TIBS 17 - OCTOBER 1992
ALL CELLS possess the capacity to receive and process information from their surroundings. External signals such as light, odorants and dietary chemicals stimulate target cells in specialized sensory organs; circulating or locally released hormones, neurotransmitters and growth factors serve as chemical messengers between neighboring or distant cells. The interaction of these messengers with specific receptors at the ceil surface represents only the first step in a cascade of molecular events that underlies transmembrane signaling. In many cases, stimulation of these receptors results in activation of effector proteins (e.g. enzymes or ion channels), which mobilize chemical 'second messengers' that initiate characteristic actions within the cell. In all eukaryotic organisms, a family of heterotrimeric GTP-binding and hydrolysing proteins (G proteins) plays an essential transducing role in linking many cell-surface receptors to effector proteins at the plasma membrane. This review will provide a brief summary of knowledge of G protein structure, function and mechanism of action. The G proteins are part of a larger superfamily of GTPases that includes factors involved in protein synthesis, for example elongation factor Tu (EF-Tu) and a large number of monomeric 20-25 kDa proteins such as p21r~s; these will not be discussed here, but have been reviewed previously TM.
Second messengergeneration and destruction
The family of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serves an essential role in transducing receptor-generated signals across the plasma membrane. Recent findings reveal unexpected functional roles for individual G protein subunits. Thus, GTP-binding o~-subunits and the [~,-subunit complex can influence the activity of effector molecules independently or simultaneously, either synergistically or in opposition, to elicit a complex constellation of cellular events.
is inactive, whereas the GTP-bound form of a dissociates from 137 and serves as a regulator of effector proteins. Aluminium tetrafluoride (AIF~), together with Mg2÷, can interact with abound GDP to mimic GTP and thereby activate a. Increasing evidence now supports the hypothesis that [~', like a, can also interact with and modulate the activity of at least some effector proteins (see below). All a-subunits are themselves enzymes. That is, these proteins possess intrinsic GTPase activity and will, at varying rates, hydrolyse the terminal phosphate of bound GTP to yield bound GDP and free inorganic phosphate (P~). In some cases, a-subunits possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyses the transfer of the ADP-ribose moiety of NAD to a specific Arg residue in certain a-subunits. Similarly, pertussis toxin ADPribosylates those a-subunits that posStructure and properties G proteins are heterotrimers, com- sess a specific Cys residue near the posed of three distinct subunits: carboxyl terminus. Modification of a by a (molecular mass = 39-46 kDa), 13 (37 cholera toxin constitutively activates kDa) and ~/(8 kDa). The properties of these proteins (by inhibiting their these polypeptides are presented in GTPase activity), whereas modification Tables I and II. The 13- and ~,-subunits by pertussis toxin prevents receptorexist as a tightly associated complex mediated activation of G proteins. that functions as a unit. Although the Although none of the G protein subsame [3~,subunit complex can apparently units contains regions that might obvibe shared among different a-subunits to ously associate with a lipid bilayer, the form the heterotrimer, the identity of heterotrimer is bound to the plasma the a-subunit is currently used to membrane. This is apparently due to the define an individual G protein oligomer. fact that 7-subunits are prenylated and The a-subunits have a single, high- at least some a-subunits (those of the affinity binding site for guanine G~ subfamily; see below) are myristoylnucleotides (GDP or GTP). The GDP- ated. These lipid modifications serve to bound form of a binds tightly to [~, and anchor the subunits to the membrane (perhaps by increasing the affinity of protein-protein interactions) and they J. R. Hepler and A. G. Gilman are at the also increase the affinity of a for [3~,5 Department of Pharmacology, Universityof (J. Ifliguez-Lluhi et al., submitted). A Texas Southwestern Medical Center, 5323 Harry Hines Bird, Dallas, TX 75235, USA. working model of the interplay between © 1992.ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00
receptors, G proteins and effectors is shown in Fig. 1 and details of the G protein activation/deactivation cycle are described in the legend (see also Refs 6,7). Relationships and functions of G protein subunits The family of G protein a-subunits can be subclassified according to functional or structural relationships 4,8 and there is reasonable congruence between such schemes. The nomenclature utilized to denote individual family members is unfortunately confusing, but knowledge of function is probably still too limited to propose a lasting alternative. G proteins were first identified functionally and purified conventionally; names were assigned with subscripts chosen to evoke functional roles. Since purification and cloning of subsequently discovered members of the family were largely accomplished with homology-based approaches, later names were chosen according to the whim of the discoverer. One now resorts to numbers. To date, cDNAs that encode 21 distinct G protein a-subunits (the products of 17 genes) have been cloned; these can be divided into four major subfamilies according to amino acid sequence relationships, i.e. those represented by Gs, G~, Gq and G12. In addition, at least four distinct [3- and six ~,-subunits have been described; it is a safe bet that these numbers will increase. The sequence relationships between different a-subunits and family groupings are shown in Fig. 2. The ubiquitous hormone-stimulated adenylate cyclase system 6 and the specialized light-activated cGMP phosphodiesterase pathway in retinal rod outer segments 7 have served as models for understanding G protein-receptor
383
Second messenger generation and destruction
TIBS 1 7 - OCTOBER 1992
Table I, Properties of mammalian G protein ~-subunits Family/subunit
GS cCs(s)(2X)c (Zs(L)(2X)c
Mass (kDa × 10 -3)
% Amino Toxinb acid identitya
Tissue distribution
Representative receptors
Effector/role
44.2 45.7
100 -
CTX CTX
Ubiquitous Ubiquitous
BARd, glucagon, ~ TSH, others j"
1" Adenylate cyclase 1" Ca 2+ channels $ Na+ channels
C~olf
44.7
88
CTX
Olfactory neuroepithelium
Odorant
1" Adenylate cyclase
Gi 0~il o¢i2 o¢i3
40.3 40.5 40.5
100 88 94
PTX PTX PTX
Nearly ubiquitous Ubiquitous Nearly ubiquitous
M2Ch°' (z2AR' / others
O¢OA c C(OBc
40.0 40.1
73 73
PTX PTX
Brain, others Brain, others
Met-Enk, c¢2AR, others
1` K÷ channels $ Ca2÷ channels $ Adenylate cyclase (?) 1` Phospholipase C (?) 1" Phospholipase A2 (?)
C% 0¢t2
40 40.1
68 68
CTX,PTX CTX,PTX
Retinal rods Retinal cones
Rhodopsin Cone opsin
C(g
40.5
67
CTX (?), PTX
Taste buds
Taste (?)
c~z
40.9
60
Brain, adrenal platelets
M2Cho (?), others (?)
