concept/ in biochemi~ try
Edited by: WILLIAM M. SCOVEU Bawling Green State University Bowling Green. Ohio 43403
Receptor-G Protein-Effector: The Design of a Elliott M. Ross Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235 The eukaryotic plasma membrane is the cell's switchboard.. res~onsible for receiving all sorts of extracellular . messages: hormones, neurotransmitters, pheromones, odors, and light. These messages must be detected, decoded, amplified, integrated with each other and with cellular me~aholiim, and conveyed to the cytoplasm as intracellular chemical messapes. Despite the diversitv of messaees and the complexity of signal transduction, cell surface receptor proteins utilize only a few biochemical mechanisms for these processes ( I ) . The vast majority of receptors depend on GTP-bindine reeulatorv proteins, or G proteins, to amplify . . and intetheir signds. The cell uses G proteins as coupling factors between the receptors that bindhormones and the ihtracellular processes that the receptors regulate. G proteins respond to receptors by modulating the activities of more than a dozen enzymes, ion channels, and transport proteins. These effector proteins eenerate intracellular second messeneers that in turn modurate the activities of both specialized and housekeeping functions within the cell. P e r h a ~ s10 different G proteins are found in mammals (see ~ l o & a r ~Each ) . G protein controls its own group of effector proteins, but each also communicates with the others to form a transmembrane signaling
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switchboard that integrates incoming signals with each other and with the needs of the cell (see Fig. 1). Receptors that act via G proteins include classical mammalian receptors for catecholamines, prostaglandins, many peptide hormones, and muscarinic receptors for acetylcholine. The family also includes visualrhodopsin (which can be thought of as the receptor for trans-retinal), olfactory receptors in the nose, and receptorsfor pheromones insuch microorganisms as yeast and slime molds. A new G protein-regulated function or a receptor that acts via a G protein is reported every few months. Several receptors may act on a single G protein, and a single receptor may regulate one or more G proteins. Branchingof signalingpathways is thus the rule. Regardless of the diversity of the signals received and the use a single intracellular messaees eenerated. " . G proteins . . molecular mechanism to couple receptors with effectors (Fie. 2). When a G protein binds GTP, it becomes activated .&h that it can bind to an effector protein and regulate its activity. Activation of the G protein is terminated by hydrolysis of GTP to GDP, which remains tightly bound but which does not activate. Receptors mediate the extent of G protein activation by promoting both the dissociation of bound GDP
Glossary: The Famlly Members and Their Roles in Life G,
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Or@naily identif8eo as tne G protein mat medales anlvatlon of adenvlate cvcia~eG..ombabiy also anivales a Ca" channe. in muscle and may be involved in the inhibition of Mg2+and glucose uptake. There are two G, a subunits. which appear to be slightly different in the stabilities of their activated. GTP-liganded states and their sensitivities to receptors. They are the products of the same gene, but their mRNA's are differentially spliced. G, is ADP-ribasylated by cholera toxin. Mare commonly called transducin, me G protein in retina that M w pies rhodopsin to a cyclic GMP phosphodiesterase. There are two transducins that are the products of two different genes, one in rod cells (which are specialized for dim light vision) and another in the three cane cells (which detect red, green. or blue light).
G, A G,-like protein that is found in the olfactory epithelium of the nose. Olfactory cells express huge amounts of adenylate cyclase and Caw: olfactory adenylate cyclase is stimulated by odorants.
molecJar we ght range One G med atesactovat on of a K' channel' memoels 0t m 5 grow prooaoy also rsgu atea pnospho pase A2, a pho~phoiipaseC, and perhaps a ~IUCOSB transporter.
