protein = MORT1
binds to MACH=MORT1 associated CED 3/ICE homologue
alias FLICE = FADD-like ICE
80 amino acid death domain to trigger cell death pathway

R.C.Duke et al., Cell suicide in health and disease, Scientific American, December, 1996, (Volume 275#6) 80-87 (and cover)
Cell death in heart attack and stroke is especially bad since these cells are not replaced by mitosis:
Initially, necrosis, but some apoptosis can follow
several interesting examples of tricks viruses use to inhibit the death of the cells they infect
Epstein-Barr (mononucleosis) - produce Bcl-2-like substances
Papillomavirus (cervical cancer) degrade p53
p53, missing or inactive in many tumors, activates apoptosis
Cowpox - prevents ICE-like proteases from working
An interesting cancer story:
Melanocytes (important since they block damaging light) have lots of Bcl-2, cancers are aggressive
This paper elaborates on the Fas signalling, and is very general
the discovery of ICE led to the elaboration of ICE-like proteasesCells can die because of deprivation of survival factors, Example disappearance of interleukin-2 (T-cell survival factor) at end of infection
on the other hand, there can be death signals like Fas
Infected cells display Fas - killer-T cells display Fas-ligand
TRANSPARENCY How T cells may be damaged in AIDS by apaptosis and involvement of Fas and ligand

J. L. Fox, Whodunit? The apoptosis conspiracy begins to unravel (news), J. NIH Research, 9, April 1997, 31-32
Mitochondrial membrane is important, oddly, and cytochrome c (oxidative phosphorylation), also oddly
order seems to be CED-9 -> CED-4 -> CED-3
there are conserved domains called BH2 and BH3 on CED-9 to mediate this interaction
CED-9 is on membranes like nuclear membrane while CED-4 is cytoplasmic
(26 kDa, transmembrane domane, see Umansky)
thus CED-9 could pull CED-4 away from being to activate "killer protein" CED-3

E. H.-Y. Cheng et al., Conversion of Bcl-2 to a Bax-like death effector by caspases, Science 278, 1966-1968, 1997
caspases are cysteine proteases which chop protein kinases, retinoblastoma protein, cytoskeletal proteins
Cleavage of a novel protein called DFF triggers DNA fragmentation
"cleavage of Bcl-2 by caspases unleash a latent proapoptotic activity"

S. R. Umansky, Apoptosis: molecular and cellular mechanisms (a review), Molecular biology 30 285-295, 1996

T. Hoey, A new player in cell death (Signal transduction), 1997, 1678-1579
TRANSPRENCY - (and a preview of coming attractions about JAK-STAT signalling)
quick trip from membrane to nucleus in transmembrane cytokine receptors which are not enzymes in and of themselves is mediated through help of Janus kinases (JAKs) and signal transducers and activators of transcription (STATs)


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The cancer outline

Cancer

Alberts et al., chapter 24

There have been some break-throughs:
E. Culotta & D.E. Koshland, Jr., p53 sweeps through cancer research (molecule of the year) Science 262, 1958-1959, 1993, also editorial "Molecule of the year" p. 1953 and cover
also 2 breast cancer genes isolated (BRCA 1 and 2, see below for where they fit into the whole regulation of cell integrity)
Remember involvement of patched (hedgehog pathway) in basal cell carcinoma => relevance of cancer in a signal transduction course

M. Barinaga, From benchtop to bedside, Science 278, 1036-1039, 1997
TRANSPARENCY - discuss how drug design might be addressed to altering something like fatty acid group added to ras

Here I duplicate some fundamental information from the eye development lecture:
tyrosine kinase signalling
small G protein
sevenless signalling pathway
Boss = bride of sevenless is 7 transmembrane domain ligand
sevenless is receptor tyrosine kinsae -
2 transmembrane subunits, 2 extracellular subunits
It is a topic of intense present interest how this signals across membrane
Drk = downstream of receptor tyrosine kinase
which is a small SH adaptor protein, SH = src homology
src = oncogene of Roux sarcoma virus
Sos = son of sevenless
which is a GNRP (guanine nucleotide releasing protein)
to exchange GTP for GDP on ras
ras = rat sarcoma [viral ras oncogene of normal protooncogene]
ras is actually linked to membrane by fatty acid
GAP (sextra in Drosophila) does opposite (GTPase activating protein)

