Molecular and Cellular E~docrj~ofo~, 49 (1987f 1-16 Elsevier Scientific Pub&hers Ireland, Ltd. MCE 01574 Review Signal transduction by guanine nucleotide binding proteins Allen M. Spiegel Molecular Pathophysiolqgv Section, National Institute of Diabetes, Digestive, and Kidney Diseuse, National institutes of Health. Bethesda, MD 20892, U.S.A. Key words: Signal transduction; Guanine nucleotide binding protein; Review Summary High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, l-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the rus gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effecters (E). A similar function has been suggested but not proven for the rus gene products. Known G-proteins include G, and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phos- phodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. l&237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and G,, a G-protein of unknown function that is highly abundant in brain (Stemweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 1380613813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. General characteristics of G-protein structure After solubilization from membranes with ap- propriate detergent-containing buffers, G-proteins behave as monomers of approximately 100 kDa. Each monomer is composed of three distinct pro- tein subunits, alpha, beta, and gamma, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under nondenatur- ing conditions, beta and gamma subunits remain tightly associated, but upon activation of a G-pro- tein in solution (see below), alpha subunits readily Address for correspondence: Allen M. Spiegel, Molecular G,. a G-protein of unknown function discovered in brain; TD, Pathophysiology Section, National Institute of Diabetes, Di- transducin, the G-protein of retinal photoreceptors; SDS- gestive, and Kidney Disease, Building 10, Room 9C101, Na- PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- tional Institutes of Health, Bethesda, MD 20892, U.S.A. phoresis; GTP, y-S-guanosine 5’-( y-thio)-triphosphate; EF-2, Abhreuiations: R, receptor; E, effector; G-protein, guanine elongation factor 2; PI, phosphoinositide; cDNA, complemen- nucleotide binding protein; G, and Gi, G-proteins associated tary DNA; p21,21 kDa products of ras genes; eIF-2, eukaryotic with stimulation (G,) and inhibition (Gi) of adenylate cyclase; initiation factor 2. 0303.7207/87/~03.50 0 1987 Elsevier Scientific Publishers Ireland, Ltd.
Transcript
Molecular and Cellular E~docrj~ofo~, 49 (1987f 1-16
Elsevier Scientific Pub&hers Ireland, Ltd.
MCE 01574
Review
Signal transduction by guanine nucleotide binding proteins
Allen M. Spiegel Molecular Pathophysiolqgv Section, National Institute of Diabetes, Digestive, and Kidney Diseuse, National institutes of Health.
Bethesda, MD 20892, U.S.A.
Key words: Signal transduction; Guanine nucleotide binding protein; Review
Summary
High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, l-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the rus gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effecters (E). A similar function has been suggested but not proven for the rus gene products. Known G-proteins include G, and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phos- phodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. l&237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and G,, a G-protein of unknown function that is highly abundant in brain (Stemweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 1380613813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins.
General characteristics of G-protein structure
After solubilization from membranes with ap- propriate detergent-containing buffers, G-proteins behave as monomers of approximately 100 kDa. Each monomer is composed of three distinct pro-
tein subunits, alpha, beta, and gamma, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under nondenatur- ing conditions, beta and gamma subunits remain tightly associated, but upon activation of a G-pro- tein in solution (see below), alpha subunits readily
Address for correspondence: Allen M. Spiegel, Molecular G,. a G-protein of unknown function discovered in brain; TD, Pathophysiology Section, National Institute of Diabetes, Di- transducin, the G-protein of retinal photoreceptors; SDS- gestive, and Kidney Disease, Building 10, Room 9C101, Na- PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- tional Institutes of Health, Bethesda, MD 20892, U.S.A. phoresis; GTP, y-S-guanosine 5’-( y-thio)-triphosphate; EF-2,
Abhreuiations: R, receptor; E, effector; G-protein, guanine elongation factor 2; PI, phosphoinositide; cDNA, complemen- nucleotide binding protein; G, and Gi, G-proteins associated tary DNA; p21,21 kDa products of ras genes; eIF-2, eukaryotic with stimulation (G,) and inhibition (Gi) of adenylate cyclase; initiation factor 2.
alpha subunit binds guanine nucleotide with affin- ities in the submicromolar range, is a GTPase, and
serves as a substrate for ADP-ribosylation by bacterial toxins. The alpha subunit of each G-pro-
tein is distinct, and may determine the specificity of a given G-protein in terms of R and E coupling.
On SDS-PAGE, known alpha subunits vary in size
from about 39 to 52 kDa.
The beta subunit migrates as a 36 kDa protein on SDS-PAGE, and is similar if not identical in
each G-protein (Manning and Gilman, 1983; Gierschik et al., 1985). A more rapidly migrating,
approximately 35 kDa species is observed in some tissues (Sternweis and Robishaw, 1984). Gamma subunits are relatively low molecular weight pro- teins (about 8-11 kDa on SDS-PAGE). The gamma subunit of transducin is distinct from other
G-protein gamma subunits (Gierschik et al., 1985) but the relationship among the gamma subunits of G-proteins other than TD is not clear.
G-proteins, including G,, Gi, and G,, are
thought to be associated with the cytoplasmic surface of the plasma membrane. TD is unique in
that it is associated with the cytoplasmic surface
of rod outer segment disc membranes. The nature
and extent of membrane insertion of G-proteins
are unclear. Detergents are necessary for solubili- zation of G,, Gi, and G,, but not of TD. G-alpha
subunits appear to be relatively hydrophilic, and behave as monodisperse species in buffers without
detergent (Neer et al., 1984; Sternweis, 1986). The beta/gamma complexes of G-proteins other than TD require detergent for solubilization. Thus, beta/gamma complexes may serve to anchor G- proteins in the membrane (Sternweis, 1986), but definitive evidence is lacking. The ability to
solubilize TD without detergents may reflect unique properties (relative hydrophilicity) of its gamma subunit (Hurley et al., 1984a; ~vchinnikov et al., 1985; Yatsuna~ et al., 1985).
General aspects of G-protein function
G-proteins function as signal transducers by acting as on-off switches. Interaction of G-pro- teins with R activated by signals (first messengers) external to the cell enables GTP to bind to and activate the G-protein. Activated G-protein in turn
interacts with E which transmits intracellular sig- nals (second messengers). G-protein activation and
interaction with E is terminated by the relatively slow GTPase activity of the alpha subunit. GDP- bound G-protein, no longer capable of interacting
with E, must reassociate with activated R to begin
another cycle.
In solution, G-protein activation is accompa-
nied by dissociation of alpha from beta/gamma
subunits. Activation by nonhydrolyzable analogs
of GTP such as guanosine (~-O-Tao)-triphos-
phate (GTP-y-S) or by aluminum fluoride
(Sternweis and Gilman, 1982), (which may form a GTP-like complex with bound GDP (Bigay et al., 1985)), is required for alpha subunit dissociation.