$ Adenylate cyclase (?) others (?)
Gq C~q (](11 c~14
42 42 41.5
100 88 79
Nearly ubiquitous Nearly ubiquitous Lung, kidney, liver
MlCh°' c¢IAR, / J others ?
$ Phospholipase C-[31, [~2, [~3 others (?)
] ~
1" cGMP-specific phosphodiesterase ?
c(15
43
57
B cells, myeloid cells
?
?
c~6
43.5
58
T cells, myeloid cells
?
1" Phospholipase C-131,
Ubiquitous Ubiquitous
? ?
? ?
612 c~12 o~13
44 44
100 67
-132,-~3
a% Amino acid identity; comparison is with the first-listed member of each family.
bCholera toxin (CTX) and pertussis toxin (PTX) catalyse the ADP-ribosylation of an Arg residue (CTX) and a Cys residue (PTX), respectively, of the indicated ~¢-subunits. CSplice variants, c~s(s),short forms of C(s; c(s(L),long forms of c¢s. ~Receptor abbreviations: ~AR, ~-adrenergic; M2Cho, M2-muscarinic cholinergic; c~2AR, c¢2-adrenergic; met-enk, met-enkephalin; M1Cho, Ml-muscarinic cholinergic; c¢IAR, ~¢l-adrenergic.
and G protein-effector interactions. In both cases, definitive evidence for the involvement of a particular G protein was achieved by reconstitution of purified components (activated a-subunit and effector protein). Such studies still provide the most rigorous criterion for the involvement of a G protein in a specific signaling pathway. Hormone and odorant receptors interact with members of the Gs family (G~ and GoIL)to stimulate adenylate cyclase and thus enhance the rate of cyclic AMP (cAMP) synthesis. Because of alternative splicing of a single precursor mRNA, G~ is expressed as four distinct polypeptides with predicted molecular masses ranging from 44 200 to 45700 [although these proteins migrate on SDS gels as two distinct bands with apparent molecular weights
384
of 45000 (Gs~.s) and 52000 (Gs~-L)]. These splice variants have not been well-distinguished functionally. Five distinct isoforms of adenylate cyclase have been described to date; all of these are activated by Gs~. The Gotf protein subunit is expressed exclusively in olfactory neuroepithelium and presumably serves to link odorant receptors with an olfactory-specific form of adenylate cyclase. In addition to activation of adenylate cyclase, purified Gs~ regulates at least two ion channels in reconstituted systems, stimulating dihydropyridinesensitive voltage-gated Ca2+ channels in excised patches from skeletal muscle 9 and inhibiting cardiac Na+channels 1°. In photoreceptor rod outer segments, light-activated rhodopsin activates transducin (Gt]) to stimulate a cGMP-specific phosphodiesterase; cyto-
plasmic concentrations of cGMP are thereby decreased 7. Although Gtl is expressed exclusively in retinal rods, a second form of transducin, Gt2, is expressed in cones and presumably links cone opsins to activation of a distinct phosphodiesterase. A novel transducin-like G protein named gusducin (Gg) has been described recently and is apparently expressed only in taste buds 11. The amino acid sequences of Gg and the transducins are remarkably similar (80%), particularly in those regions of the G protein c¢-subunit known to interact with receptors and effectors. No information is yet available about the actions of Gg, although one might predict the involvement of a phosphodiesterase. The physiological actions of a broad class of hormones, neurotransmitters
TIBS 1 7 - OCTOBER 1992
and growth factors can be explained by their capacity to activate phosphoinositide-specific phospholipase C (PLC), an effector enzyme that catalyses hydrolysis of the minor lipid phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] to form two second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol. Compelling evidence for the involvement of pertussis toxin- and cholera toxin-insensitive G proteins in receptor-mediated activation of PLC has been obtained from many laboratories. At least eight c~-subunits have been described that are not modified by these bacterial toxins (Table I) and, by default, all are candidates. Recent reports have identified members of the Gq family as regulators of this pathway. A mixture of Gq and GH has been purified from bovine brain ~2 and rat liver~3; a closely related protein has also been isolated from turkey erythrocytes 14. Reconstitution of these proteins with purified PLC-[3115,16 or turkey PLCTM results in specific and marked stimulation of the enzyme. Reconstitution of purified recombinant forms of either Gq~ or Gll ~ with purified PLC-[3~, PLC-[32and PLC-[33confirm these findings (J. R. Hepler et al., unpublished); recombinant G~4~has the same action. Although Gq, G~ and G~4 are found in many tissues, the other two members of the Gq family, G~5and G~6,are expressed only in cells of hematopoietic lineage (Table I; Ref. 8). GI5~and G~6~are also more distantly related to Gq~ (57% and 58% identity). Such limited tissue distribution suggests a specialized signalling role for these proteins. However, reconstitution studies reveal that purified recombinant G16a also activates PLC-[31, PLC-[~2 and PLC-[~ (T. Kozasa et al., unpublished). Further studies are in progress to determine the specificity of interactions between members of the Gq family and the plethora of isoforms of PLC. Pertussis toxin inhibits hormonal activation of PLC in a limited number of tissues, suggesting the involvement of a G~-like protein rather than a member of the Gq family. To date, however, conclusive evidence implicating an individual G~or Go protein in this pathway is lacking. Less is known of direct interactions between effectors and several members of the G~ subfamily. Only the transducins have been unambiguously assigned to a signaling pathway. The fact that pertussis toxin blocks many cellular responses implies the involvement of G, family members in a variety
Second messengergeneration and destruction Table II. Properties of mammalian G protein ~- and ~subunits Subunit
131
132 [33 134
Mass %Amino acid (kDa x 10 -3) identitya
37.3 37.3 37.2 37.2
100 90 83 89
Tissue distribution
Effector/role
Ubiquitous "~ Nearly ubiquitous Nearly ubiquitous Nearly ubiquitous
Required for %-receptor interaction Inhibition of % activation Modulate activation of certain adenylate cyclases by Gs~ or calmodulin Support of agonist-induced receptor phosphorylation and desensitization
T T1 T2
8.4 7.9
100 38
T3 Y4 T5
8.5 (?partial) 7.3 7.5
36 (34) 25 35
Retina, other (?) Brain, adrenal, other (?) Brain, testis [Kidney,retina (?)] Liver, other (?) Brain, other(?) j
1" Phospholipase C 1" K÷ channels (?) 1` Phospholipase A2 (?)