G,
G,
identified in brain as an abundant, pertussis toxin-sensitive G protein with an n subunit smaller than Gi (39 KDa). The "0" in %stands for "other", which describes our understanding of its function. It may regulate a Ca2+ channel. 1. G, is the name used for a "virtual G protein" whose function has been described but which has not yet been isolated. It mediates the hydrolysis of polyphosphoinositides(hence the "p" for "phosphatidylinositol" end "phospholipase") and is not ADP-ribosylated by pertussistoxin. There are recent repans both of the partial puriticationof a G protein wltha 40.000 dalton, pertussis toxin-insensitive cr Subunit and of the cloning of a cDNA that encodes a Grlike o subunit that lacks a pertussis toxin substrate site. The number of different G.' ~s, ,is- men to soeculation. 2. Tony Evans an0 .oh" horth~pp~rifiada GTP-oinomg proten of mltnown f.nction from p 81eiels and p acenta an0 namw l aner ts t S S U ~ Sof or g n ( p' I 15 s ze s mom 25.000da l o n t s also iomd in brain, which detracts from me mnemonic subscript ~
GI
A generic grouping of at least three different G proteins that were originally identifiedas being sensitive to penussis toxin and involved with the inhibition (hence Y') of adenylate cyclase. The cr subunits of G,, the products of different genes, are in the 40.000-41,000
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Fioure 1. Remotor-G ormein-eflector svstems are numerous and diverse. ,~ even in a single cer . medagram deplcts three receptors (R,. R2. an0 R Jthat eacn reg" ate separate G prolelnr (d rl'ng~ishsdby the r different0 sub~nIs. also numDere0 1.2. and 31 nahlpieal ce I , receptors lor several normones a neurotransminers may coexist and coordinately regulate e single G protein. The first path is shown leading to the control of an integral plasma membrane protein (E,), such as adenylate cyclase. The sewnd regulates a peripheral protein (Ed such as the retinal cyclic GMP phorphadiesterase. The third regulates an ion channel (Ed in the plasma membrane. Although these signailng paths are depictedasdiscrete, ail will be biochemically linked in the cell. A receotar ma" talk to more than one G omtein. and s G orotein mav simiiariv la* lo mullple effectors. me & subunits, whtcn appear to form a pool Common to ail me G protam ot the plasma membrane, Interact *llh me u s.o~nltsto rnm'ulate an0 nlsgrate the acllvatfonof all lne pathways. ~~~F
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and the association of another molecule of GTP, thus increasing the relative amount of time that the G protein is in the active state. Formally speaking, therefore, G proteins are GTPases, and the activity of the effector enzyme is proportional to the steady-state concentration of the G proteinGTP comolex. In this article I will focus on how the biochemical mechanism of signal transduction by G protein-mediated systems allows amplification, integration of multiple signals, and feedback control by the target cells. I will also introduce the structures of G proteins an2 their receptors and outline the numerous messages that they convey. For more detailed information, interested readers are referred to two excellent recent reviews on G proteins (2,3),an earlier review on the analysis of adenylate cyclase ( 4 ) , and a recent review on phosphoinositides (5). Hormone-Sensltlve Adenylate Cyclase: A Model System Hormone-sensitive adenylate cyclase is one of the two experimental prototypes for studying receptor-C proteineffertor roupling. Visual transductiun is the other. Adenylate cyclase ratalyzes the synthesis of adenosine-3'5'-cyclic monophosphate (cyclic AMP), an intracellular regulatory molecule. The activity of the cyclase is resulatrd both positively and negatively by a large numher of hormones, neurotransmitters, and related drugs-a single cell may respond to se\,eral such aeents. Over I 5 vears aao. hlartin Rodbell and his co-workers showed thathormones activate adenylate cvclase onlv if GTP or a similar nucleotide is resent. GTP ;n the adsence of hormone has little effect: Poorly hvdrolvzed - analogues of GTP, such as GTPrS1, activate the cyclase essentially irreversibly even in the absence of hormone, although hormone accelerates such activation. These findings led &any investigators to propose that GTP, not hormone, is the proximal activator of the cyclase and that GTP hvdrolvsis is involved with deactivation. In 1976. Dan asse eland i v i Selinger demonstrated the existence of a minute hormone-stimulated GTPase activity that seemed to ?
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GTPyS isa synthetic analogue of GTP in which one of the oxygen arouo is reolaced bv sulfur. atoms of the terminal (7) ohosohorvl , GTPvS is resistant to h'vdrd~vsis and therefore , ~ ~ actjvates ~~-, G broteins ~-~~ essent-ally irreversibly. Anotner widely used nonhyoro yzable GTP analogue s Gpp(NH)p, in whicn the ,j-7 phosphoanhyoride bond is replaced by an lmldodiphospnate (P-IUH-P). GDP.?S, an analogue of GDP, is frequently used as a G protein inhibitor,
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Figure 2. G proteins are GTPases. but the role of these enzymes is not to degraderubstrateor to yield product. They arespecialized to regulate the level of the enzyme-substrate compiex. G'-GTP. This activated form of the G protein can bind to an effector protein (E), thereby causing its activation (E'). Deactivation of the system occurs when bound GTP is hydrolyzed (reaction 2) to GDP, which remains tightly bound to the G protein but which does not activate it. Thus, nonhydroiyrabie analogues of GTP permanently activate G proteins and the effectorsthey regulate. The receptor-hormone compiex (R . H) promotes both the release of GDP (reaction 3) and the binding of GTP (reaction I), thereby increasing lhe steady-state level of the G'-GTP compiex. Receptors can catalyze nucieotide exchange on several G proteins within the lifetime of the W-GTP compiex, yielding considerable amplification of their signal.