G-protein activation causes a change in alpha subunit conformation, reflected in altered suscep-
tibility to tryptic proteolysis (Fung and Nash, 1983; Hurley et al., 1984b), changes in exposure of sulfhydryl groups (Ho and Fung, 1984) and
reduced affinity for the beta/gamma complex.
Mg*+ concentration is a critical determinant of G-protein activation (Iyengar and Birnbaumer,
1982; Katada et al., 1984b). At sufficiently high Mg2+ concentration (50 mM), alpha subunit dis- sociation may occur without addition of activators
such as GTP-y-S (Deterre et al., 1985). Resolved, activated alpha subunits are capable
of interacting with and activating their corre-
sponding E. This has been directly demonstrated
for G,-alpha (May et al., 1985), and for TD-alpha (Bitensky et al., 1981; Stryer, 1986). Hydrolysis of GTP to GDP returns the aIpha subunit to a state of higher affinity for the beta/gamma complex. It is not clear whether GDP-bound alpha subunits are intrinsically less capable of interacting with E or whether their reduced efficacy is due to reas-
sociation with beta/gamma to form the holopro- tein.
The beta/ga~a complex has been viewed as an ‘in~bitor’ (Gilman, 1984), keeping the G-pro- tein in its inactive form. Binding of GTP, promot- ed by activated R, is necessary to overcome inhibi- tion by beta/gamma. The beta/gamma complex, however, may be required for association between G-protein and R (Fung, 1983; Florio and Sternweis, 1985). GDP-bound alpha subunits, incapable by themselves of interacting with R, would represent a ‘dead-end’ in the activation-
TABLE I
FUNCTION AND DISTRIBUTION OF KNOWN G-PROTEINS
G-protein Receptor Effector Distribution Toxin substrate Comments
Transducin, Rbodopsin
Transducin, Cone opsins
Gs
G 'PI
Receptors for ago-
nists that stimulate
cyclase; many, in-
cluding beta-adren-
ergic, glucagon,
ACTH; may also
function with olfac-
tory receptors
Receptors for agc-
nists that inhibit
cyclase; many, in-
cluding alpha,-ad-
renergic, muscarin-
ic choline@, so-
matostatin
? (coupling to mus-
catinic cholinergic,
alpha,-adrenergic,
Da-dopaminergic has
been demonstrated)
Receptors for ago-
nists that stimulate
phospholipase C;
many, including
f-Met-Leu-Phe,
TRH, alpha,-adren-
ergic
cGMP phospho-
diesterase
Retinal rods
cGMP phospho-
diesterase ’
Retinal cones
Adenylate cyclase Ubiquitous
Adenylate cyclase
? (possibilities in-
clude ion channels
and phospholipase
C)
Phospholipase C
Ubiquitous
(but see Comments)
Abundant in brain:
immunoreactive ma-
terial also found in
heart and other tis-
sues
Ubiquitous
(but see Comments)
Cholera, pertussis
Cholera a, pertussis ’
Cholera
Pertussis
Pertussis
Pertussis: ? also chol-
era e.g. in neutro-
phils
Alpha subunit
cDNA (Tanabe et
al., 1985)
Alpha subunit
cDNA (Lochrie et
al., 1985)
Alpha subunit
cDNA (Nukada et
at.. 19X6b. Robishaw
et al., 1986a) at least
two forms of mRNA
corresponding to 45
and 52 kDa proteins
Multiple different al-
pha subunit cDNAs
found (Nukada et al.,
1986a: Itoh et al.,
1986; Reed and
Jones, personal com-
munication)~ wheth-
er these differ in
function, receptor or
tissue specificity is
undefined
Alpha subunit
cDNA (Itoh et al.,
1986; Reed and Jones, personal com-
munication)
Several distinct G-
proteins, including
G,, G,. and others
may be G.,,; pertus-
sis toxin sensitivity
is variable in differ-
ent cells: in neu-
trophils. a pertussis
toxin substrate dis-
tinct from ‘brain G, /‘Go ’ is involved
’ Not directly demonstrated.
deactivation GTPase cycle. Reassociation with beta/gamma subunits promotes R interaction and thereby binding of GTP to begin another round of the cycle.
Note that G-protein affinity for guanine
nucleotides (submicromolar) is seemingly much greater than required given the high (submilli- molar) concentrations of GTP in the cell. Such high affinity dictates a relatively slow spontaneous dissociation rate for bound GDP. Analysis of
4
G-protein alpha subunits (Ferguson et al., 1986), and of t-us gene products (Poe et al., 1985), indi- cates that they copurify with tightly bound GDP.
Thus, spontaneous activation of G-proteins does
not occur (despite GTP: GDP ratios > 1 in the
cell). Instead, GTP binding is dependent on inter-
action of G-protein with activated R. The rela- tively slow GTPase activity, moreover, permits
GTP-bound G-protein to activate E before hy-
drolysis to GDP (turn-off) occurs. Agonists bind with higher affinity to R-G-pro-
tein complexes than to free R (Lefkowitz et al., 1983). Upon binding guanine nucleotide, G-pro- tein dissociates from the R-G-protein complex. Agonist binding to R in membrane preparations
freed of endogenous guanine nucleotides is char- acterized by high and low affinity components, reflecting R complexed with G-protein and free R, respectively. Addition of guanine nucleotides re-
duces the apparent affinity of R for agonists,
presumably by dissociating G-protein from R. The
effect of guanine nucleotides on agonist affinity is
a useful indicator of R-G-protein interaction. For
some agents, e.g. angiotensin in adrenal cortex (Glossmann et al., 1974) and TRH in pituitary (Hinkle and Phillips, 1984), reduction in agonist
binding affinity by guanine nucleotides was the first indication for G-protein involvement in the
mechanism of action. This effect has also been used to monitor reconstitution of R-G-protein in- teraction in artificial phospholipid vesicles (Ceri- one et al., 1984). Uncoupling of G-protein from R
(e.g. by pertussis toxin, see below) converts R to the low affinity state for agonist binding and abolishes the effect of added guanine nucleotides.
Stimulation by agonists of G-protein GTPase activity is another measure of R-G-protein inter- action. In general, purified G-proteins show low rates of GTPase activity. G-protein interaction with activated R promotes release of bound GDP and permits binding of GTP. The net effect (indi- rect, by providing fresh substrate) is that agonists stimulate G-protein GTPase activity. This can be demonstrated in crude membrane preparations (Cassel and Selinger, 1978) when appropriate con- ditions (submicromolar concentration of GTP substrate, millimolar concentration of ATP ana- logs to block nonspecific nucleotide tri- phosphatases) are used to measure high affinity
GTPase activity. Agonist stimulation of GTPase activity has also been used to monitor R-G-pro- tein interaction in artificial phospholipid vesicles
(Pedersen and Ross, 1982; Cerione et al., 1985b).
