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The Gß DIMER as a NOVEL SOURCE of SELECTIVITY in G-Protein Signaling: GGL-ing AT CONVENTION

Department of Pharmacology and
¹ UNC Neuroscience Center and Lineberger Comprehensive Cancer Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365

	SUMMARY

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
Heterotrimeric G proteins relay information between cell surface receptors and effector molecules in diverse signaling pathways to mediate critical cellular processes in both physiologic and pathologic conditions. Multiple isoforms of each of the three G protein subunits yield enormous structural and functional diversity. G proteins are thus obvious molecular targets for the therapeutic manipulation of signaling pathways. Their ubiquity among a vast array of G protein–coupled receptor pathways, however, may at first seem to threaten the attractiveness of G proteins as drug targets for specific signaling processes; in order for G proteins to be effective targets, some degree of selectivity must be defined and exploited. Although a great deal has been determined about the functional selectivity of G subunits, relatively little is known regarding Gß selectivity. In this review, we discuss functional diversity among Gß subunits in both receptor coupling and effector activation. The novel functions of Gß₅, in complex with proteins of the GGL domain–containing R7 subfamily of regulators of G protein signaling, are discussed in detail, with specific focus on the potential of the Gß₅–RGS9-2 pair as a therapeutic target in Parkinson’s disease.

	INTRODUCTION

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
Heterotrimeric guanine nucleotide binding proteins (G proteins) transmit signals across the plasma membrane by undergoing conformational changes, activating enzymes, and participating in multiprotein complexes, thereby allowing cells to transduce diverse external stimuli (e.g., morphine and light) into signals that culminate in neurotransmission, growth, differentiation, or cell death. In the GDPbound basal state, the G protein subunits form an inactive heterotrimer (consisting of G, Gß, and G subunits) that can associate with the cytosolic domains of heptahelical G protein–coupled receptors (GPCRs). In the GTP-bound (i.e., activated) state, the G subunit dissociates, and both it and the Gß dimer can then interact with effector molecules to initiate signaling cascades [for review, see (1)]. These effectors can be enzymes such as phospholipase C (PLC) or adenylyl cyclase (AC), which produce second messengers that regulate intracellular calcium and cyclic AMP concentrations, respectively, or ion channels, such as the G protein–regulated inwardly rectifying K⁺ (GIRK/K_ir3.0) channels or N-type Ca²⁺ (Ca_v2.2) channels.

Regulation of the G subunit is determined by nucleotide exchange, wherein dissociation of GDP allows replacement by the more abundant GTP, as well as the subunit’s intrinsic GTPase activity. The exchange reaction is catalyzed by ligand activation of receptors coupled to the GDP-bound heterotrimer; once the ligand binds, the receptor induces a structural change in the subunit resulting in displacement of GDP (2). Efficient coupling of the heterotrimeric G protein to receptor requires all three subunits (3–8) and causes a conformational change in the receptor, increasing its affinity for ligand (3). Therefore, ligand-induced nucleotide exchange is the culmination of a series of complex interactions among four polypeptides and a ligand (Figure 1).

View larger version (43K): [in this window] [in a new window]   Figure 1. Standard model of heterotrimeric G-protein signaling. Heterotrimeric guanine nucleotide–binding proteins exist in the basal state as a GDP-bound G subunit (red) bound to a Gß dimer (blue and yellow). Ligand-bound receptor (green), serving as a guanine nucleotide exchange factor (GEF) for the G subunit, catalyzes the release of GDP and the binding of GTP. GTP-bound G separates from the Gß dimer and thus allows G and Gß to regulate their respective effectors. The cycle is completed upon hydrolysis of GTP to GDP, resulting in reassociation of the GDP-bound G with Gß. The G subunit has an intrinsic guanine nucleotide triphosphatase (GTPase) activity, which can be stimulated by regulators of G protein signaling (RGS proteins) and, in some cases, the G effector.