be related to cyclase activation. They correctly proposed that the bindine of GTP causes the activation of the cvclase: that hydrolysisif GTP to GDP causes deactivation; a i d that hormone, acting through its receptor, promotes either the removal of bound GDP or the binding of GTP. Bv this time. i t was clear that hormone receptors and adenylate cyclase were separate proteins. In 1977, both Ross and Gilman and Pfeutfer and Helmreich demonstrated that the regulatory GTP-hindingcomponent of thesystem isnlio a distinct protein. It was identified ai. a factor in detergent extracts of plasma memhranei that could reconstitute mtcrholamine-stimulated adenslate ryclase actkity in plasma membranes of a mutant mouse lymphoma cell that lacked assayable cyclase activity. Further work indicated that the mutant is deficient in this factor and that the factor acts by stimulating an otherwise inactive adenylate cyclase. The factor was shown to be a GTP-binding protein that is required for receptor-mediated activation of adenylate cyclase by hormones. This factor, denoted G, for the stimulatory GTP-binding protein, was characterized with respect to its nucleotide binding properties and its ability to reconstitute cyclase regulation in several systems depleted in G,. Quantitative reconstitutive assays allowed the purification of G,. Our understanding of the mechanism of regulation of G proteins is based lareelv .. . on studies of ourified G.. transducin (a retinal G prutein that was isolated at about the samr time,, and other G oroteins that were subseuuentlv identtfied. Certain features that were first associated with the hormone-sensitive adenylate cyclase system are now recognized as distinctive and diagnostic of all G protein-mediated transmembrane signaling systems. (1) GTP is required for hormonal activation of the effector protein (e.g., adenylate cyclase), although this requirement is frequently difficult to demonstrate unless the membrane ore~arationis uuite pure. (2) Non-hydrolyzable GTP analogues are themselves siimu~ ~ latorv. and the onset of their effect is accelerated bv hormone.'(3) AP+ and F- stimulate activity in the absence of added guanine nucleotide or hormone by forming GDP Al Fa on the G protein. (4) When hormone binding to its recep-
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tor can be measured, its affinity is frequently decreased by the presence of guanine nucleotides; the binding of hormone antagonists is not altered. (5) Toxins from Bordetella pertussis and Vibrio cholerae perturb regulation characteristically (see below). The role of a G protein in signaling can frequently be inferred from such behavior, even in whole cellsor poorly defined preparations and even when the effector protein is unknown. Regulation of G Protein Actlvation by Receptors The common regulatory properties of G protein-mediated signaling systems suggests a similarity of structure among their protein components. This suggestion is true for G proteins themselves and for the receptors that talk to them; it may also be true for G protein-regulated effectors. Extensive sequence homology among G proteins and among G proteincoupled receptors has also helped define their functionally important structural domains and has suggested aspects of their three-dimensional structures (2,3). G proteins are each composed of three subunits, denoted a, 0, and y. The a subunit binds GTP and, a t least in the case of G. and transducin. the GTP-activated a subunit can also actiiate its effector protein in the absence of the py subunits. The a subunit thus defines a specific G orotein in terms of its selectivity for an effector;and a subunits are named with subscriptsas are trimericG proteins (a, for the a subunit of G,, etc:; see Glossary). TG a subunit is also selective for specific receptors. I t is generally assumed that the a subunit interacts directlv with recentor. althoueh the py subunits are clearly involved mechanis~ically.The subunits are relativelv soluble. and orohable bind to the inner face of the plasma membrane. The activation of'a G nrotein bv the hindinvof GTP m the a subunit is regulated by the pyauhunits, divalent cations, and the hormone-liganded receptor. These regulatory interactions provide mechanisms for amplification of the receptor's signal, the integration of inhibitory and stimulatory inputs,-and crosstalk among separate signaling systems. GTP binding is usually a slow, first-order process that may take hours at 30 "C.Such slow binding reflects the fact that a molecule of GDP is tightly bound to a G protein a subunit, either in membranes or after many weeks of storage of the purified protein. GDP can be removed under appropriate conditions, however, and the unliganded G protein binds GTP with second-order, diffusion-controlled kinetics. The slow release of GDP is thus generallv rate-limiting in the activation of G proteins, and the acceleration of GDP dissociation is a principal mechanism of receptor action (Fig 2). The hormone-figanded receptor decreases the affinity of G proteins for guanine nucleotides, conceptually converting
a
Figure3. inhibition mediated by the By subunits.When aGprotein isactivated by the binding of GTP, the affinity of the activated (GTP-iiganded)a subunit (a') for the by subunits Is decreased such that dissociation of the a a y trimer is favored.Reciprocally, excess by subunit that has been relea& from other trimers retards the activation of any a subunit. The By subunits also stabilize the binding of GDP, further blacking activation. This panern can lead to the inhibition of one G protein by another. in the figure, when at 1s activated (to a,.). by subunits are released and can inhibit the activation of a* Such inhibition is reciprocal. its extent is dependent on the relative amounts of trimeric G1 and G2 and on the relative affinitiesof a , and a 2 for b y . The activation and concomittant dissociation of both G proteins are shown as promoted by two differentreceptors.