Such studies indicate that R-G-protein cou-
pling is somewhat promiscuous. Beta-adrenergic
R, representative of a class of R that stimulates adenylate cyclase, interacts not only with G, as expected, but also with Gi (Asano et al., 1984).
Some specificity is preserved in that G, is more efficient than G, in coupling to the beta-adren- ergic R, and TD is relatively ineffective (Cerione et al., 1985b). Rhodopsin, in contrast, couples effectively with TD, as well as Gi and G, (Cerione
et al., 1985b, 1986a). Coupling of a given R-G- protein pair in artificial phospholipid vesicles (e.g.
Okajima et al., 1986) cannot be taken as evidence
for a physiologically relevant interaction. Pro-
miscuity in R-G-protein interaction may reflect
similarities in structure in domains involved in
R-G-protein coupling.
Bacterial toxins have important effects on G-
protein function. This was first shown for diph- theria toxin and the soluble guanine nucleotide binding protein, eukaryotic elongation factor-2 (EF-2) (Collier, 1975). The toxin mono-ADP- ribosylates EF-2, and thereby blocks further pro- tein synthesis. In an analogous manner, cholera
toxin ADP-ribosylates G, and TD (and possibly other G-proteins), while pertussis toxin ADP- ribosylates Gi, G,, and TD (Gilman, 1984).
Cholera toxin appears to reduce the intrinsic GTPase activity of G, (Cassel and Selinger, 1977)
and TD (Abood et al., 1982). This causes rela- tively longer-lived activation of G-protein by GTP, and ultimately, increased activation of E. Al-
though pertussis toxin catalyzes the same covalent modification, mono-ADP-ribosylation, the func- tional result is different. Pertussis toxin causes uncoupling of G-protein from R (Hsia et al., 1984; Ui, 1984; Van Dop et al., 1984b). This also leads to reduced agonist-stimulated GTPase activity, su- perficially resembling the effect of cholera toxin. The end result of pertussis toxin action, however, is reduced signal transduction by its G-protein substrate. Both toxins have been extremely useful in identifying G-proteins in membranes and in studying G-protein function.
5
Specific G-proteins and their functions
G, and G, Adenylate cyclase, the enzyme that catalyzes
the formation of the second messenger CAMP, is coupled to receptors for agonists that either stimulate or inhibit its activity by distinct G-pro- teins, G, and Gi. G, is found in essentially all cells of vertebrates. Mutant S49 mouse lymphoma cells show abnormalities in G, function, including com- plete deficiency of G, in CYC- (Bourne et al., 1982), and inability to couple with R in the UNC mutant (Haga et al., 1977). Agonists are incapable of stimulating CAMP production in CYC- and UNC mutants. In CYC - cells, fluoride, guanine nucleotide analogs and cholera toxin are also inef- fective. Partial G, deficiency appears to cause re- sistance to multiple agonists acting via CAMP stimulation in a human genetic disease termed pseudohypoparathyroidism type Ia (Van Dop and Bourne, 1983; Spiegel et al., 1985).
Cholera toxin ADP-~bosylat~on of G,-alpha leads to persistent activation of G, and increased CAMP production. The diterpene, forskolin, can activate adenylate cyclase without G, (Seamon et al., 1984), and may bind directly to a site on the catalytic moiety, an approximately 130 kDa glyco- protein recently purified using a forskolin affinity column (Pfeuffer et al., 1985). Forskolin blinds to specific sites on brain membranes (Seamon et al,, 1984). The affinity of these sites is increased by agents that activate G,. Forskolin stimulation of adenylate cyclase is potentiated by G,, and vice versa (Darfler et al., 1982). Differential effects of chymotrypsin on adenylate cyclase activation by forskolin and G, suggest that the forskolin bind- ing site and site of G, interaction with cyclase are distinct (Gierschik and Spiegel, 1985).
Inhibitory effects of guanine nucleotides on adenylate cyclase were recognized even before stimulatory effects (Cryer et al., 1969). Kinetic and other indirect evidence suggested that the guanine nucleotide binding site mediating inhibi- tion is distinct from the site causing stimulation of adenylate cyclase (Rodbell, 1980). Through the use of pertussis toxin (islet activating protein; Ui, 1984), Gi was identified as a distinct G-protein responsible for inhibition of adenylate cyclase. Pertussis toxin ADP-ribosylates an approximately
41 kDA protein (distinct from slightly higher molecular weight cholera toxin substrates) in most cells. Intact cells treated with pertussis toxin show increased basal and/or agonist-stimulated CAMP production. In~bito~ agonists, moreover, are rendered ineffective. This was shown to be due to uncoupling of Gi from inhibitory R (Hsia et al., 1984; Ui, 1984). Guanine nucleotide-mediated in- hibition of basal and forskolin-stimulated adenyl- ate cyclase activity in CYC- cells (lacking G,) provided further evidence for a distinct G, protein (Hildebrandt et al., 1982). The inhibitory effect of agonists such as somatostatin and of guanine nucleotide analogs in CYC- cells is eliminated by pertussis toxin, again, linking G-protein-mediated adenylate cyclase inhibition to a pertussis toxin substrate.
Gi appears to be as ubiquitously distributed as G,; its concentration in cell membranes appears to be (perhaps lo-fold) higher than that of G, (Gil- man, 1984). While Gi and G, alpha subunits clearly differ, the beta (Mining and Gilman, 1983; Gierschik et al., 1985) and possibly the gamma (Hildebrandt et al., 1984) subunits of purified G, and Gi appear to be identical. Re- solved beta/gamma subunits are more effective inhibitors of adenylate cyclase than are resolved G,-alpha subunits (Katada et al., 1984b). Simi- larly, the beta/gamma subunit of TD is an effec- tive inhibitor of adenylate cyclase (Cerione et al., 1986b). The data have led to the suggestion that inhibition of adenylate cyclase is due to a direct effect on G, of beta/gamma complexes released from Gi (Gilman, 1984). Given the excess of G, over G,, activation of Gi through inhibitory recep- tor agonists should release sufficient beta/gamma complexes from G, to retard G, dissociation. This mechanism of inhibition presumes that activation of G, requires dissociation of G,-alpha from beta/gamma, or at least, a significant change in the interaction between these subunits.
