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A large body of experimental evidence exists thatlinks heterotrimeric guanosine triphosphate–bindingprotein (G protein) structure to function. The determi-nation of the crystal structures of G proteins in vari-ous activational states and, more recently, in com-plexes with effectors and other signaling partnershighlights the varied mechanisms involved in G pro-tein regulation. Signaling complexes, such as the re-cently solved complex of Gβγ and G protein receptorkinase 2 (GRK2), provide new insights into the mech-anisms underlying the regulation of these highlyconserved signaling molecules. In this Review, wediscuss the latest findings and their implications forG protein–signaling paradigms.

IntroductionA large body of experimental evidence exists that links het-erotrimeric guanosine triphosphate (GTP)–binding protein (G pro-tein) structure to function. This Review highlights the major newfindings of the past few years and discusses the implications of themost recent structural data for understanding G protein–signalingparadigms. Many of the early findings were reviewed previously ina comprehensive Review by Sprang et al. (1). Since then, a numberof new structures have affected our thinking about G protein signal-ing. Palczewski’s group solved the structure of the f irst Gprotein–coupled receptor (GPCR), dark-adapted rhodopsin cova-lently bound to its inverse agonist 11-cis retinal covalently bound(2). This structure provides a starting point for understanding GPCRstructure and function. Until the structure of an activated receptor issolved, other structural and functional studies continue to shape ourthinking about receptor activation. Many crystal structures of G pro-teins solved in recent years have provided new insights into themechanisms underlying the regulation of these highly conservedsignaling molecules.

G proteins bind to and are activated by liganded GPCRs. Adiverse array of ligands binds to and activates GPCRs, includ-ing photons, odorants, neurotransmitters, hormones, glycopro-teins, and chemokines, leading to diverse biological responses.G proteins are heterotrimers, consisting of α and βγ subunits,and each is involved in signaling to distinct effectors. The spe-cific combination of α, β, and γ subunits, in combination withother regulatory and scaffolding proteins, connects a particularreceptor to a specific effector. Although the determinants of thisspecificity are not completely known, it is likely that a combi-nation of factors (G protein–effector coupling efficiency, whicheffectors are expressed within the cell, and the relative concen-trations of other signaling molecules within the cell) stronglyinfluences G protein specificity in vivo.

There are upwards of 16 different mammalian Gα subunits(depending on alternative splicing) and 5 β and 14 γ subtypes,which are found in nature in many, but by no means all, possi-ble combinations. The variety of Gβγ dimers that can be formedadds to the large diversity among G proteins and may be anoth-er mechanism regulating receptor–G protein specificity.

G protein classes are defined according to the primary se-quences of their α subunits, resulting in four main families:Gαi/o, Gαs, Gαq/11, and Gα12/13. In many cases, these G proteinscan couple to more than one receptor subtype, with differingaffinities. GPCRs act as GEFs (guanine nuceotide exchangefactors) for their cognate Gα subunits. Gα subunits bound toactivated receptors undergo a conformational change, resultingin the release of guanosine diphosphate (GDP), the rate-limitingstep in G protein activation. The transient high-affinity emptystate is followed by binding of GTP to Gα and its dissociationfrom Gβγ and receptors. The GTP-bound Gα subunit and disso-ciated Gβγ dimer can then interact with multiple downstreameffectors, such as adenylyl cyclase, phosphodiesterases, phos-pholipase C, and ion channels. In the absence of other regulato-ry factors, the intrinsic guanosine triphosphatase (GTPase) ac-tivity of the Gα subunit returns it to the inactive state, allowingreassociation of Gα and Gβγ (Fig. 1).

The determination of the crystal structures of these proteinsin various activation states and, more recently, in complexeswith effector proteins and other regulatory proteins highlightsthe diverse mechanisms involved in G protein regulation. Theinitial crystal structures of Gα proteins revealed nucleotidesbound in a cleft between an α-helical domain and a Ras-likeGTPase domain (3–6). The core structural feature of the inter-action between Gα and GDP is based on interactions of con-served residues between the β1 strand and α1 helix in the GTPase domain and the α and β phosphates of GDP. In the rat(r) Gαi heterotrimeric structure, an additional contact betweenArg178 and Glu43 augments the binding of GDP (5), an interac-tion also seen between GDP and the homologous residues inGαt (6) (Fig. 2).