the nucleotide bindine site from a "closed" confiauration to an "open" state thatWfreelyexchanges nuc~eotideligands. Receptors have little if any effect on the actual rate of hydrolysis of bound GTP (kcat). Because the cellular GTPIGDP ratio is large, receptor-promoted exchange of bound nucleotide causes net activation of the G protein. The receptor acts catalytically in this process: it accelerates a reaction that is slow hut otherwise favored. Because receotor and G nrotein are both free to move laterally in the plane of the membrane, a sinele receotor can catalvze the activation of multinle G protein molecules.'l'he receptor-catalyeed activation wucleotide exrhanee,. nmcess is fast com~aredto GTI' hvdrolvsis. . . . and a single hormone-liganded receptor can maintain the activation of manv molecules of G protein. This leads to considerable ampification of the hormone's signal. In the case of the 8-adrenergic receptor, for example, such amplification can be >10 molecules of G, per receptor; a single bleached rhodopsin molecule can activate 1000 molecules of transducin (6).
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The By Subunits and Receptor-Mediated lnhibltlon In contrast to a subunits, the py G protein subunits are not specific for each species of G protein. Instead, each G protein appears to share a common pool of By subunits. There are two p subunits, the products of different but nearly identical genes. They both have a molecular weight of -35,000 and have nearly indistinguishable functional properties. The two 0 subunits are variably expressed in different cells. T h e y subunit is small (-8000 dalton) and hydrophilic, and several different forms may exist. The B and y subunits have not been senarated in active form: therefore. one cannot ascribe a distinct function to one orthe other. kxcept in the case of the retinal G nrotein transducin.. the b'. ? subunits are soluble only in the presence of detergent and probably help attach the a subunit to the plasma membrane. The 31 subunits regulate the binding ot'guanine nuclentides to the a subunits, and this reyulatlon provides a mechanism for the hormone-mediated inhibition of G protein signaling pathways. The 31 subunits stabilize the binding of GIlPand inhibit the hindineof(;TP hv ~ subunits.Thus. subunits slow the activation of G proteins by inhibiting thk dissociation of GDP and. orobablv less imnortantlv. bv inmere hibiting GTP binding. This description of py inhibitor of activation is oversimplified, but it is probably correct in most physiological situations (2). The commonalitv of the B r subunits and their inhibitow activity form the mechanism of inhibitory transmembrane signaling via G proteins. About 1980, it was found that py subunits tend to dissociate from a when GTP is bound (Fig. 3). This dissociation reflects the reciprocal nature of negatively cooperative hinding: if By inhibits the binding of GTP, then GTP will inhibit By binding as well. Therefore, when a G protein is activated in the plasma membrane, the local concentration of py is increased, which inhibits further activation. Because the B r subunits are common amona G oroteins, the activation bione G protein will also tend to inhibit the activation of other G proteins. For example, when a G; (see Glossary) is activated in response to hoimone, the re: lease of its py subunits causes the inhibition of adenylate cyclase. According to this mechanism, the activation'of any G orotein will cause the inhibition of activation of all other G proteins in the cell. This effect is not generally observed, for several reasons. First, the concentrations of different G proteins can vary greatly, and only the activation of a relatively abundant G protein (an abundant source of By) can significantly inhibit another for stoichiometric reasons. Second, the affinities of a subunits for Br subunits are also variable. One can estimate that a, bindspy perhaps 100-fold more tightly than does ao, making it far more sensitive to Bymediated effects. Thus, both the affinity of a, for py and its
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relatively low abundance contribute to the sensitivity of adenvlate cvclase to hormonal inhibition. It has recently been proposed that the By G protein subunits can directlv stimulate a voltaae-sensitive K+ channel in the plasma membranes of heart'cells. This observation remains controversial, but its experimental suoport is reasonably good. Other p-mediated functions hake also been proposed. The direct regulation of an effector by By is provocative because it demands that By subunits select among multiple effector proteins just as the more numerous cc subunits do. A way out of this puzzle is to suggest that By actually exerts a negative effect on an inhibitory a subunit that is tightly bound to the channel. Slgnallng Systems that UHllze G Protelns