There are several problems with this model. Subunit dissociation has not been demonstrated upon G-protein activation in native membranes. Even in solution, subunit diss~iation has not been shown to occur with the presumptive natural G-protein activator, GTP (Huff and Neer, 1986). The model, moreover, does not readily explain guanine nucleotide-mediated inhibition of adenyl-
6
ate cyclase in CYC membranes. The apparent
total lack of G, in CYC- suggests that Gi-mediat- ed inhibition involves a direct effect of Gi on the
cyclase catalytic moiety (Hildebrandt et al., 1982). Indeed, the activated G,-alpha subunit (but not
beta/gamma) does inhibit adenylate cyclase activ-
ity in CYC- membranes (Katada et al., 1984a; Roof et al., 1985). Experiments involving partially
purified preparations of adenylate cyclase, and purified G-proteins reconstituted into phospholip-
id vesicles, or in detergent solution have yielded
conflicting results. TD and Gi are capable of inhibiting adenylate cyclase, but only if G, is coinserted in the phospholipid vesicle (Cerione et
al., 1985a). TD-beta/gamma is far more inhibi- tory than is activated TD-alpha, and both are
effective only if G, is included in the reconstitu-
tion with the cyclase catalyst (Cerione et al.,
1986b). In contrast, both activated G,-alpha and beta/gamma have been reported to inhibit the
catalyst directly (i.e. without added G,) in deter- gent solution (Katada et al., 1986). The catalyst preparation used in this report, however, con-
tained G,. In summary, the mechanism of adenyl-
ate cyclase inhibition by G-proteins is prob-
lematic. Beta/gamma subunits are clearly more potent inhibitors of adenylate cyclase than are
G,-alpha subunits, but the relevance of this dif- ference to the situation in the intact cell remains to be clarified.
A further ramification of this problem is the difficulty in relating a specific G-protein to the function of inhibition of adenylate cyclase. If beta/gamma rather than alpha subunits are re- sponsible for adenylate cyclase inhibition, and if
most (?all) G-proteins possess functionally equiv-
alent beta/gamma subunits, ‘Gi’ may not be a meaningful term for a specific or unique G-pro-
tein. Also, since the general model of G-protein function postulates interaction of activated alpha subunits with E, the functional role of G,-alpha is open to question.
TD
Visual transduction in rod photoreceptor cells involves a G-protein (Wheeler et al., 1977) also termed transducin (Stryer, 1986). TD couples a light R, rhodopsin, to a distinct E, cGMP phos- phodiesterase. In rod cells, cGMP appears to keep
sodium channels in the plasma membrane open, and thereby regulates synaptic transmission. Thus cGMP hydrolysis is a key event in visual transduc- tion in rods. A similar mechanism, involving cone
pigments related to rhodopsin (Nathans et al., 1986) a G-protein, and cGMP phosphodiesterase
may also operate in cone photoreceptor cells. Polyclonal antisera raised against TD-alpha puri-
fied from bovine rod outer segments detect TD in
chick retinal rods but not cones (Grunwald et al.,
1986). This suggested that a putative cone TD
might be immunochemically distinct from rod TD. A cDNA for a unique form of TD-alpha has
recently been cloned (Lochrie et al., 1985). Anti- bodies raised against a unique peptide predicted
by this cDNA react specifically with cones in bovine retina (Lerea et al., 1986).
Unlike G, and Gi, TD is found only in photo-
receptor cells. TD-alpha is ADP-ribosylated on an arginine residue (Van Dop et al., 1984a) by cholera toxin, and on a cysteine residue (West et al., 1985) by pertussis toxin. The former modification activates TD (Abood et al., 1982); the latter
uncouples TD from rhodopsin (Van Dop et al., 1984b).
Go This protein was discovered during purification
of Gi from bovine brain (Neer et al., 1984; Sternweis and Robishaw, 1984; Milligan and Klee,
1985). The beta/gamma complex of G, is indis- tinguishable from that of Gi. In contrast, the 39 kDa Go-alpha subunit is distinct from that of
other G-proteins in susceptibility to proteolysis (Sternweiss and Robishaw, 1984; Winslow et al., 1986), and in reactivity with specific antibodies (Huff et al., 1985; Mumby et al., 1985; Pines et al., 1985; Gierschik et al., 1986b). G, is 5- to lo-fold more abundant than Gi in bovine brain (Gierschik et al., 1986b), comprises about 1% of total cerebral cortical plasma membrane protein, and accounts for the majority of high affinity guanine nucleotide binding activity in brain (Sternweis and Robishaw, 1984). Like Gi and TD, G, is a pertussis toxin substrate. Pertussis toxin ADP-ribosylates holo-G-proteins much more effi- ciently than G-alpha subunits. Go-alpha dissoci- ates more readily from beta/gamma than do other G-alpha subunits (Neer et al., 1984; Sternweis and
7
Robishaw, 1984). Pertussis toxin ADP-ribosyla-
tion of G, is, thus, particularly sensitive to condi- tions (Mg*+ concentration, guanine nucleotides)
favoring dissociation of alpha subunits. G, may not be as widely distributed as are G,
and Gi. Little, if any, G, is found in liver (Huff et
al., 1985) or neutrophils (Gierschik et al., 1986a).
Resolution by SDS-PAGE of two distinct pertus-
sis toxin substrates (39-41 kDa) in fat cells (Malbon et al., 1984) and in heart (Halvorsen and Nathanson, 1984; Malbon et al., 1985) may indi-
cate that both G, and Gi are present. G, has been identified in heart (Huff et al., 1985) and in 3T3-Ll
fibroblasts and adipocytes (Gierschik et al., 1986~) with specific antibodies.
The specific function of G, is unknown. The assumption that G, is a signal transducer rests on its association with the plasma membrane, its analogous structure to that of other G-proteins,
and its interaction with specific R. In reconstitu- tion experiments, purified G, couples with
muscarinic R (Florio and Sternweis, 1985; Kurose
et al., 1986) and with alpha,-adrenergic R (Ceri-
one et al., 1986a) with approximately the same efficiency as does purified Gi. It is not clear
whether either or both of these G-proteins couple
to these R in native membranes as opposed to in artificial phospholipid vesicles. The D, dopamine receptor from anterior pituitary copurifies with a
G-protein, tentatively identified as G, with specific antibodies (Senogles et al., submitted). This sug- gests that G, and D, dopamine R may interact specifically in membranes.
The specific E with which G, interacts is even more problematic. Dissociation of beta/gamma subunits from activated G, could inhibit adenylate
cyclase. As discussed earlier, such an action would mean that G, is a form of Gi. A specific function
for Go-alpha has yet to be elucidated. Go-alpha does not substitute for TD-alpha in activation of
cGMP phosphodiesterase, nor does it stimulate (like G,-alpha) or inhibit (like G,-alpha) adenylate
cyclase in CYC- S49 membranes (Roof et al., 1985). Immunocytochemical localization of G, in brain corresponds more closely with the distribu- tion of protein kinase C than with that of adeny- late cyclase (Worley et al., 1986). This has been interpreted to suggest involvement of G, in regu- lation of phosphoinositide (PI) breakdown. Sub-
stantial evidence links a G-protein to stimulation
of PI breakdown (see next section), but there is no evidence identifying this G-protein as G,. In the- ory, G, could act to inhibit PI breakdown, an
intriguing possibility given the possible connection
between G, and the D, R (Senogles et al., sub-
mitted) and the inhibitory effect of D, R agonists on PI breakdown and prolactin release in anterior
pituitary (Enjalbert et al., 1986).
