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
Return to Signal Transduction Syllabus