A few common features and unifying concepts come to mind when one considers effector proteins that are regulated by G proteins, but their diversity is far more obvious. The list now contains enzymes that synthesize or degrade intracellular second messeneers. ion channels. and transnort nroteins. Sorting these parhways from receptor through G protein to effector is a maior challenge in the biochemistrv of cellular regulation. Adenvlate Cvclase. This svstem is nearlv ubiouitous. and regulates both differentiated-(cell-specificjand';housekeeping" functions. The cyclase is stimulated by G,; its inhibition is generically ascribed to the Gi's, of which there are three known species, but inhibition is predominantly mediated by the By subunits. Cyclic GMP Phosphodiesterase. This is a specialized encvclic zvme of retinal ohotoreceotor cells. It hvdrolvzes " , GMP, another second messenger, to the inactive product 3GMP. Cvclic GMP activates a Ka' channel in the retina. where th:ls system has been studied in great detail. In othe; cells, cyclic GMP acts primarilv bv stimulating .. a orotein. . kinase.-stimulation of the retinal ~hosphodiesteraseis mediated t ) transducins ~ ( G , ) ,one transducin in the color-sensitive cone cells and another in the rods. In other organs, G, or other G proteins stimulate a phosphodiesterase activitv that has not been well cbaracter&ed. Phospholipase C. This enzyme, which has not yet been purified, cleaves polyphosphoinositides (PI'S), a minor group of phospholipids in the plasma membrane. The reaction products are both second messeneers. .. . diacvlelvcerol ,u, and ik,nitol phosphates. r)iacylglycerol promotes the acrivalion of a Ca-'-stimulated orotein kinase. orotein kinase C. which regulates numerous~cellularfunctions. Inositol phos: phates cause the release of Ca2+ from intracellular organelles. Ca2+in turn regulates numerous enzymes, contractile proteins, and ion channels. The receptor-stimulated phospholipase C is probably regulated by one or more of the Gi's and by a pertussis toxin-insensitive G protein that is probably similar to the Gi's. I t has been referred to as G, (see Glossary). Phospholipase As. This enzyme also cleaves phospholipids, but its reaction yields arachidonic acid, the precursor of prostaglandins, thromboxanes, leukotrienes, and many other regulatory molecules. The spectrum of bioactive metabolites of arachidonic acid is diverse and highly tissue-specific. This phospholipase, which also has not been purified, is regulated by one or more Gi's. Voltage-Sensitiue K+ Channel. This plasma membrane channel causes hyperpolarization of the plasma membrane in muscle cells via the efflux of K+, thus inhibiting coutraction. The channel is stimulated by a Gi, although the mechanism remains controversial. Ca2+ Channel. Several Ca2+-specific channels allow the influx of extracellular Ca2+or the release of Ca2+ from the endoplasmic reticulum. Elevated cytoplasmic concentrations of Ca2+regulate diverse cellular responses, as discussed above. A Ca2+channel in the heart is stimulated by G,, and
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other Ca2+ channels are also probably regulated by G proteins. Glucose and Mg2+Transport. Indirect but fairly convincing data suggest that a glucose carrier in the plasma membrane and a Mg2+uptake system are stimulated by a Gi and inhibited by G,. Neither observation has been verified by reconstitution of regulation using pure oroteins. As one can see from this list;most G protein-regulated effectors are membrane proteins, but the retinal cvclic GMP phosphodiesterase is only weakly bound to the disc membrane and the phosphatidylinositol-specific phospholipase C may only be transiently membrane-bound. Few of these effector proteins have been purified, and reconstitution of a complete signal transduction pathway using purified proteins has only been achieved in the case of catecholaminestimulated adenylate cyclase and rhodopsin-stimulated cyclic GMP phosphodiesterase. I t is interesting to note that adeuylate cyclase, cyclic GMP phosphodiesterase and phospholipase C all break O-P bonds, perhaps suggesting an evolutionary relationship among effectors. A group of closely related protein encoded by the ras oncogenes are homologous to G protein a subunits and are evidently regulated by similar mechanisms. These proteins, called p21's according to their molecular size (21,000 dalton), cause carcinogenic transformation of cells. In numerous tumors, p21 is either overexpressed or its amino acid sequence has been mutated to retard its ability to hydrolyze bound GTP (and thus deactivate). I t is widely assumed that p21's regulate an effector protein or proteins that control cell proliferation, although such a protein has not been identified. I t is also unclear if normal cellular p21's regulate this putative effect in response to a