Comparison of Gα structures in the heterotrimeric and inac-tive states to those in the active state demonstrates the confor-mational changes in switch regions that occur as a result of acti-vation (3, 7, 8). The inactive state of Gα is the GDP-boundform and, in vivo, the active state is the GTP-bound form,which is similar to a form bound to both GDP and AlF4

−. AlF4−

forms a complex with GDP, which mimics the transition statetoward GTP hydrolysis in the Gα•GDP-AlF4

− structure (7, 8).GTP-bound subunits display Mg+2 coordinated to oxygens ofthe β and γ phosphates of the bound nucleotide. The activatedstate is also stabilized by the nonhydrolyzable GTP analogGTPγS. In the GTPγS-Mg bound state, interactions between ba-sic residues in the switch II region and acidic residues in theswitch III regions stabilize the active state, along with interac-tions between switch II and the α3 helix (Fig. 3) (9).

G Protein Signaling: Insights from New Structures

Anita M. Preininger and Heidi E. Hamm*(Published 3 February 2004)

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Department of Pharmacology, Vanderbilt University Medical Center,Nashville,TN 37232–6600, USA.

*Corresponding author. E-mail: heidi.hamm@vanderbilt.edu

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The interactions observed for GTPγS-stabilized Gα are similarto those in the Gα•GDP-AlF4

− bound state of Gα. The transitionstate stabilizing interactions seen in the Gα•GDP-AlF4

− structureare similar to those in the crystal structure of Gαι•GDP-AlF4

−

bound to RGS4 (regulator of G protein signaling 4) (10). This simi-larity revealed that RGS4 is a GTPase activating protein (GAP),which stabilizes this transition state toward hydrolysis. In the crystal, the core RGS domain is in complex with Gαil•GDP-AlF4

−

in a conformation that stabilizes switch II of Gα (11) in a favorableorientation resembling the transition state for GTP hydrolysis. Mutational analysis suggested that a conserved Asn [position 128 inhuman (h) RGS4] is a critical mediator of affinity between Gαi andRGS4 (12, 13). Mutation of this residue impaired binding of RGS4to Gαi (12). Supporting this result, a mutational study used Gαt, aGαi family member, to identify residues in Gα that are importantfor RGS binding to Gα subunits. Because the crystal structure ofrRGS4/Gαi (10) shows Asn128 of RGS4 in contact with Ser206 ofGαil, it is not surprising that mutation of the homologous Ser inhGαt, Ser202, prevented binding of Gαt to hRGS [retinal (13)].

What Are the Regions of G Proteins That Interact withReceptors?Numerous biochemical studies with rhodopsin and other GPCRshave demonstrated that activation results in a change in the relativeorientation of transmembrane domains (TMs) 3 and 6, which isthought to affect the conformation of the intracellular loops thatmay contact the G protein. Hubbell et al. measured light-activatedchanges in rhodopsin. They employed internitroxide distance mea-surements between labeled residues at cytoplasmic ends of TM1(where a nitroxide label was attached as a reference point) andTM2, and another labeled residue at the cytoplasmic end of TM8.Residues in TM2 were displaced relative to the position of TM8 up-on light activation (14) when compared to the position of the refer-ence residue. This group used the same approach to show that dis-tances between a labeled residue in TM1 and TM7 increase uponlight activation of rhodopsin, which is consistent with a 2 to 4 Åmovement of TM7 away from TM1 (15). Together these studiessuggest that conformational changes occur in several regions ofrhodopsin’s cytoplasmic face.

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βγ

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Fig. 1. Overview of the G protein cycle. Gα, blue; Gβ, brown; Gγ, gray; GDP, yellow, with smaller circles representing the two phos-phates; GTP, green, with smaller circles representing three phosphates; RGS (regulator of G protein signaling), magenta. See(http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/218/re3/DC1) for an interactive animation of G protein cycling in the pres-ence of various regulators of G protein activity.

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The structure of bovine (b) rhodopsin by Palczewski and co-workers reveals that in the dark, Glu134 and Arg135 in the intra-cellular side of TM6 are inaccessible (2). Mutation of theseresidues in (b) rhodopsin [Glu134→Gln134, Arg135→Gln135

(E134Q, R135Q)] strongly influenced receptor-mediated activa-tion of transducin. The E134Q rhodopsin mutant was more effi-cient at stimulating transducin than the wild-type receptor,whereas the R135Q rhodopsin mutant was less efficient at stim-ulating transducin than wild-type receptors (16). These resultssuggest that these residues maybecome accessible to trans-ducin upon light activation ofrhodopsin.