Other types of signal transduction by G-proteins
PI breakdown R-stimulated breakdown of PI leads to forma-
tion of a calcium-mobilizing second messenger,
IP3, and an activator of protein kinase C, di- acylglycerol (Berridge and Irvine, 1984). Many different agonists utilize this signal transduction
pathway for which the relevant E appears to be phospholipase C. Two lines of evidence suggest that one or more G-proteins (Gomperts, 1983)
couples R to phospholipase C (reviewed in Wil-
liamson, 1986). First, guanine nucleotides aug- ment agonist stimulation of phospholipase C or directly activate the enzyme in many types of cell
membranes, including blowfly salivary glands
(Litosch et al., 1985) and human neutrophils (Cockroft and Gomperts, 1985; Smith et al., 1986).
Second, in certain cell types, including neutrophils
(Okajima and Ui, 1984; Becker et al., 1985; Verghese et al., 1985a, b; Volpi et al., 1985) and
mast cells (Nakamura and Ui, 1984) pertussis toxin blocks agonist stimulation of PI breakdown. This suggests that a G-protein pertussis toxin sub- strate couples R such as that for the chemotactic peptide, f-Met-Leu-Phe, to phospholipase C. The G-protein(s) responsible for coupling R to phos- pholipase C has not been definitively identified. The effect of pertussis toxin in cells such as neu-
trophils (ADP-ribosylation of an approximately 40 kDa protein correlates with inhibition of agonist-stimulated PI breakdown) has led to the
suggestion that Gi activates phospholipase C. The evidence, however, is inconclusive, since several G-proteins serve as pertussis toxin substrates. Im- munochemical studies (Gierschik et al., 1986a) suggest that two of these, G, and TD, are either absent or present at too low a concentration to represent the pertussis toxin substrate of human
8
neutrophils. An antibody readily capable of de-
tecting Gi in brain, however, also shows minimal reactivity with the neutrophil pertussis toxin sub-
strate (Gierschik et al., 1986a). This may be ex- plained by the recent cloning of several related but distinct forms of cDNA for G,-alpha (Reed
and Jones, personal communication). Rather than a single G,, there appear to be several, perhaps tissue-specific, forms. Further evidence that the
G-protein linked to phospholipase C in white cells
may be unique derives from studies with cholera
toxin. In a macrophage (Acksamit et al., 1985) and T-cell line (Imboden et al., 1986) cholera toxin
ADP-ribosylates a protein of about 41 kDa and
inhibits agonist-stimulated PI breakdown. This ef- fect is independent of G, activation and is similar
to that of pertussis toxin. One can speculate that
the G-protein linked to phospholipase C in white cells is both a cholera and pertussis toxin sub- strate, and that unlike for G, and TD, cholera toxin ADP-ribosylation deactivates the G-protein.
There is good evidence for R-phospholipase C
coupling by a G-protein in many other cell types
(e.g. GH, pituitary, pancreatic acinar, liver; Wil- liamson, 1986). In these cells, however, the G-pro-
tein appears to be insensitive to treatment of the
cell with pertussis toxin. This suggests either that
at least two distinct G-proteins (a pertussis toxin- sensitive and an insensitive form) mediate phos-
pholipase C stimulation in different cell types, or that a single G-protein with varying susceptibility to pertussis toxin is involved. Accessibility to per-
tussis toxin ADP-ribosylation could be masked by tissue-specific post-translational modifications. Another relevant factor is the ratio of alpha to beta/gamma subunits. Pertussis toxin favors the G-holoprotein as substrate; free G-alpha subunits would be relatively insensitive to the toxin. This
may be particularly relevant for G-proteins, e.g. G, (Huff and Neer, 1986), whose alpha subunit shows relatively low affinity for beta/gamma.
Many agonists (e.g. alpha-adrenergic and muscarinic cholinergic agents, angiotensin) both inhibit adenylate cyclase and stimulate PI break- down. Distinct R (e.g. alpha,-adrenergic for cyclase and alpha,-adrenergic for PI) and distinct G-proteins may be involved. Pertussis toxin un- couples a G-protein from alpha,-adrenergic R in kidney, without affecting the coupling of alphai-
adrenergic R (Boyer et al., 1984). Treatment with
pertussis toxin or N-ethylmaleimide blocks muscarinic cholinergic agonist inhibition of adenylate cyclase without altering stimulation of PI breakdown (Martin et al., 1985). Thus two distinct G-proteins may mediate signal transduc-
tion through these two pathways (Hughes et al., 1984). f-Met-Leu-Phe action in neutrophils pro-
vides another example of the dichotomy between
agonist-stimulated PI breakdown and cyclase in-
hibition. The chemotactic peptide stimulates PI
breakdown, without inhibiting (perhaps even
stimulating) adenylate cyclase (Smith et al., 1985). Stimulation of PI breakdown, rather than
cGMP hydrolysis, may be the second messenger pathway mediating phototransduction in in- vertebrates (Fein et al., 1984). A G-protein links photosensitive R to E in invertebrate photorecep- tors (Saibil and Michel-Villaz, 1984; Vandenberg and Montal, 1984; Blumenfeld et al., 1985; Fein, 1986). Its structure has not been elucidated but its
alpha subunit may, like vertebrate TD, be a per- tussis toxin substrate (Tsuda et al., 1986). It is not clear if invertebrate G-proteins are heterotrimeric;
a report (Tsuda et al., 1986) of a 35 kDa protein in octopus photoreceptors showing cross-reactivity
with beta subunit antibodies requires confirma-
tion.
Control of meiosis in Xenopus oocytes
CAMP inhibits meiosis in Xenopus oocytes. Progesterone acts at a site on the cell membrane to decrease CAMP formation and promote meio- sis. Progesterone action appears to involve a G- protein (Sadler and Maller, 1981). Several groups have shown that oocyte membranes contain a substrate for pertussis toxin ADP-ribosylation, but that modification of this protein does not abolish the inhibitory effect of progesterone on adenylate cyclase (Goodhardt et al., 1984; Olate et al., 1984). The nature of the ‘Gi’ in Xenopus oocyte mem-
branes remains to be clarified.