receptor or whether they are otherwise regulated. Novel effector proteins mav exist in veast and other sim--ple eukaryotes, where G proteins are i&olved with the control of the mating response, cell proliferation. and adantation to changes in nutrients. he sequence of the yeait G protein that stimulates adenylate cyclase is more similar to that of the mammalian p21mr than it is to mammalian a., although it is significantly larger. Regardless, mammalian p21's can function as an n , in yeast. An a,-likeprotein is also involved in the stimulation of yeast cyclase, and another 0 , like protein modulates the cell's response to mating factors, pheromones that promote fusion between cells of opposite sex. Its effector protein is unknown. This . ohdoeeneric di. ~~versity suggests that we may have much more to learn about G proteiu-mediated functions in animals as well. Other G protein-mediated functions probably exist, awaiting only the right probes for their discovery. GTPbinding regulatory proteins have recently been shown to mediate secretion, transport of newly synthesized proteins through the Golgi apparatus, and Ca2+uptake by the endoplasmic reticulum. I t is not clear whether these and similar findings suggest the existence of new G proteins, new functions for old ones, or new classes of GTP-binding proteins that mediate intracellular events as receptor-coupled G proteins mediate signaling.
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Proteln Sequence Homologles: Selectlvlty and Crosstalk The structural similarities among G proteins and among their receptors made it no surprise that a subunits and the receptors that talk to them are also homologous at the level of their primary amino acid sequences. The genes or cDNA's for about a dozen a subunits have now been cloned and sequenced. They display considerable similarity of sequence, including large regions of near identity. The maintenance of such identity through evolution suggests the importance of the conserved regions. Three stretches of highly conserved sequence make up the guanine nucleotide binding site, which was identified by analyzing the three-dimension-
such that its amino terminus lies on the luminal side of the membrane and its carhoxyl terminus lies on the cytoplasmic side. The membrane-spanning segments, which are quite hydrophobic, are connected by relatively more hydrophilic "loops" (see Fig. 4). Several rhodopsin cDNA's have been sequenced, including the three human color-selective rhodonsinr. -~. When our group and those of Lefltowitz and Dixon cloned the cDNA's for two 8-adrenergic catecholamine receptors, it became apparent that this receptor shares both strone sequence homology and overall shilarity of structure with rhodopsin. Moreover, sequence similarity to rhodopsin is not uniform throughout the receptor; i t is preferentially conserved in the membrane-spanning domains. The suhsequent cloning of the genes for several muscarinic cholinergic receptors, an an-adrenergic catecholamine receptor, and receptors for serotonin (a biogenic amine) and substance K (a regulatory peptide) has extended this family and enhanced our ability to generalize about common or distinctive structural elements. Such speculations must be tested, however. For example, three of the larpe, nonconserved, hvdrophilic . . regions of the 8-adrenergic receptor can he removed, either proteolytically or by mutation, without impairing the receptor's ligand binding activity or G, regulatory activity. These data stress the importance of the conserved hydrophobic core of the receptor in regulation. Similarly, a 0-adrenergic photoaffinity ligand labels a tryptophan residue in the seventh membrane svan of the B-adrenereic recentor a t anmoximately the sameiocation aB the lysine residie in rhozpsin to which retinal binds. Such similarities helo locate the sites at which ligands drive receptors from inacti've to active conformations. Other exriting hypotheses to be tested soon con-
al structures of a hacterial GTP-binding protein, elongation factor Tu, and a p21T". The overall homology among a subunib suggests that there are also conserved regions of primary sequence that are responsible for interactions with receptors and effectors. By such an argument, the receptor-binding domain of a specific a subunit will have a structure that is grossly similar to the homologous domain on other a subunits but is uniquely specialized to recognize only its own family of receptors. Similarly, its effector-binding domain should differ from that of other a subunits in its selectivity for effector protein. This simplistic prediction seems likely to be correct. For example, proteolytic and immunologic studies of a subunits, their modification by pertussis toxin (which uncouples G proteins from receptors), and the location of an uncoupling mutation in the a subunit of G, all point to