Ser338, Ser343, and Ser 344 ofrhodopsin are phosphorylatedupon prolonged agonist stimu-lation, which results in arrestinbinding and steric inhibition ofactivation of G proteins. In the(inactive) rhodopsin structure(Fig. 4A), these serines are notexposed to solvent, which sug-gests that critical changes oc-cur in this region upon receptoractivation. Other contact re-gions implicated in numerousbiochemical studies between areceptor and Gα include the re-ceptor C terminus and the sec-ond and third intracellularloops of many GPCRs.

In the Gα molecule, experi-ments with proteins containingmutations in residues in theα4/β6 loop (Fig. 4B), especial-ly residues at the C-terminalend of the α4 helix, have sug-gested a role for these residuesin receptor-mediated activationof Gα subunits. A number ofbiochemical studies have alsoimplicated the N terminus ofGα subunits in receptor inter-actions [reviewed in (17)]. So-lution-based electron paramag-netic resonance biophysicalstudies with a nonmyristoylatedGαil protein suggest that the Nterminus does not interact withinactive rhodopsin (darkrhodopsin), nor are changes de-tected in selected N-terminalresidues upon light activationof the G protein–receptor com-plex (18). There are structural data indicating the interaction be-tween the extreme C terminus of Gαt and rhodopsin. High-reso-lution nuclear magnetic resonance (NMR) revealed a helicalconformation for hGαt peptide (residues 340 to 350) upon bind-ing light-activated rhodopsin and a 40° angle between the axisof this helix with respect to the membrane face. The C terminuswas capped with an αL type C-cap encompassing a glycine at a

critical position along the reverse turn that makes up the cap(19). (An αL type C-cap is characterized by a reverse turn at theC terminus of the peptide, which allows interaction between C-terminal residues and the α-helical amino-terminal portion ofthe peptide.) Gαt residues that make close contact with activat-ed rhodopsin in this solution-based structure include Asn343,Asp346, Gly348, and Leu349 (Fig. 5B) (19). Consistent with thisresult, cross-linking studies demonstrate that residue 240 in thethird intracellular loop of rhodopsin cross-links to a region on

Gαt encompassing residues 342 to 345 upon light activation ofrhodopsin (20). Furthermore, Ridge and co-workers used a sol-uble receptor-mimetic peptide based on a four-helix bundle de-sign to examine interactions between the second and third intra-cellular loops, as well as the C terminus of rhodopsin and Gαtpeptides. Transfer nuclear Overhauser enhancement spec-troscopy revealed that the C-terminal residues of Gα undergo

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Fig. 2. Space-filling views of G proteins from Protein Data Bank (PDB) file 1GOT(http://www.rcsb.org/pdb/cgi/explore.cgi?pid=108601067965969&pdbId=1GOT) (42). (A) Space-fillingoverview of Gαβγt. Blue, Gα; gray, Gβ; green, Gγ. (B) Gαt•GDP in the context of heterotrimer; Gβγ is notshown. (C) Enlarged view of GDP binding site, with GDP rendered as a space-filling representation withCorey-Pauling-Koltin (CPK) coloring. Ionic interactions between residue Arg174 (cyan) and Glu39 (pink)span a 2.7 Å distance over the nucleotide binding site. CPK coloring defines carbon as light gray; oxy-gen, red; hydrogen, white; nitrogen, blue; sulfur, yellow; and phosphorous, orange. (D) Sequence align-ment of human Gαt and Gαil created with GeneStream (44). [See movie (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/218/re3/DC2) showing a computerized interpretation of the transition of theGTP- to GDP-bound form of Gα.]

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significant ordering upon interaction with the receptor mimet-ic (21). In these NMR studies, the C terminus again adopts acap conformation, consistent with the structure of the C-terminal region of Gαt upon interaction with the light-acti-vated form of rhodopsin, metarhodopsin II (21). Ordering ofthe receptor mimetic’s intracellular loops was not seen uponinteraction of the Gt peptide, suggesting a very dynamic cyto-plasmic surface for this rhodopsin mimetic. These flexible re-gions may require structural constraints from the entirerhodopsin molecule in order to be able to exert effects on Gtbinding. The next steps toward understanding receptor-medi-ated G protein activation await determination of the structuresof the complex of the activated receptor and the G protein.