Olfaction
Olfactory transduction involves sensitive detec- tion of a vast array of odorants, and generation of relevant second messengers leading to synaptic
9
transmission. There are anatomic and functional
parallels between olfactory (specialized cilia) and
visual (photoreceptor outer segments) transduc- tion. Stimulation of adenylate cyclase (Pace and
Lancet, 1986) and of PI breakdown (Huque and Bruch, 1986) may both be involved in olfactory
transduction. A G-protein is likely linked to both second messenger pathways. It is not yet clear if odorant stimulation of adenylate cyclase in olfac-
tory cilia involves G, (as in other cells) or a
protein related to but distinct from G, (Pace and
Lancet, 1986).
Insulin action
Receptors for insulin and many other growth factors are tyrosine kinases. As such, they may
function as complete R-E units. Nonetheless, in- teraction with G-proteins could also be relevant to
agonist action on tyrosine kinase type R. Sugges- tions that rus gene products may couple to growth factor R will be discussed in a later section. There
are also suggestions that a specific G-protein in- teracts with and mediates certain actions of the insulin R (Houslay and Heyworth, 1983). Under certain conditions, insulin inhibits adenylate
cyclase in a guanine nucleotide-sensitive manner
(Heyworth and Houslay, 1983). Insulin, moreover,
reduces cholera toxin-catalyzed ADP-ribosylation of a 25 kDa protein in liver membranes. The latter
is suggested to be a G-protein based on photoaf- finity labeling with a GTP analog (Heyworth et al., 1985). Further characterization of the structure
and function of this putative ‘g-insulin’ is needed.
Ion channels
G-protein(s) appear to link muscarinic R di- rectly to a potassium channel in frog (Breitweiser and Szabo, 1985) and chick (Pfaffinger et al.,
1985) heart. In the latter, a pertussis toxin sub- strate is implicated. Ion channels are known to be modified by second messengers generated by G- protein-linked E. The present data suggest that ion channels themselves may act as E linked to G-proteins, and that G-proteins may modulate other types of E in addition to enzymes. In dorsal root ganglion neurons, a pertussis toxin-sensitive G-protein links R for norepinephrine and GABA
to inhibition of voltage-sensitive Ca*+ channels.
The E in this case may not be the ion channel
itself, since diacylglycerol released from PI breakdown mimics the effect of R agonists (Holz et al., 1986).
Exocytosis
IP3, a second messenger derived from PI breakdown, releases Ca*’ from stores in endo-
plasmic reticulum (Berridge and Irvine, 1984).
Elevation of cytosolic Ca*+ is thought to be the
signal for exocytosis. Recent evidence suggests
that guanine nucleotides act, not only to modulate
formation of IP3, but directly to release Ca*+
from endoplasmic reticulum (Barrowman et al.,
1986; Henne and Solling, 1986). The effect is guanine nucleotide-specific, but unlike for other
G-protein-mediated effects, nonhydrolyzable GTP analogs are ineffective (Gill et al., 1986). The
nature of the G-protein involved is unclear. There
are also data suggesting Ca*+-independent effects of guanine nucleotides on exocytotic secretion (Gomperts, 1986).
Specific features of G-protein structure
Recent work on purification of G-proteins, cloning of cDNAs coding for G-protein subunits,
and X-ray crystallography of EF-Tu, a guanine
nucleotide binding protein with sequence ho- mology to G-proteins has dramatically increased
our understanding of G-protein structure and function.
Alpha subunits
Purification of TD- and Go-alpha in sufficient quantity for amino acid sequencing yielded partial sequences of several tryptic peptides (the amino- termini of both alpha subunits are blocked). The sequences obtained revealed regions of diversity, as well as regions identical in TD- and Go-alpha (Hurley et al., 1984b). Oligodeoxynucleotides cor- responding to the amino acid sequence of con- served regions proved useful in screening cDNA libraries for clones coding for G-alpha subunits. Antibodies against TD-alpha were also successful in screening expression vector libraries.
Two distinct cDNA clones thought to represent TD-alpha were obtained by screening bovine reti- nal libraries with either antibody (Medynski et al.,
1985; Tanabe et al., 1985; Yatsunami and Khorana, 1985) or oligodeoxynucleotide (Lochrie
et al., 198.5) probes. The cDNAs code for proteins 350 and 354 amino acids long, respectively, and
differ by about 20% in amino acid sequence. Re-
cent evidence indicates that the 350 amino acid
polypeptide is rod TD-alpha, and that the 354
amino acid protein is the corresponding alpha
subunit in cones (Lerea et al., 1986). On Northern
blots, the two cDNAs hybridize to distinct mRNAs
in retina but not in other tissues. This is consistent
with the restricted distribution of TD-alpha (ret-
inal photoreceptors only). Multiple cDNAs coding for G,-alpha have been
obtained by screening bovine (Harris et al., 1985;
Nukada et al., 1986b) and human (Bray et al., 1986) brain libraries with oligodeoxynucleotides corresponding to conserved sequences of G-alpha subunits. This is surprisingly given the lower abundance of G, relative to other G-proteins in brain. Antibodies raised against unique peptides
predicted by the putative G,-alpha cDNA react on immunoblot with purified G,-alpha (Harris et al.,
1985). Further proof that the cDNA codes for G,-alpha comes from Northern blots; the cDNA
hybridizes to a 1900 base mRNA in all tissues but
fails to hybridize to RNA from mutant CYC-
cells, deficient in G, activity (Harris et al., 1985;
Bray et al., 1986).
The first cDNA for G,-alpha obtained from bovine (Nukada et al., 1986b; Robishaw et al., 1986a) and rat (Itoh et al., 1986) brain libraries codes for a 394 amino acid protein. Subsequently, a cDNA for a 380 amino acid protein was ob- tained from a bovine adrenal library (Robishaw et al., 1986b). The latter is identical to the former except for a ,small region between amino acids 71-87. G,-alpha is known to exist in at least two forms (Gilman, 1984), variously designated 42 and 47, or 45 and 52 kDa. Expression of the two G,-alpha cDNAs in cos cells, and use of peptide- specific antibodies led to the conclusion that the two cDNAs correspond to the two forms of G,-al- pha protein (Robishaw et al., 1986b). The latter are derived from distinct mRNAs rather than being products of post-translational modifica-
tions. Screening of a human brain library (Bray et
al., 1986) yielded four distinct forms of G,-alpha cDNA, differing in the identical (71-87) region. Two correspond to the two bovine cDNAs de- scribed. All four forms may be derived from alter- native splicing of a single precursor mRNA (Bray
et al., 1986). G,-alpha cDNA sequence is extraor- dinarily conserved (95%) between rat, cow and
man. Conservation is high not only in the protein
coding region, but also (91%) in the 3’ untrans- lated region. The latter may reflect a regulatory
role for this region of G,-alpha mRNA.