	Gß SIGNALING: DIVERSITY AND SELECTIVITY

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
The requirement for G proteins in the propagation of critical signals across the plasma membrane suggests that pharmacologic manipulation of these proteins may be a lucrative therapeutic endeavor. Further, the diversity of downstream pathways and cellular responses suggests that G proteins must exhibit selectivity in their signaling coupling; such selectivity, moreover, is essential to therapeutic intervention. Gß dimers provide a great potential for diversity and selectivity. There are five genes for Gß and at least twelve for G (Figure 2), yielding an enormous diversity of potential dimer combinations. Most of the combinations can form dimers (14); however, there are exceptions, such as the Gß₂ protein that can pair with G₂ but not G₁ (15). Nevertheless, the numerous possible Gß dimer combinations suggest functional selectivity. Selectivity could occur in at least four different levels: at the G protein–receptor interface, at the G protein–effector interface, via other unidentified Gß-interacting cellular components, or due to post-translational modification (e.g., C-terminal lipidation).

View larger version (55K): [in this window] [in a new window]   Figure 2. The relationship between the human G subunit proteins and R7 subfamily of RGS proteins. A. An unrooted dendrogram depicts the degrees of similarity among proteins. As shown in the inset, R7 subfamily members contain a DEP domain, GGL domain, and RGS domain; only the GGL domain (blue) sequences were used for the dendrogram analysis. G subunits are post-translationally modified with a geranylgeranyl (green) lipid or a farnesyl (red) lipid moiety. The unrooted dendrogram was generated using TreeView X from a multiple sequence alignment created with ClustalW using default settings. B. Multiple sequence alignment of human G subunits and GGL domains of human R7 subfamily of RGS proteins. Black boxes depict identical amino acids shared by at least 60% of sequences within alignment. Sequence alignment between human G subunits and GGL domains was computed by the Wisconsin GCG Pileup program using default parameters. GenBankTM gi identifier numbers for human sequences used: RGS7, 17380284; RGS6, 31742476; RGS9, 8475983; RGS11, 34452688; 11, 4758448; 1, 11386179; 8, 3023844; 7, 4826746; 12, 40254926; 4, 4758450; 2, 11277005; 3, 6912394; 5, 4885287; 10, 4758446; 13, 7706567.

	Gß SPECIFICITY IN RECEPTOR COUPLING

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
Coupling of Gs-coupled receptors to G protein heterotrimers appears to be influenced by Gß subunit composition. Using purified recombinant Gß dimers containing the ₂ and the ß₁, ß₂, ß₃, ß₄, or ß₅ subunits, McIntire and colleagues measured EC₅₀ values for nucleotide exchange induced by ligand binding to ß1-adrenergic receptors (19). Whereas dimers containing ß₁, ß₂, ß₃, or ß₄ subunits (i.e., Gß_1-42) tend to display similar EC₅₀ values (0.5–2.7 nM), the Gß₅₂ dimer coupled poorly (17.1 nM). Clearer differences were detected when the investigators examined the A2a adenosine receptor: The most efficient coupling occurred with Gß₄₂ (1.3 nM), and the least efficient occurred with Gß₅₂ (232 nM); Gß₁₂ coupled poorly (15.7 nM), whereas Gß₂₂ (5.7 nM) and Gß₃₃ (5.9 nM) coupled with moderate efficiency. The poor ability of Gß₅₂ to couple to the G_s-coupled ß1-adrenergic and A2a receptors is likely indicative of its selectivity for G_q-coupled receptors (20).

Compared to the Gß subunit, the G exists in a more extensive array of isoforms (Figure 2), suggesting a potentially greater role in Gß dimer selectivity. The suppression of G₇ expression in HEK 293 cells results in the coincident degradation of Gß₁ but not Gß_2–5 monomers (21); prostaglandin E1-induced adenylyl cyclase activity and carbachol- and ATP-induced phosphoinositide turnover were unaffected. Isoproterenol-induced adenylyl cyclase activity, however, was abated, suggesting a specific role for G₇ in ß-adrenergic receptor signaling in HEK 293 cells (22). These studies led Robishaw’s group to generate G₇ knockout mice, which, although fertile and of normal weight, demonstrate an increased startle response (23). Most notably, G_olf expression in the striatum is reduced 82%, whereas G_s, G_o, G₁₃, and G_q are expressed normally. The mice also exhibit reduced D₁ dopamine receptor-induced adenylyl cyclase activity specifically in the striatum, mimicking the reduced D₁ receptor- induced adenylyl cyclase activity demonstrated previously in G₇-suppressed HEK 293 cells (24). Taken together, these results indicate specific functions for G₇: Gß₁₇ dimers couple to ß-adrenergic receptors in HEK 293 cells to mediate activation of adenylyl cyclase activity; the G₇ subunit also appears to couple with G_olf and the D₁ dopamine receptor to regulate adenylyl cyclase activity in the striatum. Further studies are required to determine if selective functions exist for additional G subunits.