How Do Activated Gα Subunits Recognize and Acti-vate Effectors?One emerging paradigm is the concept of nucleotide-depen-dent effector binding occurring in Gα in a manner specific toeach effector. The structure of the γ subunit of phosphodi-esterase (PDE) (Pγ), a Gαt-like chimera (Gαt/i), and of RGS9provide an example of this paradigm (22). RGS binding increases the rate of GTP hydrolysis of Gα, and Pγ is the inhibitory subunit of holoPDE, which binds to Gα upon activation of PDE. The structure of the Pγ/Gα/RGS complexsuggests (i) that the mechanism of PDE activation may lie inthe sequestration of the C terminus of Pγ within the Gα bind-ing site, and (ii) that Pγ promotes Gα inactivation through po-tentiation of RGS9’s GTPase activating protein (GAP) activity(22). This 2.0 Å structure is a complex con-sisting of residues 46 to 87 of bPγ, residues26 to 350 of bGαt, and the RGS domain ofbRGS9, residues 276 to 422. In the activatedG protein, Pγ binds to a region in switch IIand the α3 helix in the Gα subunit. Thisstructure reveals a pocket of hydrophobicresidues on the surface of Gα that helps tobury ~1500 Å of surface area between Pγ(residues 46 to 87) and Gα in an interfacenearly devoid of solvent (22). After GTP hydrolysis, Pγ’s binding site is no longer avail-able (Fig. 5B), releasing Pγ to reassociatewith the catalytic subunit of PDE, preventingcGMP hydrolysis and terminating the visualsignal. This theme was also repeated in thestructure of Gαs•GTPγS•adenylyl cyclase(23), which shows similar hydrophobic con-tacts between Gαs and adenylyl cyclase.Again, the binding sites are no longer avail-able upon GTP hydrolysis, providing an effi-cient termination mechanism.

Determination of the Pγ/Gαt/RGS9 struc-ture confirmed results obtained earlier in so-lution-based studies that revealed the switch IIand α3 regions as major binding interfaces forPγ on Gα (24–26). Although not part of thePγ/Gα/RGS9 complex, previous solution-based studies suggested that Pγ also interactswith the Gα α4/β6 loop (27, 28). Because this structure (22)only includes the C-terminal portion of Pγ, it neither confirmsnor rules out the interaction detected in the solution studies. Thesurface rendering of the complex (Fig. 5A and top panel of Fig.

5B) demonstrates a radically different topology for Gα in thecomplex as compared to the topology of Gα•GDP (Fig. 5B, bot-tom panel). Clearly, a large number of critical interactions alongthe binding interface stabilize the active conformation of the

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Fig. 3. Gαil•GTPγS from PDB file 1BH2 (http://www.rcsb.org/ pdb/cgi/ex-plore.cgi?pid=111681067966089&pdbId=1BH2) (45). (A) Overview ofhelical and switch domain interactions, backbone trace (top panel), andspace-filling diagram (bottom panel). (B) Enlarged view of (A). (Top pan-el) Asp237 in switch III is 3.6 Å from Arg205 in switch II, and Glu245 in theα3 helix is 2.8 Å from Arg208 in the α2 helix of switch II. (See Fig. 2D foralignment of Gα family residues.) [See Movie (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/218/re3/DC2) showing a computerizedinterpretation of the transition of the GTP- to GDP-bound form of Gα.]

Fig. 4. GRASP (http://ltrantor.bioc.columbia.edu/grasp) (17) views of the interacting sur-faces between rhodopsin’s cytoplasmic face (A) and Gt’s rhodopsin-interacting surface(B), using PDB files 1HZX (http://www.rcsb.org/pdb/cgi/explore.cgi?pid=199901067968307&pdbId=1HZX) for rhodopsin (ground state) and 1GOT(http://www.rcsb.org/pdb/cgi/explore.cgi?pid=108601067965969&pdbId=1GOT)for Gt.Imaginary folding on the dotted line will dock the two molecules, whose orientation isbased in part on residues on Gα known to interact with receptor loops. Charge comple-mentarity between the acidic (red) and basic residues (blue) is evident, especially inresidues Lys341, Lys248, Lys141, and Arg147 on rhodopsin and Asp311 and Glu212 on Gαt.When 11-cis retinal is bound to rhodopsin, critical activating residues, such as the Glu-Arg-Tyr sequence, are buried in the structure. Rhodopsin reveals interhelical spaces orpockets on the surface that may provide an interaction site for residues in Gα, such as theflexible C terminus known to interact with rhodopsin (46, 47 ).

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Gα molecule, allowing binding to RGS and Pγ simultaneously.Interestingly, the surface of Gα•GDP (Fig. 5B, bottom panel)appears to have several linear grooves of residues on the sur-face, which suggest the possibility of a linear or α-helical bind-ing site for another partner on the surface of Gα.