Go-alpha cDNA was obtained by screening a
rat CG-glioma library with synthetic probes (Itoh
et al., 1986). The identity of the cDNA was con-
firmed by comparing predicted amino acid se- quence with that of tryptic peptides of purified
Go-alpha. The protein is 354 amino acids long (Reed and Jones, personal communication).
cDNAs coding for G,-alpha subunits have been
obtained from bovine (Nukada et al., 1986a), rat (Itoh et al., 1986), and human (Bray, personal communication) brain libraries. The identity of the bovine cDNA, coding for a 354 amino acid protein, was verified by comparing predicted
amino acid sequence to that of tryptic fragments of bovine brain G,-alpha (purified and separated from G,,-alpha). The human cDNA is highly ho-
mologous ( > 90%) to the bovine in both the cod-
ing and 3’ untranslated region. The rat cDNA,
however, codes for a 355 amino acid protein,
differs slightly from the bovine sequence in the
coding region, and differs substanti~ly in the 5’ and 3’ untranslated regions. R. Reed and D. Jones
(personal communication) screened a rat olfactory cilia library with oligodeoxynucleotide probes and
obtained six distinct cDNA clones coding for G- alpha subunits. Two correspond to G,- and Go-al- pha. One corresponds to the rat Gi-alpha de- scribed by Itoh et al. (1986). At least two of the three remaining cDNAs code for Gi-alpha-type proteins. These differ only subtly in the coding region but are distinct in untranslated portions of the mRNA. Apparently then, there are multiple versions of G,-alpha. The significance of these diverse forms of G,, in terms of function and tissue distribution is at present obscure,
Analysis of the predicted amino acid sequences of cDNAs coding for G-alpha subunits reveals
11
several noteworthy features: (1) TD, Gi, and G,
show approximately 60% homology; G,, at 40% homology to TD, is more distantly related. (2)
Four regions strongly conserved within the G-al-
pha family also show significant homology to cor- responding regions of other guanine nucleotide
binding proteins, including the ras gene products, and elongation and initiation factors (see e.g. Hal-
liday, 1983/1984). X-Ray crystallographic studies of EF-Tu (Jurnak, 1985; McCormick et al., 1985)
reveal that amino acids in three of these regions participate in binding to the phosphoryl groups
and guanine ring of GDP. (3) The site of cholera
toxin ADP-ribosylation, arginine 174 in rod TD- alpha, is not directly linked to guanine nucleotide
binding, but borders on a site (arginine 204) whose
sensitivity to tryptic cleavage is determined by the
nature of the bound guanine nucleotide (GDP vs. GTP-y-S). Interestingly, not only G,- and TD-,
but also Gi- and Go-alpha subunits have arginine
in the corresponding position. The basis for pref- erential ADP-ribosylation of G, and TD by cholera toxin is not clear, but may relate to differences in secondary and tertiary structure. (4) G,-, G,,-, and TD-alpha subunits (but not G,) all contain a
cysteine as the fourth amino acid from the carbox- yl-terminus. This is the site of pertussis toxin ADP-ribosylation (West et al., 1985), and likely of
N-ethylmaleimide alkylation (Reichert and Hoff- man, 1984; Asano and Ogasawara, 1985), that
leads to uncoupling of Gi, G,,, and TD from R.
This, and other indirect evidence, suggests that the
carboxyl-terminus of these G-alpha subunits may be involved in R interaction.
The domains involved in other G-alpha subunit
functions have not been identified. An amino-
terminal l-2 kDa tryptic fragment may be re- quired for beta/gamma interaction (Watkins et al., 1984; Medynski et al., 1985). The region roughly comprising amino acids loo-140 is par- ticularly divergent between the G-alpha subunits. One might speculate that this region defines the
specificity of G-protein-E interaction.
Beta subunit
The amino-terminus of the 36 kDa beta subunit is blocked; tryptic peptides were sequenced, and oligodeoxynucleotide probes corresponding to
these sequences used to screen bovine retinal
cDNA libraries (Sugimoto et al., 1985; Fong et al., 1986). The cDNA obtained codes for a 340 amino acid protein. On Northern blots, two forms of
mRNA (possibly related to two potential poly- adenylation sites in the 3’ untranslated region) are detected in retina and other tissues. This is con- sistent with the observations (Manning and Gil-
man, 1983; Gierschik et al., 1985) that the beta subunits of TD and other G-proteins are related.
Using the retinal cDNA (presumed to code for
TD-beta), Sugimoto et al. (1985) screened a bovine
brain library and obtained a cDNA presumed to code for G-beta. The two cDNAs are identical in
the protein coding region, at least through nucleo-
tide residue 264 (the remaining sequence has not
yet been reported). The two cDNAs, however, differ in the 5’ untranslated region. Thus there
appear to be at least two distinct mRNAs for TD-
and G-beta subunits. It is unclear if these are
separate gene products or arise by alternative splicing.
On SDS-PAGE, TD-beta appears as a single band of 36 kDa. Other G-betas, e.g. in liver and
brain, consist of a doublet of 36 and 35 kDa (Sternweis and Robishaw, 1984). Immunochemical
studies suggest that the 36 and 35 kDa forms may be distinct, and that 35 is not a proteolytic prod- uct of 36 (Mumby et al., 1985). Further work is needed to define the structural basis for beta subunit heterogeneity, and its possible functional
significance.
Gamma subunit
Gamma subunits of TD and other G-proteins are similar in size on SDS-PAGE (Hildebrandt et al., 1984) but the gamma subunit of TD is im- munochemically distinct from that of other G-pro- teins (Gierschik et al., 1985). Peptide mapping
studies confirm the difference between TD- and other G-gamma subunits, and suggest that G,-, G,-, and Go-gamma subunits may be identical (Hildebrandt et al., 1985). The amino acid se- quence of bovine TD-gamma has been determined (Ovchinnikov et al., 1985) and cDNA clones have been obtained (Hurley et al., 1984a; Yatsunami et al., 1985). The protein contains 73 amino acids and is relatively acidic and hydrophilic. On North-
12
ern blots, the cDNA hybridizes only to retinal mRNA, consistent with the differences observed
between TD- and G-gamma subunits.
Ras and rus-related gene products
Three distinct mammalian rus genes encode
three closely related proteins of about 21 kDa
(~21) (Ellis et al., 1981). The rus gene products differ primarily in a small region (amino acids
165-185) near the carboxyl-terminus (Taparowsky et al., 1983). Ru.s gene products like G-protein
alpha subunits, bind guanine nucleotides, show GTPase activity, and are associated with the cyto-
plasmic surface of the cell membrane (Gibbs et
al., 1985). A cysteine residue (fourth from the
carboxyl-terminus) is critical for membrane at-
tachment of p21 (Willumsen et al., 1984). Fatty
acid acylation of this residue (Sefton et al., 1982)
apparently precedes membrane insertion (Willum- sen et al., 1984). Interestingly, TD-gamma, and G,-, G,-, and TD-alpha subunits contain a cys- teine in the identical position. This has led to
speculation (e.g. Baehr and Applebury, 1986) that fatty acid acylation of the corresponding cysteine in G-protein subunits occurs, and leads to mem- brane insertion. It is not cIear how to reconcile this suggestion for G,-, G,-, and TD-alpha sub- units with the availability of the same cysteine
residue for ADP-ribosylation by pertussis toxin.