General and fundamental points:
carcinoma - epithelial cell
sarcoma - muscle or connective tissue
leukemia - hemopoeitic cells
carciongenesis and chemicals like aflatoxin (sometimes modified by metabolism) alter DNA in a single cell
other chemicals promote tumors by encouraging cell division or discouraging terminal differentiation - like phorbol esters which activate PKC
tumor=neoplasm, benign vs malignant, metastases to secondary tumors
carcinoma crosses basal lamina to metastasize
angiogenesis is very important
although there is not much mention in the chapter, regulation of apoptosis and immune surveillance are also very inportant

The retinoblastoma story
1 / 20,000 children have it
hereditary form has multible tumors in both eyes from neural precursor cells
non-hereditary - one eye - one tumor
both have deletion in chromosome 13. deletion => loss of a tumor suppressor
TRANSPARENCY - Fig. 24-29, p. 1282 - shows genetics
unphosphorylted Rb binds to proteins, keeping them from allowing DNA replication
Of course, cell division and its regulation should be involved
Tumor suppressor - these would be harder to find than genes that cause cancer (below)

The p53 story (another tumor suppressor)
discovered in 1979, molecule of the year in 1993
hard to see missense mutations on Southern blots
6.5 million cancer diagnoses per year, half have mutations in p53
p53 mutant in many types of cancers, aggressive ones
"53" signifies 53 kDa
p53 is a transcription factor, binds DNA, induces transcription of gene for 21kDa protein
p21 binds and blocks kinase activity of complex (G1 cyclin - Cdk2)
p53 (&p21) thus halt cell cycle at G1 checkpoint (before going onto S phase)
p53 is high in cells insulted with UV or ionizing radiation
human papilloma virus (cervical cancer) targets p53
p53 triggers programmed cell death in cells with DNA damage
with p53 mutation there is abnormal cell growth
knockout is born normal but has tumors or dies by 6 mo

E.R.Fearon, Human cancer syndromes: Clues to the origin and nature of cancer, Science 278, 1043-1050, 1997
"syndromes" -- this paper is dealing with genetic tendencies
gain of function = oncogenme, both alleles loss of function = tumor suppressor
There is an informative table, and the cloned genes include RB1 (retinoblastoma), p53, APC (adenomatous polyposis coli), BRCA 1 & 2 (breast cancer)
colorectal cancer - other tumor suppressors:
APC (familial adenomatous polyposis coli)
DCC (deleted in colon carcinoma)
HNPCC (hereditary non-polyposis colorectal cancer)
recalling the wnt signalling pathway:
TRANSPARENCY - WNT->Frizzled->DSH (dishevelled)->GSK3 (glycogen synthase kinase)->APC->beta-Catenin

Virus and its relation to cell
(chicken) Rous sarcoma virus - this is a retrovirus - has src gene inactivated
viral form (v-src) was apparently picked up from a cellular form (c-src)
The hallmark of retroviruses is their ability to put and take from the genome
TRANSPARENCY - Table 24-4, p. 1276, demonstrates why cancer is a topic in signal transduction course- kinases, GTP-binding proteins, growth factors, gene regulation proteins
TRANSPARENCY Table 24-5, p. 1277 some other familiar signalling molecules
like bcl-2 and RAR-alpha
Genes that cause cancer (oncogenes) but the idea is that the normal gene should subserve a normal function which is only disrupted if the gene (proto-oncogene) becomes abnormal
TRANSPARENCY Fig. 24-26, places where signalling molecules can be altered in cells in cancer
(including ras, raf, steroid-like receptors, membrane growth factor receptors, growth factors)
TRANSPARENCY elaborates ras material covered earlier with the suggestion that a drug which interferes with farnesyl transferase could help to regulate ras
From M. Barinaga,From bench top to bedside (News), Science 278, 1036-1039
TRANSPARENCY very complex (from Fearson, see above) genes involved:
including hedgehog, patched, smoothened, APC, and many others

From M. Peifer, Cancer, catenins, and the cuticle pattern: A complex connection, Science 262, 1667-1668, 1993
also B Rubinfeld et al., Association of the APC gene product with beta-catenin, Science 262, 1731-1734, 1993
and
L.-K. Su et al., Association of the APC tumor suppressor protein with catenins, Science 262, 1734-1737, 1993
TRANSPARENCY showing earlier model with APC-catenin interaction involved in cadherin of adhering junction involving armadillo = beta-catenin, wingless, etc
reminder from earlier lectures, including Beth Burgwyn's wnt lecture, wnt pathway interacts with notch and hedgehog pathways
Also there are a lot of interactions outside the cell where the ligand interacts with the receptor that make wnt signalling very complex
otherwise, beta-catenin helps transcription by a factor called Tcf-Lef
involvement of APC and GSG which phosphorylate beta-catenin to mark it for ubiquitinization for degradation by the proteasome