This structure sheds light on the mechanism of Gα inactiva-tion by Pγ through potentiation of RGS9 GAP activity (22). Pγaccomplishes this by binding to Gα and inducing conforma-tions favorable for GTP hydrolysis, thereby augmenting RGSbinding. RGS binding stabilizes this conformation, leading toan increased rate of GTP hydrolysis. In the complex, Pγ bindsto this Gαt-like chimera in the Gα switch II/α3 cleft, with theindole nitrogen of Pγ’s Trp70 contacting Ser248 in the α3 helix ofGα . This pushes the aromatic ring of Trp70 into the switch II re-gion of Gα, thereby restricting the position of switch II residueTrp207. This in turn constrains nearby switch II residues on Gαand promotes hydrogen bond formation between Gln200 andArg204 on Gα. Together, these interactions position the Gαbackbone favorably for subsequent hydrolysis of GTP. Thus, Pγcontributes to Gα inactivation by binding to Gα and inducingconformations favorable for GTP hydrolysis, thereby augment-ing RGS binding. The binding of RGS to the Gα/Pγ dimerburies 1800 Å2 of solvent-accessible surface area, with manywater molecules at the binding interface (22). The structure reveals a conformational change that occurs in RGS upon binding to the dimer, notably rotation of the α5/α6 helix, whichorients the RGS’s critical GAP residue Asn364, consequentlystabilizing the conformation of Gln200 in the Gα molecule.Gln200 is involved in stabilizing both the nucleophilic water andthe planar transition state required for GTP hydrolysis (22).Comparison to a previous RGS4/Gαil structure (10) revealssmall differences, which may indicate some plasticity in thebinding site. This plasticity may assist in effectively fine-tuningbinding kinetics to quickly amplify and attenuate visual signals.

A polycationic stretch on Pγ, which was not part of this struc-ture and which has been implicated in effector binding (25), is notaddressed by the Pγ/Gαt/RGS9 structure. It remains to be seenwhether this segment, or other parts of the Pγ molecule, binds to theGα/RGS complex. If both Gα and RGS contribute residues to thisbinding interface, this would create a nucleotide-dependent bindingsite for additional residues of Pγ on the Gα/RGS interface, which isconsistent with a paradigm of nucleotide-dependent changes thatregulate the activity and specificity of G protein signaling. Becausethese structures contain only the RGS domain of RGS9, a Gα witha small N-terminal truncation, and a portion of the Pγ molecule, itremains to be seen whether other regions of Pγ are involved in bind-ing to Gα and RGS. In this complex, Gα serves as a nucleotide- dependent scaffold for Pγ and RGS, which could conceivably be ex-tended to include the RGS9 binding partner, the G protein β5 sub-unit (Gβ5).

Scaffolding in G Protein SignalingMutational studies have shown that distinct, but partially overlap-ping, regions in Gβγ activate different effectors (25), as well as bindto Gα. This association with Gα prevents Gβγ signaling in the ab-sence of G protein activation. The activation-dependent change inGα releases Gβγ from this inhibitory interaction, thus revealing (in-directly) an activation-dependent binding site on Gβγ.

The recently solved structure of G protein receptor kinase 2(GRK2) and Gβγ (29) gives new details of Gβγ binding to aneffector protein and possibly to other pleckstrin homology (PH)

domain–containing proteins. In addition to a kinase domain,which is responsible for phosphorylation of rhodopsin, GRK2contains an RGS domain and a PH domain. The geranylgerany-lated C-terminal residues of bGγ, residues 62 to 68, as well asresidues 52 to 61 upstream from this region, adopt a uniqueconformation when compared to other Gβγ structures (29) thatlack this modification. However, interactions of the geranylger-anyl group with the aqueous environment containing detergentresults in disordering of this acyl group. This may indicate arole for acylation of Gγ in protein-protein interactions. Acyla-tion may induce a conformation on nearby residues and mayplay a role in ordering binding interfaces between proteins, inaddition to contributing directly to membrane binding of Gβγand its ability to efficiently recruit GRK to the membrane. Thisacylation-induced conformational change would not occur innonacylated proteins. Binding of the C terminus of the PH do-main of GRK2 to Gβγ orders the C terminus of Gγ and posi-tions residues in β3 and β4 of GRK to contribute positivelycharged residues to a putative membrane-binding interface,along with the acyl group on Gγ that would likely participate inmembrane interactions (29).