a% Amino acid identity: comparison is with the first-listed member of each family. of signaling pathways. The most widely studied of these include inhibition of adenylate cyclase and activation or inhibition of several ion channels roles that are attributed to the three G~ subunits and to the two splice variants of Go~. Stimulation of many hormone receptors results in inhibition of adenylate cyclase activity. This response is blocked by treatment with pertussis toxin, and there is ample evidence for involvement of the G~ proteins. Nevertheless, attempts to inhibit adenylate cyclase activity using purified G~ polypeptides have met with limited success at best 17,18.These observations led to the idea that [3y derived from the G~ oligomer might inhibit adenylate cyclase indirectly by interaction with its activator, Gs~19. Alternative explanations include the ability of [3~'to inhibit certain types of adenylate cyclase directly2° and the possibility that effects of G~ proteins on adenylate cyclase activity are mediated indirectly21. Pertussis toxin-sensitive G proteins also regulate ion channels in muscle, neurons and elsewhere. For example, muscarinic cholinergic receptors activate K+ channels in cardiac myocytes in a membrane-delimited fashion. The pathway can be blocked by pertussis toxin, and channel activity is responsive to guanine nucleotides. Addition of activated G~ proteins to membrane patches excised from these cells stimulates K+ channel activity; G~I, Gia2 and Gia3 work equally well 22. Similarly, certain neuronal K+ channels are activated by purl-
fled Go~. However, demonstration of direct interactions between the G protein a-subunits and K+ channel proteins in lipid bilayers has not yet been reported (the relevant channels have not been purified) and some believe that G protein [3y-subunits may also play an important role 23. Cellular concentrations of the G~ and G proteins are much higher than are those of Gs and Gq. In brain, Go~ is 1-2% of membrane protein. It seems unlikely that the protein is used only to regulate K~ and Ca2+channels. If so, why is Go so concentrated in neural growth cones24? Recent findings suggest that members of the G~ family may regulate pathways deep within the cell. In a kidney cell line, G~3 has been localized to the Golgi complex. Overexpression of Gi~3 in these cells slows secretion of packaged proteins and associated vesicular transport, and this effect is reversed by treatment with pertussis toxin 25. Further evidence comes from studies with the fungal toxin brefeldin A, which stimulates release of the Golgi membrane coat protein [3-COP to cause a pathological fusion of Golgi membranes with those of the endoplasmic reticulum. Treatment of perforated cells with GTPyS or AIF4 antagonizes the actions of brefeldin A26,27.Furthermore, addition of G protein [3y-subunits to a cell-free system prevents GTPTS-mediated binding of B-COP to Golgi membranes 28. Taken together, these findings indicate that a heterotrimeric G protein of the G~ subclass serves an important
385
Second messenger generation and destruction Basal state
jr
Receptor activation
H
(a)
(b) GTP GDP
1 (e)
(e)
GTPase
eo~.N
/
Subunit dissociation
(d)
S
P
Effector activation
Figure 1 G protein-mediated transmembrane signaling. In the basal state (a), G proteins exist as heterotrimers with GDP bound tightly to the c(-subunit; the hormone receptor (R) is unoccupied and the effector (E) is inactive. Upon hormone binding and receptor activation (b), the receptor interacts with the heterotrimer to promote a conformational change and dissociation of GDP from the guanine nucleotide-binding site; at normal cellular concentrations of guanine nucleotides, GTP fills the site immediately. (Under experimental conditions where GTP is absent, the hormone has high affinity for the receptor and the H-R-G protein complex is stable.) Binding of GTP to c~ induces a conformational change with two consequences (c). The G protein dissociates from the H-R complex, reducing the affinity of hormone for receptor and, in turn, freeing the receptor for another liaison with a neighboring quiescent G protein. GTP binding also reduces the affinity of (z for 13)', and subunit dissociation occurs. This frees (z-GTP to fulfill its primary role as a regulator of effectors (d). At least in some systems, the free J3ysubunit complex may also interact directly with an effector (El) and modulate the activity of the active complex, or it may act independently at a distinct effector (E2). The c(-subunits possess an intrinsic GTPase activity (e). The rate of this GTPase determines the lifetime of the active species and the associated physiological response. The c(-catalysed hydrolysis of GTP leaves GDP in the binding site and causes dissociation and deactivation of the active complex. The GTPase activity of (z is, in essence, an internal clock that controls an on/off switch. The GDP bound form of (z has high affinity for [37; subsequent reassociation of c~GDPwith I~Y returns the system to the basal state (a).
regulatory role in membrane trafficking. Information regarding a receptor-like analog in this pathway is lacking, although mastoparans (peptides that mimic the actions of G protein-coupled receptors) stimulate the binding of [~COP to Golgi membranes and block the effects of brefeldin A in a pertussis toxin-sensitive manner 27. These findings provide the most compelling evidence to date that the actions of heterotrimeric G proteins are not limited to transmembrane signaling at the cell surface. Yet, one is left uncomfortable with the assignment of the same G protein to play seemingly unrelated regulatory roles in presumably different cellular pathways. It seems likely that an unsuspected common theme will emerge.