An alternative suggestion would be that acylation
of amino-terminal glycine by myristic acid, as in
the src gene product (Sefton et al., 1982) occurs in G-alpha subunits.
The products of cellular rus genes (protoonco- genes) are involved in regulation of cell division. Mutant forms of ~21, particularly involving amino acids 12, 13, 59, and 61, cause malignant transfor-
mation of NIH 3T3 cells. Similar oncogenic pro- teins are encoded in retroviral genomes, e.g. Kirs- ten and Harvey sarcoma viruses. Many human tumor cell lines, e.g. T24 bladder cancer, contain DNA sequences coding for oncogenic forms of ~21. The substituted amino acids are either di- rectly implicated in guanine nucleotide binding, or as in glycine 12, may indirectly influence hydroly- sis of GTP by altering the secondary structure of a guanine nucleotide binding region. Site-directed mutagenesis of other amino acids related to
guanine nucleotide binding, e.g. aspartic 119 also can confer transforming activity (Sigal et al., 1986).
Many (McGrath et al., 1984; Sweet et al., 1984;
Gibbs et al., 1985), but not all (Der et al., 1986; Lacal et al., 1986) transforming p21 proteins show
reduced GTPase activity. By analogy with G-pro- teins and specifically with cholera toxin-ADP-
ribosylated G,, reduction in p21 GTPase may lead to longer-lived activation and to second messenger
generation, independent of agonist. Mutant forms of p21 with other defects in guanine nucleotide
binding, e.g. enhanced dissociation of GDP, could also cause transformation because of agonist-inde- pendent signal transduction (Sigal et al., 1986;
Walter et al., 1986).
These explanations of the oncogenic potential of mutant p21 assume that rus gene products
function in a manner analogous to G-proteins. As
yet, this remains unproven. In particular, the puta-
tive R and E with which mammalian p21 interacts
have not been identified. In yeast, two genes closely related to mammalian t-us genes code for proteins of 309 and 322 amino acids. In S. cerevisiue (Kataoka et al., 1985; Toda et al., 1985), but not
in S. pombe (Fukui et al., 1986) t-us gene prod- ucts are linked to regulation of adenylate cyclase activity. Although mammalian p21 can substitute for yeast ras gene products in reconstituting
adenylate cyclase activity (Broek et al., 1985) there is no evidence directly linking p21 to mam-
malian adenylate cyclase. Cell lines transformed
by ras (and other oncogenes) show reduced
adenylate cyclase activity, but p21 neither stimu- lates nor inhibits adenylate cyclase directly (Beck-
ner et al., 1985). Indirect evidence suggests that R for growth factors, e.g. EGF (Kamata and Fera- misco, 1984), may be linked by rus gene products to stimulation of PI breakdown (Fleischman et al., 1986).
The analogy between ras gene products and G-proteins extends only to the alpha subunits of the latter. G-beta/gamma subunits apparently do not interact with p21 (Broek et al., 1985), nor have ras-specific equivalents of G-beta/gamma sub- units been identified. This does not exclude the possibility that ~21 could act as a R-E coupler. p21, unlike G-alpha subunits, may not require the equivalent of G-beta/gamma subunits for interac- tion with R. Guanine nucleotide-dependent alter-
13
ation in ~21 (and G-alpha) conformation may control signal transduction without participation of beta/gamma subunits.
Several cDNAs coding for p21-related proteins have recently been identified. These show se- quence homology in regions linked to guanine nucleotide binding, but differ in other regions that may specify protein target interactions. One exam- ple is the rho gene family (Madaule and Axel, 1985), originally identified in aplysia, but also present, and highly conserved, in man. The func- tion of rho and other ras-related gene products is unknown.
Two other mammalian guanine nucleotide binding proteins have recently been identified. One, termed ARF, was originally identified as a protein cofactor required for cholera toxin-cata- lyzed ADFribosylation of G,-alpha. This protein of about 21 kDa is itself a guanine nucleotide binding protein (Kahn and Gilman, 1986). As such, it likely serves a purpose over and above enabling cholera toxin to modify G,. Another protein of approximately 21 kDa was purified from placental membranes (Evans et al., 1986). This protein (ambiguously termed Gr for placenta rather than PI breakdown) app~ently copurifies with beta/gamma subunits comparable to those of other G-proteins. The relationship of this pro- tein to ARF, and to ras and ras-related gene products is unknown at present.
Questions
The recent accumulation of data on G-protein structure and function poses many additional questions: (1) How is tissue-specific expression of G-proteins, e.g. TD, regulated, and how is the synthesis of individual G-protein subunits coordi- nated? (2) What interactions (‘cross-talk’) occur between parallel G-protein transduction path- ways, and between G-protein and other signal transduction pathways? Recent evidence suggests that G,-alpha may serve as a protein kinase C substrate (Katada et al., 1985), and that TD-alpha is both a C kinase and insulin R substrate (Zick et al., 1986). Phorbol esters, C kinase activators, modulate agonist-stimulated CAMP formation negatively (perhaps through phosphorylation of R (Sibley et al., 1984)), and positively (Bell et al.,
1985; Pines et al., 1986), perhaps through G-pro- tein phosphorylation. Phosphorylation of another guanine nucleotide binding protein, eIF-2 (Matts et al., 1983), has important consequences for its function. The physiologic relevance, if any, of G-protein phospho~lation remains to be proven. (3) What additional factors modulate R-E cou- pling by G-proteins? Recent evidence extends the analogy (Bitensky et al., 1981) between neuro- transmitter and photo-transduction. In both sys- tems, a specific kinase phosphorylates the cyto- plasmically located carboxyl-terminus of trans- membrane R. Phosphorylation requires the activated form of R and by reducing R-G-protein interaction may mediate desensitization (Benovic et al., 1986). In the photoreceptor system, an additional 48 kDa protein competes with TD for binding sites on phosphorylated rhodopsin (Wilden et al., 1986). The 48 kDa protein shows sequence homology to TD-alpha (Wistow et al., 1986). This may reflect similarity in domains specifying rhodopsin interaction. Is there a coun- terpart to the 48 kDa protein mediating desensiti- zation in the neurotransmitter and hormone signal transduction pathways?
Acknowledgements
I am grateful to present and former fellows, collaborators, and colleagues who have contrib- uted to my underst~ding of this field. I particu- larly thank Mark Bitensky for first pointing out the symmetry between visual and hormonal trans- duction, and Gerald Aurbach for support and encouragement.
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