A complex of GRK2, Gβγ, GPCR, and Gαq was predictedusing the rhodopsin structure as a rough model for the GPCR.In the model, the C terminus of rhodopsin was positioned sothat it was able to dock at the polypeptide binding cleft ofGRK2, and residues on rhodopsin known to bind GRK2 were

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Fig. 5. Molecular surface of Gαt (gray) bound to the RGS domainof RGS9 (red) and Pγ (orange) (A and B) versus GDP-bound Gαt

monomer [(B), lower panel] (45 ) from PDB fi les 1FQJ(http://www.rcsb.org/pdb/cgi/explore.cgi?pid=219351067968795&pdbId=1FQJ) and 1TAG (http://www.rcsb.org/pdb/cgi/explore.cgi?pid=220771067968840&pdbId=1TAG), respectively. Acontact surface was generated and decorated with bonded atoms.Red dots are atoms close enough to the surface to be noncova-lently bonded. Dark areas are too far away from any atoms outsidethe surface to be bonded. White or light areas are close enoughfor hydrophobic van der Waals interactions. Magenta areas areclose enough for hydrogen bonds. Oxygens or nitrogens that arelikely to be hydrogen-bonded are shown as red dots.

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aligned with the kinase domain of GRK2 (29). This structuralmodel docks Gβγ, GPCR, and Gα at distinct binding sites onGRK2, resulting in a complex that may mediate G protein sig-naling. Modeling Gαq from homology with the Gαi/RGS4 com-plex and omitting the flexible N terminus of Gαi allowed posi-tioning of Gαq onto the GRK/Gβγ complex. Interactions withGβγ seen in the heterotrimeric structures guided positioning ofGβγ, which abuts the Gαq binding residues of GRK2 (30). Thismodel describes how GRK2’s kinase domain phosphorylatesactivated receptors, whereas GRK2’s RGS homology (RH) do-main binds Gαq, and the PH domain of GRK2 binds Gβγ. Teth-ering thus may play a role in preventing the interaction withother downstream signaling partners (29).

This structure suggests that GRK may also act as a signalingscaffold involved in rapid signal attenuation (Fig. 6). GRK, positioned at the membrane with the aid of Gβγ, can phospho-rylate activated receptors. A potential binding of Gα•GTP to thecomplex’s RGS domain may tether the Gα to the complex, wellpositioned to reassociate with Gβγ upon GTP hydrolysis. Acti-vation-dependent changes of the switch region as a result ofGTP hydrolysis regulate the interaction of Gα with the RGS do-main of GRK and, after GTP hydrolysis, position the Gα•GDPin close proximity to Gβγ for fast, efficient signal termination.

Because the binding sites for the interaction of Gβγ with Gαand GRK are overlapping, the binding is thought to be mutuallyexclusive (29).

Scaffolded signaling complexes may also be involved in theregulation of ion channels, such as G protein–coupled inwardlyrectifying K+ channels (GIRKs). Given the fact that a complexof RGS9 and Gβ5 can regulate GIRK channel activity (31), it ispossible that a complex of receptor, Gα, RGS, Gβ5, and theGIRK channel may form to maintain signaling. Supporting thisidea, several groups have immunoprecipitated complexes of re-ceptor, G protein, and Ca2+ or K+ channels (32, 33).

The signaling scaffold paradigm is likely to receive in-creased scrutiny as interactions are sought within a larger sig-naling context, including membrane-bound receptors, receptorsubdomains, soluble signaling partners, and other accessoryproteins.

Novel Regulation of G Protein SignalingThe fundamental paradigm describing G protein signalingthrough activated Gα subunits can be enlarged to include anumber of novel regulatory mechanisms. Gβγ acts as a negativeregulator of Gα signaling by binding to Gα and by reducing therate of spontaneous GDP release. RGS proteins act as GAPs for

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A B C

D E

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domain

Fig. 6. Schematic of the GRK:Gα:Gβγ interaction with activated receptor. GRK (green). Activation through endocytosis of the receptor is shownin panels A to E.

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many G proteins, capable of promoting GTP hydrolysis and re-turning Gα to the inactive GDP-bound state. GAPs may never-theless promote steady-state signaling through a mechanismwhereby activated Gα•GTP bound to RGS hydrolyzes Gα•GTPto Gα•GDP, thus promoting repeated rounds of high-affinitycoupling to the receptor (the GDP-bound form of Gα has ahigher affinity for the receptor than the GTP-bound form). Intheory, a complex formed between G protein, RGS, and recep-tor through a scaffolding mechanism may allow Gα to undergoGTP hydrolysis at an accelerated rate while maintaining steady-state signaling (34).