386
Traditionally, the 0(-subunit has been viewed as the 'business end' of a G protein because it binds and hydrolyses GTP and interacts with effectors. In contrast, the ]3~'-subunit complex has been viewed as a regulatory component for 0( which stabilizes the GDP-bound form of 0(, 'presenting' 0( to receptors, serving as a membrane anchor for the oligomer. Yet the activation of G proteins yields two subunits (0(/137) that could act on downstream targets. A growing body of evidence now supports the idea that free ~Y can itself interact functionally with effector proteins. Genetic studies first demonstrated that responses to mating factors in budding yeast are dictated by [~yrather than 0(29. In mammalian systems, direct biochemi-
TIBS 17 - OCTOBER 1992 cal evidence for interactions of [37 with effectors has come from study of different isoforms of adenylate cyclase. Although the adenylate cyclases expressed in most peripheral tissues appear to be largely immune to regulation by 137, at least some of the enzymes found largely in brain are regulated by [~7 in a type-specific fashion2°. Type I (Gs~- and calmodulin-activated) adenylate cyclase is inhibited by 97, apparently directly. Type I! and type IV adenylate cyclases are activated conditionally by ~7; that is, ~7 is stimulatory only when Gs~ is also present. This appears to represent a mechanism for crosstalk between signaling pathways. Concentration requirements suggest that 137would arise by dissociation of G~ or Go, while Gs~ would obviously be liberated by activation of G~-linked receptors. Other effectors also appear to be subject to regulation by ~7. An undefined form of PLC in HL-60 granulocytes is markedly activated by 137~°. In addition, ~7 appears to have effects on K÷ channel activity, although it is unknown if these effects are mediated directly or indirectly23. Recent findings also indicate that 137plays an obligatory role in agonist-induced receptor phosphorylation and desensitization3L Taken together, these observations suggest that dissociation of G protein subunits in the membrane can generate parallel and/or interactive signals via both 0(and [37-subunits. Investigators should have an open and cautious attitude about which G protein subunit (and whether one or both subunits) is mediating the effect in question. It is clear that many more G proteinregulated pathways exist, and several poorly understood cellular responses appear to be dependent on guanine nucleotides, activated by A1F;, and/or sensitive to pertussis toxin. Likely effectors include phospholipases A 2 and D and a plethora of ion channels, transporters and exchangers. Cellular responses under consideration include growth control, effects of tyrosinekinase growth factor receptors, exocytotic secretory events and protein translocation. The receptors, G proteins and effectors involved in many of these pathways remain unknown. Conversely, there is little knowledge of the actions of some 'orphan' G proteins, such as 612 , 613, 615 and G~. Furthermore, we have discussed examples where a single G protein 0(-subunit (e.g. G~) can regulate more than one eflector; thus, the assignment of a given G
Second messengergeneration and destruction
TIBS 17 - OCTOBER 1992
40 ¢,t
I
60 '
I
'
I
80 '
I
'
I
100 '
I
'
I
I-- s 1 Gs 0~ol f
~
Oql ~i3
__
- -
f
0~i2
I OoA Gi - -
O~oB
F~
O~tl (Zt2
O~g ~z
-// ~ 1
O~q 0('11 ~14
4
0(,16 0~12 0~13
I
,
40
I
,
I
60
,
I
I
I
I
Gq
I
I
80
G12
I
100
% Amino Acid Identity Figure 2
Sequence relationships between mammalian G. subunits and family groupings(modified,with permission,from Refs8 and 4).
[37-subunits pre- References 1 Hall, A. (1990) Science 249, 635-639 sumably interact 2 Bourne, H. R., Sanders, D. A. and McCormick, F. to dictate the spe(1990) Nature 348, 125-132 cificities of these 3 Bourne, H. R., Sanders, D. A. and McCormick, F. (1991) Nature 349, 117-127 choices and to 4 Kaziro, Y. et al. (1991) Annu. Rev. Biochem. 60, control, synergis349-400 tically or in op5 Linder, M. E. eta/. (1991) J. Biol. Chem. 266, position, the acti4654-4659 6 Gilman, A. G. (1987) Annu. Rev. Biochem. 56, vities of effectors. 615-649 Thus, activation 7 Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119 of a given recep8 Simon, M. I., Strathmann, M. P. and Gautam, N. tor in different (1991) Science 252,802-808 9 Mattera, R. eta/. (1989) Science 243, 804-807 cells can produce a highly varied 10 Schubert, B., VanDongen, A. M. J., Kirsch, G. E. and Brown, A. M. (1989) Science 245, constellation of 516-519 activated G pro- 11 McLaughlin,S. K., McKinnon, P. J. and Margolskee, R. F. (1992) Nature 357, 563-569 teins and effecI-H. and Sternweis, P. C. (1990) J. Biol. tors, and cellular 12 Pang, Chem. 265, 18707-18712 responses will 13 Taylor, S. J., Smith, J. A. and Exton, J. H. (1990) J. Biol. Chem. 265, 17150-17156 change with development or with 14 Waldo, G. L., Boyer, J. L., Morris, A. J. and Harden, T. K. (1991) J. Biol. Chem. 266, cellular history of 14217-14225 exposure to regu- 15 Smrcka, A. V., Hepler, J. R., Brown, K. O. and Sternweis, P. C. (1991) Science 251, 804-807 latory signals. Such vast 'com- 16 Taylor, S. J., Chae, H. Z., Rhee, S. G. and Exton, J. H. (1991) Nature 350, 516-518 binatorial power' 17 Katada, T. et al. (1984) J. Biol. Chem. 259, endows cells and 3586-3595 organisms with 18 Linder, M. E., Ewald, D. A., Miller, R. J. and Gilman, A. G. (1990) J. Biol. Chem. 264, extraordinary ca8243-8251 pacity for fine 19 Gilman, A. G. (1984) Cell 36, 577-579 tuning both the 20 Tang, W-J. and Gilman, A. G. (1991) Science 254, 1500-1503 magnitude and the nature of 21 Federman, A. D. eta/. (1992) Nature 356, 159-161 their responses 22 Yatani, A. et al. (1988) Nature 336, 680-682 to the environ- 23 Logothetis, D. E. et al. (1987) Nature 325, 321-326 ment. 24 Strittmatter, S. M. et al. (1990) Nature 344,
protein to a specific effector does not end 'the game'. Conclusions
As the number of identified G protein subunits continues to grow, the task of unraveling the complexity of the G protein-regulated cellular switchboard expands exponentially. The number of identified (z-, ~- and 7-subunits indicates a limit of nearly 1 000 possible oligomeric combinations; reasonable extrapolation suggests that this number could grow to roughly 5 000 (with further discovery). Multiplication by the number of G protein-linked receptors and effectors indicates that an individual cell must sift through a very large number of choices to complete its own customized, G protein-controlled regulatory network. Further complexity at each step in the signal transduction cascade compounds the problem. Thus, there is both divergence and convergence of protein-protein interactions at both the receptor-G protein level and the G protein-effector level, and (z- and
Acknowledgements
The authors would like to thank M. Linder, T. Kozasa, J. l~iguez-Lluhi and N. Ueda for hlepful comments. Work from the authors' laboratory was supported by NIH Grant GM34497, American Cancer Society Grant BE30N, The Perot Family Foundation, The Lucille P. Markey Charitable Trust and The Raymond and Ellen Willie Chair of Molecular Neuropharmacology. J. R. H. is the recipient of a National Research Service Award F32 GM13569.
836-841 25 Stow, J. L. et al. (1991) J. Cell Biol. 114,
1113-1124 26 Donaldson, J. G., Lippincott-Schwartz,J. and Kiausner, R. D. (1991) J. Cell Biol. 112,
579-588 27 Ktistakis, N. T., Linder, M. E. and Roth, M. G. (1992) Nature 356, 344-346 28 Donaldson, J. G., Kahn, R. A., LippincottSchwartz, J. and Klausner, R. D. (1991) Science
254, 1197-1199 29 Blumer, K. J. and Thorner, J. (1991) Annu. Rev. Physiol. 53, 37-57 30 Camps, M. et al. (1992) Eur. J. Biochem. 206,
821-831 31 Haga, K. and Haga, T. (1992) J. Biol. Chem.