the carhoxyl terminus as the probable site for receptor binding. Speculations about an effector binding domain are also being tested. Many laboratories are testing the effects of specifically engineered mutations in a subunits or the functions of chimeric a subunits-the products of genetically fused components of two a subunit cDNA's. These constructs should test and begin to clarify which regions of the a subunit interact with receptors and effectors. A similar pattern of homologous yet specialized structures characterizes the receptors that act on G proteins. In this case, detailed biochemical and biophysical studies of rhodopsin and molecular genetic analysis of other receptors have combined dramatically to improve our understanding of the three-dimensional structures of the receptors. Rhodopsin, for example, is an integral membrane protein whose polypeptide chain spans the membrane bilayer seven times,
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EXTRACELLULAR SPACE
Flgde 4 Gproteineoupiedrecsptorsare arrayed " W e plasma membranasuchtnatM y spanths phospnoliptd 0 layer seven times Their carboxy termmi ie onthe cytoplasm c s ds of m e msmorans and mew ammo lermonl lie on the enracel u ar face tTn8s 0s the umlnai stde ot the ols* memorane in photoreceptor ce 5 in tne case of rhodopsin.) me amino terminal region isgiycosyiatedone or more times (CHO). The hydrophobic membrane spansare presumably bundledsuch that the few charged residues are oriented inwardand are paired with appropriate caunterions, thus directinga hydrophobic surfacetoward !he hydrocarbon phase of the biiayer. Sequence similarity among the G protein-coupled receptors is most pronounced in the membranw-spanning hydrophobic domains. The hydmphiiic domains (in the dashed boxes) display the least sequence similarity with other receptors and can be removedwithout loss of function. The figure shows the sequence of !he k d r e nergic receptor from the turkey erythrocyte (modified from Yarden st ai. Proc. Natf. Acad. Sci. USA 83.6795-6799). Single iener code for amino acid residues is used, with the cetionic amino acids (iysine, arginine, and histidine: K, R, and H) in small squares andthe anionic amino acids (giutamicacid and aspartic acid. E and D) in circles. One of two sites at which the protein can be labeled with a padrenergic photoalfinity ligand, tryptophanssa(W in the dark circle), lies in the sevenlh m e w brane span at a site homologous to the retinal attachment site on apsin. Volume
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cern the site of G protein binding and the mechanism of selectivitv amona G rotei ins. ~ l t h o u g hthe &&ity of receptor-(; protein and G protein-effeccor interactions is strikine, the homologv of the interactive domains suggests that shectivity shouid not be absolute. It is not. AsingleG protein may regulate more than one effector. G,, for example, was identified as the G protein that activatesadenylatecyclase, but it alsostimulatesa Ca2channel and probably inhibits transporters for Mg2+ and glucose. It is also likelv that a certain amount of intersvstem "crosstalk" goes on & the plasma membrane-a receptor that is relativelv selective for one G protein mav also interact with others d i t h lower efficiency. For exampie, purified muscarinic cholinereic receptors can activate both Gi and G, in reconstitutedkembri&es, and one or both of these G proteins mediates muscarinic inhibition of adenylate cyclase. The muscarinic receptor also activates a phospholipa& C in several cells, acting via a third G protein that has so far been identified onlv accordine to its insensitivitv to oertussis toxin (which atticks Gi an; G,; see below a i d ~iossary).Yet, when a recombinant muscarinic receptor was overexpressed in a cultured cell line by Avi Ashkenazi and co-workers, i t activated phospholipase C via a pertussis toxin-sensitive pathway. Other examples of crosstalk exist. The B-adrenereicreceotor can reaulate a t least one of the G,'s nearlv as well pro;iding a means to restrain oversti&ulation as G,, of adenvlate cvclase or to allow 8-adrenereic stimulation of another effect-or. John Exton and co-work& observed that the effector protein regulated by the nl-adrenergic receptor in rat liver chanees duiine develooment..arminkthat r&eotor-G protein-effector coupling is a regulated process. The limited selectivitv of receotors for C proteins and of G proteins for effectors forces us t o consider the plasma membrane as an inteerated information-transducine oreanellei a switchboard OF the sort sketched in Figure 5 r ~ hswitchboard allows a single hormonal input to variably stimulate several intracellular effectors in characteristic patterns that allow appropriate control of the metabolism in specialized e By exchange, stimulation of onesignaling cells. ~ i k i r i u of pathway is felt by all the others.