In an opposing mode, GDIs (guanine nucleotide dissociationinhibitors) inhibit GDP release, thereby inhibiting signaling(Fig. 7). In this respect, Gβγ functions as a GDI. Other classesof GDI’s are known, such as AGS3 protein, which stabilizes in-active Gα•GDP by binding to Gα subunits and preventing thedissociation of GDP. This can result in enhanced Gβγ signaling,because the binding sites for AGS3 and Gβγ on the Gαmolecule overlap. AGS3 contains four GoLoco repeats (aGoLoco repeat is a 19–amino acid stretch found in regulators ofG protein signaling, also known as GPR motifs). RGS12 andRGS14 each contain one GoLoco repeat, and proteins involved

in spindle reorientation, PINS and LGN, both contain GoLocomotifs. A structure by Kimple and co-workers (35) consists ofresidues 469 to 530 of the rRGS14L, which includes the GoLoco motif and residues C-terminal to the GoLoco motif(but lacks a RGS domain), bound to Gαi•GDP in a complexthat cannot be activated by an activated receptor and is stable inthe presence and absence of Mg+2 (needed for high-affinityGTPγS binding). A 2.7 Å resolution crystal structure of the complexwas solved, showing that 1900 Å2 of peptide surface area wasburied by Gαi binding. Interactions that stabilized the GDP-boundstate include a 6 Å movement of switch II and changes in switch IIIin GoLoco complexed to Gαi. The switch II changes result in Arg208

moving out by 6 Å, relative to the heterotrimeric structure, to blockGβγ access, making the bindings of Gβγ or the GoLoco motif toGα•GDP mutually exclusive.

The GoLoco/Gαi•GDP complex provides insight into themechanism of dissociation inhibition. In the Gαi•GDP/Gβγstructure, Arg178 on Gα makes interactions with the α and βphosphates of GDP (36), and Arg178 also makes a salt bridgewith Glu43. These same interactions are also present in the ho-mologous residues of the Gαt•GDP/Gβγ structure, where Arg174

of Gαt (Arg178 in Gαi) makes a salt bridge with Glu39 of Gαt

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Fig. 7. Hypothetical model of the G protein cycle, including novel signaling partners. Gα, blue; Gβ, brown; Gγ, gray; GDP, yellow; GTP, green;RGS, magenta; GDI, purple; proposed heterotrimeric GEF, orange. Note that no GEFs for G proteins have yet been identified. See(http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/218/re3/DC1) to explore how these various partners regulate G protein signaling.

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(Glu43 in Gαi) (Fig. 2), an interaction missing in the structure ofGα•GDP alone. In the GoLoco/Gαi•GDP complex, a new con-tact is formed between Arg178 on Gα to a hydroxyl group on theribose of GDP. In the GoLoco/Gαi•GDP structure, Arg516 of theGoLoco domain inserts into the Gα’s nucleotide-binding pock-et, replacing the analogous contacts made by Arg178 of Gα andmaking contact with the α and β phosphates of the bound nu-cleotide. In this structure, interactions between Gln147 andAsn149 on Gαi and the Gln515 residue of the GoLoco domainconstrain residue Arg516, inserting it into the nucleotide-bindingsite and displacing the Gα Arg178. A conserved triad in GoLocodomain–containing proteins (Asp-Gln-Arg) results in the inser-tion of an arginine finger into the GDP-binding site. The re-modeling of switch II architecture at Gα residue Arg178 and theinsertion of Arg516 on GoLoco into the nucleotide-binding siteare two essential features involved in the inhibition of nu-cleotide release.

The αB-αC loop of the Gαi helical domain (sometimes referredto as switch IV) moves away from the GTPase domain to accom-modate binding of the C terminus of the GoLoco motif–containingpeptide. The helical domain serves not only as a point of attachmentfor the peptide, but may also play a role in regulation of specificityof the interaction with Gαi proteins. RGS14 interacts with Gi, butnot Go, family members; a chimeric approach revealed that the re-gion C-terminal to the GoLoco motif was essential for GDI activitytoward Gi, but not Go, proteins. The N-terminal region of the pep-tide contacts the GTPase domain, which is more highly conservedthan the helical domain among Gα family members. Likewise, theregion C-terminal to the GoLoco motif is not well conserved be-tween RGS14 and other GoLoco motif–containing proteins, whichallows for specificity. Thus, the helical domain of Gα proteins mayplay a role in defining specificity with other signaling partners (37).Because RGS proteins can interact with G proteins in the absenceof receptor activation, this adds a new twist to the current G proteinregulation paradigm: Conformational changes in switch regionsmay regulate G protein activity in the absence of Gα activation.