267, 2222-2227
387
ALL CELLS possess the capacity to receive and process information from their surroundings. External signals such as light, odorants and dietary chemicals stimulate target cells in specialized sensory organs; circulating or locally released hormones, neurotransmitters and growth factors serve as chemical messengers between neighboring or distant cells. The interaction of these messengers with specific receptors at the ceil surface represents only the first step in a cascade of molecular events that underlies transmembrane signaling. In many cases, stimulation of these receptors results in activation of effector proteins (e.g. enzymes or ion channels), which mobilize chemical 'second messengers' that initiate characteristic actions within the cell. In all eukaryotic organisms, a family of heterotrimeric GTP-binding and hydrolysing proteins (G proteins) plays an essential transducing role in linking many cell-surface receptors to effector proteins at the plasma membrane. This review will provide a brief summary of knowledge of G protein structure, function and mechanism of action. The G proteins are part of a larger superfamily of GTPases that includes factors involved in protein synthesis, for example elongation factor Tu (EF-Tu) and a large number of monomeric 20-25 kDa proteins such as p21r~s; these will not be discussed here, but have been reviewed previously TM.
Second messengergeneration and destruction
The family of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serves an essential role in transducing receptor-generated signals across the plasma membrane. Recent findings reveal unexpected functional roles for individual G protein subunits. Thus, GTP-binding o~-subunits and the [~,-subunit complex can influence the activity of effector molecules independently or simultaneously, either synergistically or in opposition, to elicit a complex constellation of cellular events.
is inactive, whereas the GTP-bound form of a dissociates from 137 and serves as a regulator of effector proteins. Aluminium tetrafluoride (AIF~), together with Mg2÷, can interact with abound GDP to mimic GTP and thereby activate a. Increasing evidence now supports the hypothesis that [~', like a, can also interact with and modulate the activity of at least some effector proteins (see below). All a-subunits are themselves enzymes. That is, these proteins possess intrinsic GTPase activity and will, at varying rates, hydrolyse the terminal phosphate of bound GTP to yield bound GDP and free inorganic phosphate (P~). In some cases, a-subunits possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyses the transfer of the ADP-ribose moiety of NAD to a specific Arg residue in certain a-subunits. Similarly, pertussis toxin ADPribosylates those a-subunits that posStructure and properties G proteins are heterotrimers, com- sess a specific Cys residue near the posed of three distinct subunits: carboxyl terminus. Modification of a by a (molecular mass = 39-46 kDa), 13 (37 cholera toxin constitutively activates kDa) and ~/(8 kDa). The properties of these proteins (by inhibiting their these polypeptides are presented in GTPase activity), whereas modification Tables I and II. The 13- and ~,-subunits by pertussis toxin prevents receptorexist as a tightly associated complex mediated activation of G proteins. that functions as a unit. Although the Although none of the G protein subsame [3~,subunit complex can apparently units contains regions that might obvibe shared among different a-subunits to ously associate with a lipid bilayer, the form the heterotrimer, the identity of heterotrimer is bound to the plasma the a-subunit is currently used to membrane. This is apparently due to the define an individual G protein oligomer. fact that 7-subunits are prenylated and The a-subunits have a single, high- at least some a-subunits (those of the affinity binding site for guanine G~ subfamily; see below) are myristoylnucleotides (GDP or GTP). The GDP- ated. These lipid modifications serve to bound form of a binds tightly to [~, and anchor the subunits to the membrane (perhaps by increasing the affinity of protein-protein interactions) and they J. R. Hepler and A. G. Gilman are at the also increase the affinity of a for [3~,5 Department of Pharmacology, Universityof (J. Ifliguez-Lluhi et al., submitted). A Texas Southwestern Medical Center, 5323 Harry Hines Bird, Dallas, TX 75235, USA. working model of the interplay between © 1992.ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00
receptors, G proteins and effectors is shown in Fig. 1 and details of the G protein activation/deactivation cycle are described in the legend (see also Refs 6,7). Relationships and functions of G protein subunits The family of G protein a-subunits can be subclassified according to functional or structural relationships 4,8 and there is reasonable congruence between such schemes. The nomenclature utilized to denote individual family members is unfortunately confusing, but knowledge of function is probably still too limited to propose a lasting alternative. G proteins were first identified functionally and purified conventionally; names were assigned with subscripts chosen to evoke functional roles. Since purification and cloning of subsequently discovered members of the family were largely accomplished with homology-based approaches, later names were chosen according to the whim of the discoverer. One now resorts to numbers. To date, cDNAs that encode 21 distinct G protein a-subunits (the products of 17 genes) have been cloned; these can be divided into four major subfamilies according to amino acid sequence relationships, i.e. those represented by Gs, G~, Gq and G12. In addition, at least four distinct [3- and six ~,-subunits have been described; it is a safe bet that these numbers will increase. The sequence relationships between different a-subunits and family groupings are shown in Fig. 2. The ubiquitous hormone-stimulated adenylate cyclase system 6 and the specialized light-activated cGMP phosphodiesterase pathway in retinal rod outer segments 7 have served as models for understanding G protein-receptor
383
Second messenger generation and destruction
TIBS 1 7 - OCTOBER 1992
Table I, Properties of mammalian G protein ~-subunits Family/subunit
GS cCs(s)(2X)c (Zs(L)(2X)c
Mass (kDa × 10 -3)
% Amino Toxinb acid identitya
Tissue distribution
Representative receptors
Effector/role
44.2 45.7
100 -
CTX CTX
Ubiquitous Ubiquitous
BARd, glucagon, ~ TSH, others j"
1" Adenylate cyclase 1" Ca 2+ channels $ Na+ channels
C~olf
44.7
88
CTX
Olfactory neuroepithelium
Odorant
1" Adenylate cyclase
Gi 0~il o¢i2 o¢i3
40.3 40.5 40.5
100 88 94
PTX PTX PTX
Nearly ubiquitous Ubiquitous Nearly ubiquitous
M2Ch°' (z2AR' / others
O¢OA c C(OBc
40.0 40.1
73 73
PTX PTX
Brain, others Brain, others
Met-Enk, c¢2AR, others
1` K÷ channels $ Ca2÷ channels $ Adenylate cyclase (?) 1` Phospholipase C (?) 1" Phospholipase A2 (?)