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The signalling in plants lecture

Integrative systems in plants

"intro textbook-type background" (Chap 35 in 3rd edition, 39 in 6th)

"motor" movements - fast, action potentials
fast movements-turgor changes-mimosa
also Venus fly trap
not so fast, introduce "tropism"
geotropism (gravitropism)
also "sensory"
statoliths in cells
geotropism (gravitropism)

HORMONES

TRANSPARENCY (new) shows molecules
Auxins indole (3 acetic acid)
phototropism - Darwin expts.communication with tip for grass to grow
to light TRANSPARENCY
Went 1927 agar expts TRANSPARENCY
make cells grow more
apical dominance (pinch back flowers)
polar transport (active) from apical meristem of terminal bud
rooting hormones
Weed-be -Gone, Ortho, Scotts plus 2, 2,4-D; weed and feed, deadly agent orange
used to have 2,4,5-T but this was contaminated with dioxin
monocots resist, works on dicots, broad leafed

Ethylene - ripen fruit - kerosene heaters, blueberies, fruit rots
dropping of leaves in deciduous perennials
TRANSPARENCY (new) effects of ethylene in mutanta, seedlings

Cytokinins - contain adenine coconut milk- cell division
Miller expts on aged herring sperm - degraded DNA
interact with auxin in callus vs. root
" " " in apical dominance
Gibberellins - cell elongation - dwarf corn and peas lack
foolish [rice] seedling disease fungus
flower earlier, better Thompson seedless grapes
barley (cereal) seed germination - break dormancy
mRNA for a-amylase act through 2nd messenger
involved in "bolting" with huge internode
Abscisic acid - inhibit growth prepare for winter
rain washes out of desert seed thus germinate

Phytochrome - photodormant lettuce seeds germ after 660,
not 730 - also photomorphogenesis
TRANSPARENCY (new) dimer of photoreceptor with chromophore plus kinase
photoperiodism - dark period important
Pr->Pfr
daylight hits red, slow reconversion TRANSPARANCY
long short day plants
TRANSPARENCIES red and far red in flowering
TRANSPARENCY (new) phytochrome signalling via G protein, calmodulin, transcription factors
Florigen - timing of flowers in short and long day plants
In Summary, there are signal transductions in plants
TRANSPARENCY

Recent work
A. M. Jones, Surprising signals in plants (perspectives) Science 263 183-184, 1994
TRANSPARENCY
Salicylic acid
SAR = systemic acquired resistance, local pathogen->whole plant
bind catalase gets rid of ROS (reactive oxygen species)
turns on pathogenesis related genes
Ethylene
Etr gene homology with prokaryotic two-component signal system
downstrean CTR1 is like Raf serine-threonine protein kinase
Blue light
phototropism (growth)
receptor should be flavoprotein
hy4 mutant is insensitive
use T-DNA to find a tagged allele to get gene
like a prokaryotic flavoprotein that repairs UV damage (photolyases)
Auxin
not expect mutants since auxin is so important
axr1 has same amount of auxin so it is receptor
clone - AXR1 is like E1, an enzyme involved in ubiquinization
rapid turnover of short-lived transcription factors

S. Cutler et al., A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidosis. Science 273 1239-1241, 1996
sesquiterpine which arrests growth, controls stomata and seed dormancy
secondary messengers like IP3 and Ca2+ are involved
Arabidosis thaliana abi gene reduce sensitivity -
serine-threonine phosphatase
transcriptional activator
seek enhanced response genes (era) (less ABA to inhibit seed)
transferred DNA method (T-DNA) allows isolation of genomic region
era1 13 introns - 404 a.a. like beta subunit of farnesyl transferase
add near COOH-terminal at a CaaX motif
note that Ras and gamma of heterotrimeric G protein are farnesylated (for membrane localization - recall paper about ras in cancer lecture)
also rhodopsin kinase must be farnesylated to attenuate receptor activation