Emerging is a theme whereby scaffolding may play a role in theregulation of G protein activity. Several proteins, such as AGS3,contain multiple GoLoco repeats, and these may be involved inbinding more than one Gα subunit, again functioning to quickly andefficiently attenuate signaling. These could be located at specific in-tracellular membranes or as a part of rafts or other membrane sub-domains involved in the spatial regulation of signaling.

Another example of novel regulation of G protein signalingcomes from the recent determination of the structure of thephospholipase C–β (PLC-β) C-terminal region (38). Activationof PLC-β results in the generation of inositol trisphosphate (IP3)and diacylglycerol (DAG). PLC-β is activated by Gαq and alsoacts as a GAP on Gαq, increasing the rate of GTP hydrolysis(39). In this structure, the dimeric nature found in full-lengthPLC-β is seen in a dimer of PLC-β C termini, which forms anextensive coiled-coil region burying 3200 Å2 of surface area atthe dimer interface. The resulting dimer is theoretically capableof binding two Gαq molecules, a possibility also suggested indocking experiments (38). This dimerization may indirectly fa-cilitate the oligomerization of Gα molecules, which may in turnbe tied to dimerization (or oligomerization) of GPCRs, whichhas been reported in recent years [reviewed in (40)]. This struc-ture leads to a speculative model of a dimeric PLC-β bindingtwo Gα’s and two Gβγ’s. Another intriguing possibility is thatthe localization of Gα complexed with PLC-β may allow the

cycling of Gα activation and deactivation seen in functionalstudies (39).

Interestingly, PDE also has a dimeric structure, and thisraises the possibility that two Gα subunits can activate it; how-ever, the nature of this dimerization is unclear. In the crystalstructure of the catalytic domain of hPDE 4B2B, the twomolecules in the asymmetric unit appear to make contactthrough an α helix consisting of residues 496 to 508 of onemolecule making contact with the neighboring molecule (41).However, these authors believe this to be an artifact of crystal-lization. This is in contrast to a recent structure of PDE2 byMartinez et al., which shows dimerization occurring through N-terminal domains (42).

The GEF for the small GTPase Rho, p115RhoGEF, also hasan RGS domain and serves as a GAP toward Gα12/13 subunits.The RGS domain and residues C-terminal to the RGS domain(involved in binding Gα12/13) were crystallized, and the result-ing structure was superimposed on the structure of Gαil boundto RGS4 (43). Although there are subtle differences in the ar-chitecture of p115RhoGEF and RGS4, the central region of theRGS domains of p115RhoGEF (residues 67 to 112 of humanp115RhoGEF) and RGS4 are structurally similar and can beoverlaid with a relative mean square deviation of 1.1 Å. TheAsn128 in RGS4 is homologous to the Pro113 residue in p115RhoGEF and is part of a hydrophobic cluster that may aid inbinding Gα12/13. In this model, the side chain of Glu71 onp115RhoGEF forms an ion pair with Lys204 of Gαi. Mutagene-sis of residues 71 and 113 on p115RhoGEF suggests their in-volvement in the GAP activity of p115RhoGEF, because muta-tions did not abolish Gα12/13 binding but did decrease GAP ac-tivity. The presence of the N-terminal region of p115RhoGEFaugments GAP activity in biochemical studies, but this region isnot present in this structure. It may directly or indirectly stabi-lize binding of the RGS domain to the Gα•GDP-AlF4

− in thetransition state toward hydrolysis in Gα12/13 units.

A number of key issues remain unresolved, such as recep-tor:G protein stoichiometry and the possible functional rele-vance of oligomerization of receptors and G proteins in G pro-tein regulation. The known GEFs for small GTPases lead tospeculation regarding the existence of soluble GEFs for het-erotrimeric G proteins (Fig. 7). Other issues include how acti-vated receptors, located some 20 Å away from the Gα nu-cleotide binding pocket, can trigger the GDP release requiredfor G protein activation and downstream signaling. Importantstructures still to be solved at high resolution include the struc-ture of cGMP phosphodiesterase, rhodopsin kinase, and an acti-vated GPCR. The molecular details of interactions of Gα withholoPDE and RGS9/Gβ5 also remain to be determined. Thecurrent structures extend paradigms to include the possibility ofsignaling complexes that may regulate G protein activity bybringing together key molecules or acting as negative regulatorsby sequestering critical components of signaling machinery.

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Citation: A. M. Preininger, H. E. Hamm, G protein signaling: Insights fromnew structures. Sci. STKE 2004, re3 (2004).

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