C% 0¢t2
40 40.1
68 68
CTX,PTX CTX,PTX
Retinal rods Retinal cones
Rhodopsin Cone opsin
C(g
40.5
67
CTX (?), PTX
Taste buds
Taste (?)
c~z
40.9
60
Brain, adrenal platelets
M2Cho (?), others (?)
$ Adenylate cyclase (?) others (?)
Gq C~q (](11 c~14
42 42 41.5
100 88 79
Nearly ubiquitous Nearly ubiquitous Lung, kidney, liver
MlCh°' c¢IAR, / J others ?
$ Phospholipase C-[31, [~2, [~3 others (?)
] ~
1" cGMP-specific phosphodiesterase ?
c(15
43
57
B cells, myeloid cells
?
?
c~6
43.5
58
T cells, myeloid cells
?
1" Phospholipase C-131,
Ubiquitous Ubiquitous
? ?
? ?
612 c~12 o~13
44 44
100 67
-132,-~3
a% Amino acid identity; comparison is with the first-listed member of each family.
bCholera toxin (CTX) and pertussis toxin (PTX) catalyse the ADP-ribosylation of an Arg residue (CTX) and a Cys residue (PTX), respectively, of the indicated ~¢-subunits. CSplice variants, c~s(s),short forms of C(s; c(s(L),long forms of c¢s. ~Receptor abbreviations: ~AR, ~-adrenergic; M2Cho, M2-muscarinic cholinergic; c~2AR, c¢2-adrenergic; met-enk, met-enkephalin; M1Cho, Ml-muscarinic cholinergic; c¢IAR, ~¢l-adrenergic.
and G protein-effector interactions. In both cases, definitive evidence for the involvement of a particular G protein was achieved by reconstitution of purified components (activated a-subunit and effector protein). Such studies still provide the most rigorous criterion for the involvement of a G protein in a specific signaling pathway. Hormone and odorant receptors interact with members of the Gs family (G~ and GoIL)to stimulate adenylate cyclase and thus enhance the rate of cyclic AMP (cAMP) synthesis. Because of alternative splicing of a single precursor mRNA, G~ is expressed as four distinct polypeptides with predicted molecular masses ranging from 44 200 to 45700 [although these proteins migrate on SDS gels as two distinct bands with apparent molecular weights
384
of 45000 (Gs~.s) and 52000 (Gs~-L)]. These splice variants have not been well-distinguished functionally. Five distinct isoforms of adenylate cyclase have been described to date; all of these are activated by Gs~. The Gotf protein subunit is expressed exclusively in olfactory neuroepithelium and presumably serves to link odorant receptors with an olfactory-specific form of adenylate cyclase. In addition to activation of adenylate cyclase, purified Gs~ regulates at least two ion channels in reconstituted systems, stimulating dihydropyridinesensitive voltage-gated Ca2+ channels in excised patches from skeletal muscle 9 and inhibiting cardiac Na+channels 1°. In photoreceptor rod outer segments, light-activated rhodopsin activates transducin (Gt]) to stimulate a cGMP-specific phosphodiesterase; cyto-
plasmic concentrations of cGMP are thereby decreased 7. Although Gtl is expressed exclusively in retinal rods, a second form of transducin, Gt2, is expressed in cones and presumably links cone opsins to activation of a distinct phosphodiesterase. A novel transducin-like G protein named gusducin (Gg) has been described recently and is apparently expressed only in taste buds 11. The amino acid sequences of Gg and the transducins are remarkably similar (80%), particularly in those regions of the G protein c¢-subunit known to interact with receptors and effectors. No information is yet available about the actions of Gg, although one might predict the involvement of a phosphodiesterase. The physiological actions of a broad class of hormones, neurotransmitters
TIBS 1 7 - OCTOBER 1992
and growth factors can be explained by their capacity to activate phosphoinositide-specific phospholipase C (PLC), an effector enzyme that catalyses hydrolysis of the minor lipid phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] to form two second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol. Compelling evidence for the involvement of pertussis toxin- and cholera toxin-insensitive G proteins in receptor-mediated activation of PLC has been obtained from many laboratories. At least eight c~-subunits have been described that are not modified by these bacterial toxins (Table I) and, by default, all are candidates. Recent reports have identified members of the Gq family as regulators of this pathway. A mixture of Gq and GH has been purified from bovine brain ~2 and rat liver~3; a closely related protein has also been isolated from turkey erythrocytes 14. Reconstitution of these proteins with purified PLC-[3115,16 or turkey PLCTM results in specific and marked stimulation of the enzyme. Reconstitution of purified recombinant forms of either Gq~ or Gll ~ with purified PLC-[3~, PLC-[32and PLC-[33confirm these findings (J. R. Hepler et al., unpublished); recombinant G~4~has the same action. Although Gq, G~ and G~4 are found in many tissues, the other two members of the Gq family, G~5and G~6,are expressed only in cells of hematopoietic lineage (Table I; Ref. 8). GI5~and G~6~are also more distantly related to Gq~ (57% and 58% identity). Such limited tissue distribution suggests a specialized signalling role for these proteins. However, reconstitution studies reveal that purified recombinant G16a also activates PLC-[31, PLC-[~2 and PLC-[~ (T. Kozasa et al., unpublished). Further studies are in progress to determine the specificity of interactions between members of the Gq family and the plethora of isoforms of PLC. Pertussis toxin inhibits hormonal activation of PLC in a limited number of tissues, suggesting the involvement of a G~-like protein rather than a member of the Gq family. To date, however, conclusive evidence implicating an individual G~or Go protein in this pathway is lacking. Less is known of direct interactions between effectors and several members of the G~ subfamily. Only the transducins have been unambiguously assigned to a signaling pathway. The fact that pertussis toxin blocks many cellular responses implies the involvement of G, family members in a variety
Second messengergeneration and destruction Table II. Properties of mammalian G protein ~- and ~subunits Subunit
131
132 [33 134
Mass %Amino acid (kDa x 10 -3) identitya
37.3 37.3 37.2 37.2
100 90 83 89
Tissue distribution
Effector/role
Ubiquitous "~ Nearly ubiquitous Nearly ubiquitous Nearly ubiquitous
Required for %-receptor interaction Inhibition of % activation Modulate activation of certain adenylate cyclases by Gs~ or calmodulin Support of agonist-induced receptor phosphorylation and desensitization
T T1 T2
8.4 7.9
100 38
T3 Y4 T5
8.5 (?partial) 7.3 7.5
36 (34) 25 35
Retina, other (?) Brain, adrenal, other (?) Brain, testis [Kidney,retina (?)] Liver, other (?) Brain, other(?) j
1" Phospholipase C 1" K÷ channels (?) 1` Phospholipase A2 (?)