J. Q. Wilkinson et al., An ethylene-inducible component of signal transduction encoded by Never-ripe. Science 270 1807-1809, 1995.
ethylene C2H4 - seed germination, flower initiation, fruit ripening, tissue senescence,
organ abscission
Never-ripe (Nr)
CTR1 gene is serine-threonine kinase like Raf kinase
ETR1 encodes membrane protein which dimerises and binds ethylene (receptor)
Nr is mutant of new gene (TXTR-14) encodes 635 aa, 71 kD
lacks C-terminal 103 aa of ETR1
sensor histidine kinase
Nr has C->T at nucleotide 411 => Pro -> Leu

G. E. Schaller & A. B. Bleecker, Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene, Science 270 1809-1811, 1995
ETR1 seems to be ethylene receptor 14 C binding studies, expression in yeast.
mutants are insensitive to ethylene. etr1-1 mutant has no binding
Cys65->Tyr => metal coordination (Cys, His Met)
ETR1 gene cloned N-terminal is for membrane localization
C-terminal is like histidine kinase of bacteria
(total is 738 amino acids), dimer with disulfide bonds
hard to reconcile with tomoto homologue Never-ripe
in terms of if receptor is inactive or active with ethylene

J. Marx. Plants, like animals, may use peptide signals (research news), Science 273 1338-1339, 1996 Previously thought that the cell wall was too thick for peptides
1991 systemin to fight off insect pests
ENOD40 (ENOD gene = early nodulation) regulates formation of root nodules (for nitrogen fixation in legumes)
release cells from growth inhibition - makes auxin tolerant
real odd: ORF = 12 or 13 a.a. (in animals, peptides cleaved from precursor)
another gene - Crinkly abnormalities in corn leaf and seed
like receptor for tumor necrosis factor, that peptide being 157 aa
cr4 mutant cloned => receptor kinase
Xa21 is receptor kinase gives resistance in rice to bacterial pathogen
S = self-incompatability another receptor kinase in some plants
pistol recognizes signal on pollen

B. Lacombe et al., The identity of plant glutamate receptors, Science 292, 1486-1487, 2001
In animals, glutamate receptors are ligand gated channels.
There are 20 glutamate receptors in plants in 3 clades.
Some similarity with those of animals, but not much.May be involved in light signal transduction or Ca2+ homeostasis.

R. Hooley, Plant steroid hormones emerge from the dark (comment), Trends in Genetics 12 281-283, 1996
Brassica (mustards, cauliflowers, cabbages, turnips)
brassinosteroids discovered as a growth stimulator from pollen
works in light, not in dark (hence title of paper)
stimulate cell elongation and division (interaction with auxin)
etoliation fast growth underground then photomorphogenesis (make photosynthetic pigments)
there are mutants
TRANSPARENCY -
steroid synthesis:determine which replacements rescue
Arabidopsis DET2 gene and det2 mutation homology with 5 alpha-reductase
use T-DNA technique Agarobacterium tumefaciens transferred DNA insertion
cpd mutant of CPD gene and tomato Dwarf homology to steroid hydroxylase
dim several possible homologies
also insensitive mutants: cbb2 and bri1 receptor or post brassinolide conversion

J. Li et al., A role for brassinosteroids in light-dependent development of Arabidosis, Science 272, 398-401, 1996
also
D.W.Russell, Green light for steroid hormones, Science 272, 370, 1996
mutants of Arabidosis that seem light grown even if grown in dark
DET2 codes for 262 amino acid like mammalian 5alpha-reductase
several types of mutants, but, importantly, glutamate->lysine at 204
using NADPH, in mammals, testosterone->dihydrotestosterone
hereditary male pseudohermaphroditism from equivalent glutamate ->aspartate at 197
from campesterol to brassinolide, 10 steps, DET2 may be 1->2, CPD may be 5->6

Z. He et al., Perception of Brassinosteroids by the extracellular domain of the receptor kinase BRI1, Science, 288, 2360-2363, 2000
In animals, there are RTKs
In plants, they are always serine-threonine kinases and they are called RLKs (receptor-like kinases).
Steroids perdeived at membrane

J. Li and K. H. Nam, Regulation of brassinosteroid signalling by a GSK3/SHAGGY-like kinase, Science 295, 1299-1301, 2002
GSK3/SHAGGY is serine-threonine kinase (Recall the Wnt pathway.)
There are some in plants (10 in Arabidopsis).
BIN2 (brassinosteroid insensitive encodes a GSK3/SHAGGY kinase