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Toxins and G Proteins: Biological Warfare on Transmembrane Slgnallng G protein-mediated signaling systems are conspicuous targets of a wide ranee of bioloeical toxins. Fortunately, these toxins have proved to be useful tools in the study of G proteins. Characteristic toxin sensitivity can often suggest the involvement of a G protein in a newly described receptor-effector system, and toxins have proven to be effective labeling reagents in the identification and assay of G proteins. Cholera toxin was the first toxin that was found toact on a G protein. Cholera toxin activates adenylate cyclase by causing it to respond to GTP as if i t were a nonhydrolyzable analogue such as GTPyS. Michael Gill showed that cholera toxin catalyzes the transfer of an ADP-ribosyl group from nicotinamide adenine dinucleotide (NAD) to a plasma membrane protein that is now known to be as.ADP-ribosylation of a,blocks its ability to hydrolyze bound GTP, leading to its persistent activation. Strangely, a, is a good substrate for cholera toxin only in the presence of "ADP-ribosylation factor", a protein from the target cell that is itself a GTPbindine protein of the ras oncoaene * - family. Its ~hvsioloeical .function is unknown. Pertussis toxin is also an ADP-ribosyl-transferase that attacks acysteine residue near the carboxy terminusof the a subunits of G:, Go, and transducin. G, lacks this cysteine.
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942
Journal of Chemical Education
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€1 €2 €3 E4 Es Flgure 5. A G proteirrmedlated swhchboard. Thisdiagram depicts hypothetical G protelnmedlated signaling pathways in a single cell. Receptors (R) are shown as stimulating G proteins (GI, but G proteins may either stimulate effectors (El (solid arrows) or inhibit them (stippledarrows). Several receptors m y Stimulate morethan oneG protein. Gproteins can a i m regulate more than one effector, either patentlatlvely (G, and G2stimulating E d or antagonistically (G, and G4 muhlally antagonistic on E, and E.). Multiple classes of receptors act On a single G protein. R, is shown as stimulating E,via both GI and 4.Such interlockinO - .Dathwavs of rmulation are Woical. The monitudes of the effects wii depend on tne &ncentk on of thehdvia.al prot&r, on tneir intrnsfc sniv ties m a all,"toss, and on the concentratms of the normonas that a"'"ate the 'eCeDtOrS.
ADP-ribosylation by pertussis toxin does not inhibit GTP hydrolysis nor does i t appreciably alter the G protein's response to GTP. Rather, pertussis toxin-treated G proteins become insensitive to regulation by receptors ("uncouled"), an effect consistent with the idea that the carboxvl ierminus of G,'s IS tnvolved wtth receptor binding. The experimental utilitv ot these toxlns has been ereat. If a signaling pathway is sensitive to either toxin, it suggests the involvement of a G protein. Furthermore, the use of 32Plabeled NAD allows one to label toxin substrates even in crude membrane fractions. This allows a sensitive but nonquantitative assessment of the G protein complement of a cell or tissue. Pertussis toxin has also provided the simplest assay for the py subunits. Because isolated a; is a poor pertussis toxin substrate in the absence of the py subunits, the concentration of By in a sample can be estimated by measuring its enhancement of the [32P]ADP-ribosylationof an excess amount of purified w. Several peptide toxins from wasp venom attack G proteins noncovalently by mimickins the action of receptors. These toxins, called m&toparans,~werefirst identified as causing release of histamine from mast cells. When this process was shown to be inhibited by pertussis toxin, Tsutomu Higashijima and co-workers tested the effects of mastoparans on isolated G proteins. Thev showed that masto~aranscatalvze guanine nucleotide exchange, leading to G protein activation, bv a mechanism that is strikinelv - similar to that of hormone-liganded receptor. I t is provocative that mastoparans have a structure similar to one of the intracellular loops of G protein-coupled receptors. Other toxins attack G protein systems by mimickine effector proteins. Bacillus anthracis ithe cause of anthrax) and Bordetella pertussis (the cause of whooping cough and the source of the pertussis toxin describedabove) both secrete adenylate cyclases. Thesle bacterial cyclases are specialized to penetrate mammalian cells. where thev are activated bv the Ca2+-bindingprotein calmodulin. 0 t i e r toxic ''effect& proteins" are the snake venom phospbolipases, which mimic the signaling phospbolipases. Just as revolutionaries attack a country's communication networks, the G protein switchboard sekms to he a favorite target for toxins.
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Literature Cited 1. Alberts, B. etal. Moleular Biology ol Tho Cell: 1983: Chapter 13. 2. 0ilrnan.A.G.Ann.Reu.Bioehrm. 198'7,56,615. 3. Stryer, L.;Bourne, H.R. Ann. Re". Cell B i d L986.2.391. I . Ra8s.E. M.:0ilrnsn.A. G. Ann.Rm.Biochom. 1980,49,533. 5. Berrldge,M.J. Ann. Reu.Biocham. 1987,56,159. 6. Stryer, L.Ann. Rau. Neumsci. 1986.9, 81.