a% Amino acid identity: comparison is with the first-listed member of each family. of signaling pathways. The most widely studied of these include inhibition of adenylate cyclase and activation or inhibition of several ion channels roles that are attributed to the three G~ subunits and to the two splice variants of Go~. Stimulation of many hormone receptors results in inhibition of adenylate cyclase activity. This response is blocked by treatment with pertussis toxin, and there is ample evidence for involvement of the G~ proteins. Nevertheless, attempts to inhibit adenylate cyclase activity using purified G~ polypeptides have met with limited success at best 17,18.These observations led to the idea that [3y derived from the G~ oligomer might inhibit adenylate cyclase indirectly by interaction with its activator, Gs~19. Alternative explanations include the ability of [3~'to inhibit certain types of adenylate cyclase directly2° and the possibility that effects of G~ proteins on adenylate cyclase activity are mediated indirectly21. Pertussis toxin-sensitive G proteins also regulate ion channels in muscle, neurons and elsewhere. For example, muscarinic cholinergic receptors activate K+ channels in cardiac myocytes in a membrane-delimited fashion. The pathway can be blocked by pertussis toxin, and channel activity is responsive to guanine nucleotides. Addition of activated G~ proteins to membrane patches excised from these cells stimulates K+ channel activity; G~I, Gia2 and Gia3 work equally well 22. Similarly, certain neuronal K+ channels are activated by purl-
fled Go~. However, demonstration of direct interactions between the G protein a-subunits and K+ channel proteins in lipid bilayers has not yet been reported (the relevant channels have not been purified) and some believe that G protein [3y-subunits may also play an important role 23. Cellular concentrations of the G~ and G proteins are much higher than are those of Gs and Gq. In brain, Go~ is 1-2% of membrane protein. It seems unlikely that the protein is used only to regulate K~ and Ca2+channels. If so, why is Go so concentrated in neural growth cones24? Recent findings suggest that members of the G~ family may regulate pathways deep within the cell. In a kidney cell line, G~3 has been localized to the Golgi complex. Overexpression of Gi~3 in these cells slows secretion of packaged proteins and associated vesicular transport, and this effect is reversed by treatment with pertussis toxin 25. Further evidence comes from studies with the fungal toxin brefeldin A, which stimulates release of the Golgi membrane coat protein [3-COP to cause a pathological fusion of Golgi membranes with those of the endoplasmic reticulum. Treatment of perforated cells with GTPyS or AIF4 antagonizes the actions of brefeldin A26,27.Furthermore, addition of G protein [3y-subunits to a cell-free system prevents GTPTS-mediated binding of B-COP to Golgi membranes 28. Taken together, these findings indicate that a heterotrimeric G protein of the G~ subclass serves an important
385
Second messenger generation and destruction Basal state
jr
Receptor activation
H
(a)
(b) GTP GDP
1 (e)
(e)
GTPase
eo~.N
/
Subunit dissociation
(d)
S
P
Effector activation
Figure 1 G protein-mediated transmembrane signaling. In the basal state (a), G proteins exist as heterotrimers with GDP bound tightly to the c(-subunit; the hormone receptor (R) is unoccupied and the effector (E) is inactive. Upon hormone binding and receptor activation (b), the receptor interacts with the heterotrimer to promote a conformational change and dissociation of GDP from the guanine nucleotide-binding site; at normal cellular concentrations of guanine nucleotides, GTP fills the site immediately. (Under experimental conditions where GTP is absent, the hormone has high affinity for the receptor and the H-R-G protein complex is stable.) Binding of GTP to c~ induces a conformational change with two consequences (c). The G protein dissociates from the H-R complex, reducing the affinity of hormone for receptor and, in turn, freeing the receptor for another liaison with a neighboring quiescent G protein. GTP binding also reduces the affinity of (z for 13)', and subunit dissociation occurs. This frees (z-GTP to fulfill its primary role as a regulator of effectors (d). At least in some systems, the free J3ysubunit complex may also interact directly with an effector (El) and modulate the activity of the active complex, or it may act independently at a distinct effector (E2). The c(-subunits possess an intrinsic GTPase activity (e). The rate of this GTPase determines the lifetime of the active species and the associated physiological response. The c(-catalysed hydrolysis of GTP leaves GDP in the binding site and causes dissociation and deactivation of the active complex. The GTPase activity of (z is, in essence, an internal clock that controls an on/off switch. The GDP bound form of (z has high affinity for [37; subsequent reassociation of c~GDPwith I~Y returns the system to the basal state (a).
regulatory role in membrane trafficking. Information regarding a receptor-like analog in this pathway is lacking, although mastoparans (peptides that mimic the actions of G protein-coupled receptors) stimulate the binding of [~COP to Golgi membranes and block the effects of brefeldin A in a pertussis toxin-sensitive manner 27. These findings provide the most compelling evidence to date that the actions of heterotrimeric G proteins are not limited to transmembrane signaling at the cell surface. Yet, one is left uncomfortable with the assignment of the same G protein to play seemingly unrelated regulatory roles in presumably different cellular pathways. It seems likely that an unsuspected common theme will emerge.