J. Gewolb How seedlings see the light, Science 293, 1237-1238, 2001
photomorphogenesis
COP1 cause degradation of transcription factors
blue receptors are cryptochromes and they interact with COP
phytochromes may interact with cryptochrome

S. Ikeda et al., An aquaporin-like gene required for the Brassica self-incompatibility response. Science 276, 1564-1566, 1997
Brassica - self-pollination always inhibited in field of wild mustard, cross pollination usually works.
Self-incompatibility insures cross-polination, pollen tube development is disrupted
Background:
There is a cluster of genes at the S locus
ligand on pollen interacts with receptor tyrosine kinase -> phosphorylation cascade in the stigma's epidermal cell to "reject" pollen
New work:
outside S locus, mod mutation affects
probe shows mRNA in MOD/MOD, much less transcript in mod/mod, so allele is hypomorphic
286 aa like MIP (major intrinsic protein) like aquaporin, 6 membrane spanning
(see channel lecture, Dean et al. paper)

A. Kachroo et al. Allele-specific receptor-ligand interactions in Brassica self-incompatibility, Science293, 1824-1826, 2001
serine-threonine kinase (SRK) in stigma
cysteine-rich peptide (SCR) in pollen binds SRK

(The following was covered in channels, so it won't be repeated here.)
R. Hedrich & P. Dietrich, Plant K+ channels: similarity and diversity, Bot. Acta. 109, 1-8, 1996
This paper refers to "green" (from plants) and "red" (from animals)
this work is electrophysiological, and application of the patch clamp has contributed greatly
Of course, nutritionally, for plants and herbivores, potassium is very relevant (macronutrient)
several rapid volume change responses
for instance, here is a TRANSPARENCY from this department's introductory biology text to show the involvement of K+ in guard cell responses, responsible for opening and closing of stomata
an overall outline:
Inward rectifying voltage dependent K+ channels for K+ uptake
Outward rectifying voltage dependent K+ channels for K+ release
Also there is a high affinity K+ transporter (HKT1) about which little is known though it is thought that protons are cotransported. Such a system would be used to get K+ in from low concentration in soil.
This paper concentrates on uptake channels like those cloned from:
Arabidosis thaliana (KAT1)
Solanum tuberosum (KST1)
channel conductance is 5-30 pS
for a 10 x change in K+ gradient, voltage changes 56-58 mV in accord with the Nernst potential (see earlier this semester, discussion of Paramecium)
not many insect, scorpion, snake, frog or dinoflagellate toxins which affect animal channels affect plant channels
But external Cs+ and TEA+ do block, but weakly
In contrast with animal channels, KST1 and KAT1 have ATP and cyclic nucleotide cassettes and several channels are ATP dependent
Like shaker in S1-S6 and H5 or P (pore forming) - TRANSPARENCY with 21 conserved amino acids except that plant channel has extra 14 amino acids
No N-terminal ball and chain and no inactivation
there are AKT1 = Arabidosis K+ transporters which have ankyrin binding domains

E. Pennisi, Plants decode a universal signal (Research News) Science 278, 2054-2055, 1997
also
Y. Wu et al., Abscisic acid signaling through cyclic ADP-ribose in plants, Science 278, 2126-2130, 1997
TRANSPARENCY
abscisic acid (ABA) turns on stress responsive genes (stress = cold, drought and salinity)
acts through releasing calcium in burst from intracellular stores
in humans, disorders in cyclic ADP-ribose signaling -
heart arrythmias (bad contractions if too little calcium)
and diabetes (glucose-stimulated insulin release from pancreas)
the assay is the stress genes which ABA turns on --
rd29A (dessication-responsive gene)
and kin (cold-inducible gene)
a control gene was turned on by calcium but not by cADPR
another interesting control - IP3 could turn on genes. heparin blocks this but not if ABA turns on genes implicating another signaling pathway
may also be involvd in closing of stomata in response to drought
3 ways calcium may be regulated: (1) IP3, (2) cADPR, and (3) nicotinic acid adenine dinucleotide phosphate (NAADP+) [For (2) and (3), receptor is not known.]
ryanodine receptor (RyR) may be receptor for cADPR