386
Traditionally, the 0(-subunit has been viewed as the 'business end' of a G protein because it binds and hydrolyses GTP and interacts with effectors. In contrast, the ]3~'-subunit complex has been viewed as a regulatory component for 0( which stabilizes the GDP-bound form of 0(, 'presenting' 0( to receptors, serving as a membrane anchor for the oligomer. Yet the activation of G proteins yields two subunits (0(/137) that could act on downstream targets. A growing body of evidence now supports the idea that free ~Y can itself interact functionally with effector proteins. Genetic studies first demonstrated that responses to mating factors in budding yeast are dictated by [~yrather than 0(29. In mammalian systems, direct biochemi-
TIBS 17 - OCTOBER 1992 cal evidence for interactions of [37 with effectors has come from study of different isoforms of adenylate cyclase. Although the adenylate cyclases expressed in most peripheral tissues appear to be largely immune to regulation by 137, at least some of the enzymes found largely in brain are regulated by [~7 in a type-specific fashion2°. Type I (Gs~- and calmodulin-activated) adenylate cyclase is inhibited by 97, apparently directly. Type I! and type IV adenylate cyclases are activated conditionally by ~7; that is, ~7 is stimulatory only when Gs~ is also present. This appears to represent a mechanism for crosstalk between signaling pathways. Concentration requirements suggest that 137would arise by dissociation of G~ or Go, while Gs~ would obviously be liberated by activation of G~-linked receptors. Other effectors also appear to be subject to regulation by ~7. An undefined form of PLC in HL-60 granulocytes is markedly activated by 137~°. In addition, ~7 appears to have effects on K÷ channel activity, although it is unknown if these effects are mediated directly or indirectly23. Recent findings also indicate that 137plays an obligatory role in agonist-induced receptor phosphorylation and desensitization3L Taken together, these observations suggest that dissociation of G protein subunits in the membrane can generate parallel and/or interactive signals via both 0(and [37-subunits. Investigators should have an open and cautious attitude about which G protein subunit (and whether one or both subunits) is mediating the effect in question. It is clear that many more G proteinregulated pathways exist, and several poorly understood cellular responses appear to be dependent on guanine nucleotides, activated by A1F;, and/or sensitive to pertussis toxin. Likely effectors include phospholipases A 2 and D and a plethora of ion channels, transporters and exchangers. Cellular responses under consideration include growth control, effects of tyrosinekinase growth factor receptors, exocytotic secretory events and protein translocation. The receptors, G proteins and effectors involved in many of these pathways remain unknown. Conversely, there is little knowledge of the actions of some 'orphan' G proteins, such as 612 , 613, 615 and G~. Furthermore, we have discussed examples where a single G protein 0(-subunit (e.g. G~) can regulate more than one eflector; thus, the assignment of a given G
Second messengergeneration and destruction
TIBS 17 - OCTOBER 1992
40 ¢,t
I
60 '
I
'
I
80 '
I
'
I
100 '
I
'
I
I-- s 1 Gs 0~ol f
~
Oql ~i3
__
- -
f
0~i2
I OoA Gi - -
O~oB
F~
O~tl (Zt2
O~g ~z
-// ~ 1
O~q 0('11 ~14
4
0(,16 0~12 0~13
I
,
40
I
,
I
60
,
I
I
I
I
Gq
I
I
80
G12
I
100
% Amino Acid Identity Figure 2
Sequence relationships between mammalian G. subunits and family groupings(modified,with permission,from Refs8 and 4).
[37-subunits pre- References 1 Hall, A. (1990) Science 249, 635-639 sumably interact 2 Bourne, H. R., Sanders, D. A. and McCormick, F. to dictate the spe(1990) Nature 348, 125-132 cificities of these 3 Bourne, H. R., Sanders, D. A. and McCormick, F. (1991) Nature 349, 117-127 choices and to 4 Kaziro, Y. et al. (1991) Annu. Rev. Biochem. 60, control, synergis349-400 tically or in op5 Linder, M. E. eta/. (1991) J. Biol. Chem. 266, position, the acti4654-4659 6 Gilman, A. G. (1987) Annu. Rev. Biochem. 56, vities of effectors. 615-649 Thus, activation 7 Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119 of a given recep8 Simon, M. I., Strathmann, M. P. and Gautam, N. tor in different (1991) Science 252,802-808 9 Mattera, R. eta/. (1989) Science 243, 804-807 cells can produce a highly varied 10 Schubert, B., VanDongen, A. M. J., Kirsch, G. E. and Brown, A. M. (1989) Science 245, constellation of 516-519 activated G pro- 11 McLaughlin,S. K., McKinnon, P. J. and Margolskee, R. F. (1992) Nature 357, 563-569 teins and effecI-H. and Sternweis, P. C. (1990) J. Biol. tors, and cellular 12 Pang, Chem. 265, 18707-18712 responses will 13 Taylor, S. J., Smith, J. A. and Exton, J. H. (1990) J. Biol. Chem. 265, 17150-17156 change with development or with 14 Waldo, G. L., Boyer, J. L., Morris, A. J. and Harden, T. K. (1991) J. Biol. Chem. 266, cellular history of 14217-14225 exposure to regu- 15 Smrcka, A. V., Hepler, J. R., Brown, K. O. and Sternweis, P. C. (1991) Science 251, 804-807 latory signals. Such vast 'com- 16 Taylor, S. J., Chae, H. Z., Rhee, S. G. and Exton, J. H. (1991) Nature 350, 516-518 binatorial power' 17 Katada, T. et al. (1984) J. Biol. Chem. 259, endows cells and 3586-3595 organisms with 18 Linder, M. E., Ewald, D. A., Miller, R. J. and Gilman, A. G. (1990) J. Biol. Chem. 264, extraordinary ca8243-8251 pacity for fine 19 Gilman, A. G. (1984) Cell 36, 577-579 tuning both the 20 Tang, W-J. and Gilman, A. G. (1991) Science 254, 1500-1503 magnitude and the nature of 21 Federman, A. D. eta/. (1992) Nature 356, 159-161 their responses 22 Yatani, A. et al. (1988) Nature 336, 680-682 to the environ- 23 Logothetis, D. E. et al. (1987) Nature 325, 321-326 ment. 24 Strittmatter, S. M. et al. (1990) Nature 344,
protein to a specific effector does not end 'the game'. Conclusions
As the number of identified G protein subunits continues to grow, the task of unraveling the complexity of the G protein-regulated cellular switchboard expands exponentially. The number of identified (z-, ~- and 7-subunits indicates a limit of nearly 1 000 possible oligomeric combinations; reasonable extrapolation suggests that this number could grow to roughly 5 000 (with further discovery). Multiplication by the number of G protein-linked receptors and effectors indicates that an individual cell must sift through a very large number of choices to complete its own customized, G protein-controlled regulatory network. Further complexity at each step in the signal transduction cascade compounds the problem. Thus, there is both divergence and convergence of protein-protein interactions at both the receptor-G protein level and the G protein-effector level, and (z- and
Acknowledgements
The authors would like to thank M. Linder, T. Kozasa, J. l~iguez-Lluhi and N. Ueda for hlepful comments. Work from the authors' laboratory was supported by NIH Grant GM34497, American Cancer Society Grant BE30N, The Perot Family Foundation, The Lucille P. Markey Charitable Trust and The Raymond and Ellen Willie Chair of Molecular Neuropharmacology. J. R. H. is the recipient of a National Research Service Award F32 GM13569.
836-841 25 Stow, J. L. et al. (1991) J. Cell Biol. 114,
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579-588 27 Ktistakis, N. T., Linder, M. E. and Roth, M. G. (1992) Nature 356, 344-346 28 Donaldson, J. G., Kahn, R. A., LippincottSchwartz, J. and Klausner, R. D. (1991) Science
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