H. Ullah et al., Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis, Science 292, 2066-2069, 2001
X-Q Wang et al., G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells, Science 292, 2070-2073, 2001
BEEllis and GFMiles One for all? (Perspectives) Science 292, 2022-2023, 2001
TRANSPARENCY
G protein - mammal genomes have 20 alpha, 5 beta and 12 gamma, plants 1:2(?):1
silence GPA1 indicates that auxin acts through Galpha (in part), also abscisic acid's closing of stomata

E.M.Meyerowitz, Plants compared to animals: The broadest comparative study of development, Science 295, 1482-1485, 2002
If common ancestor were unicellular (very probable), development should be very different
TRANSPARENCY
2.7 billion years ago, eukaryotes split off
After that, mitochondria become intracellular symbiotes ("uptake of alpha proteobacterium")
1.6 billion years ago, last common ancestor of plants and animals
After that, chloroplasts become intracellular symbiotes ("uptake of cyanobacterium")
0.6 billion years ago, multicellular plants and animals (but fossil record is not complete)
Animals - "spatially specific transcriptional activation of master regulatory genes, the HOX homeobox genes"
Plants - "master regulatory genes are transcriptionally activated in a radial pattern" (for "radial pattern of floral development") but for plants, MADS box (no homology to HOX)
EGF (receptor tyrosine kinase) in Ras pathway important in Drosophila development, but Arabidopsis genome has no RTK or Ras.
Others (like I-kappa B, NF-kappaB, SMADs) have no relatives in Arabidopsis.
No nuclear receptors, Smo, Ptc, Notch in plants
Processes are similar, but genes are not homologous. (convergent evolution)
There is cell-cell signalling similar to boss-sev in shoot apical meristem (CLAVATA3 is ligand, CLAVATA1 is receptor).
Plants have important domains like serine/threonine kinases and leucine-rich repeats, but organization is different.
Plant steroid receptors are different from animal nuclear receptors - leucene rich-repeat receptor kinase.
Ethylene inactivates receptors. 5 receptors like bacterial receptors. At least one is histidine kinase like in bacteria. Act through Raf (MAPKKK)
5 phytochrome genes in plants homologous with cyanobacterial except serine/threonine kinase instead of histidine kinase.Probably horizontal transfer from uptake of protochloroplast.

References:

S. Cutler et al., A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidosis. Science 273 1239-1241, 1996

J. Gewolb How seedlings see the light, Science 293, 1237-1238, 2001

Z. He et al., Perception of Brassinosteroids by the extracellular domain of the receptor kinase BRI1, Science, 288, 2360-2363, 2000

R. Hooley, Plant steroid hormones emerge from the dark (comment), Trends in Genetics 12 281-283, 1996

S. Ikeda et al., An aquaporin-like gene required for the Brassica self-incompatibility response. Science 276, 1564-1566, 1997

A. M. Jones, Surprising signals in plants (perspectives) Science 263 183-184, 1994

A. Kachroo et al. Allele-specific receptor-ligand interactions in Brassica self-incompatibility, Science293, 1824-1826, 2001

B. Lacombe et al., The identity of plant glutamate receptors, Science 292, 1486-1487, 2001

J. Li et al., A role for brassinosteroids in light-dependent development of Arabidosis, Science 272, 398-401, 1996
also
D.W.Russell, Green light for steroid hormones, Science 272, 370, 1996

J. Li and K. H. Nam, Regulation of brassinosteroid signalling by a GSK3/SHAGGY-like kinase, Science 295, 1299-1301, 2002

J. Marx. Plants, like animals, may use peptide signals (research news), Science 273 1338-1339, 1996

E.M.Meyerowitz, Plants compared to animals: The broadest comparative study of development, Science 295, 1482-1485, 2002

E. Pennisi, Plants decode a universal signal (Research News) Science 278, 2054-2055, 1997
also
Y. Wu et al., Abscisic acid signaling through cyclic ADP-ribose in plants, Science 278, 2126-2130, 1997

G. E. Schaller & A. B. Bleecker, Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene, Science 270 1809-1811, 1995

H. Ullah et al., Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis, Science 292, 2066-2069, 2001
X-Q Wang et al., G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells, Science 292, 2070-2073, 2001
BEEllis and GFMiles One for all? (Perspectives) Science 292, 2022-2023, 2001

J. Q. Wilkinson et al., An ethylene-inducible component of signal transduction encoded by Never-ripe. Science 270 1807-1809, 1995.

This page was last updated on April 30, 2002

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