	Gß SPECIFICITY IN EFFECTOR ACTIVATION

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
In addition to demonstrating a role for Gß in receptor coupling specificity as described above, McIntire et al. also examined the ability of the various Gß₂ dimers to activate adenylyl cyclase II (ACII) and inhibit adenylyl cyclase I (ACI) (19). The Gß₅₂ dimer activated ACII poorly (EC₅₀ =76 nM) and had no measurable effect on ACI, whereas Gß₁₂, Gß₃₂, and Gß₄₂ activated ACII with EC₅₀values of 3.5–5.5 nM. In contrast, Gß₂₂ activated ACII relatively poorly (EC₅₀ = 12.9 nM). Adenylyl cyclase I was inhibited equally well by Gß₁₂, Gß₂₂ and Gß₄₂ (IC50 = 10.5–16.8 nM) but was inhibited very poorly by Gß₃₂ (IC50 = 110 nM). Thus, Gß dimers containing various Gß subunits differentially interact with adenylyl cyclase effector enzymes.

More recently, Paula Barrett’s laboratory has demonstrated that the Gß₂ isoform inhibits the low-voltage-activated (LVA) T-type calcium channel _1H (Ca_v3.2) with exquisite selectivity (25). This inhibition does not appear to depend on any particular geranylgeranylated G isoform; however, G₁₁ fails to inhibit the channel when paired with Gß₂, which may reflect the farsenylation of G₁₁ (Figure 2). Interestingly, the _1G channel (Ca_v3.1) does not exhibit this Gß dimer selectivity, demonstrating differential regulation among effector isoforms, a useful feature for potential pharmacologic targeting.

Although differences in Gß signaling may be a direct result of G protein–receptor and G protein–effector interactions, it is likely that a number of in vivo cellular factors also play a role. Diversé-Pierluissi and colleagues have measured the inhibition of N-type Ca²⁺ currents that ensues when endogenous PLC-ß is activated following the introduction of recombinant Gß₁-containing, but not Gß₂-containing, dimers into avian sensory neurons (26). Surprisingly, such selective activation of PLC-ß is not seen using in vitro assays, suggesting that additional cellular factors play a role in Gß dimer selectivity.

	Gß SIGNALING: NOVEL ASPECTS OF THE Gß3 AND Gß5 SUBUNITS

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
THE Gß₃ SUBUNIT
Alternative splicing of the five genes that encode Gß subunits introduces even greater potential for the functional diversity of Gß dimers. A novel splice variant of Gß₃, resulting from a single nucleotide polymorphism (i.e., C825T), has been associated with essential hypertension. The alternative splicing results in the deletion of nucleotides 498–620 from exon 9, putatively resulting in a six-bladed propeller structure, instead of the seven-bladed wild-type Gß3 protein (Figure 3) (27). The shorter Gß₃ protein, Gß_3s, is biologically active and likely increases signals generated from pertussis toxin–sensitive G proteins (i.e., G_i/o proteins) (27). This increased G_i/o signal transduction is believed to increase smooth muscle cell proliferation and hypertrophy, resulting in an increased susceptibility to hypertension. Gß_3s has also been associated with increased susceptibility to ischemic stroke in Caucasian patients (28) and left ventricular hypertrophy (29).

View larger version (24K): [in this window] [in a new window]   Figure 3. Deleted regions of variant Gß3 subunits. The Gß1 structure (PDB accession number 1TBG) is depicted, with sequences homologous to Gß3 shown in green; sequences that are deleted to result in shorter Gß3 variants are shown in red. Left, Gß3s; right, Gß3s2.

More recently, another biologically active polymorphism in the gene for Gß3, resulting in Gß_3s2, a putative six-bladed protein structure (Figure 3), appears to associate with expression of the C825T polymorphism (32). The relative contributions of Gß_3s and Gß_3s2 to the above disease processes, as well as the mechanisms underlying these contributions, remain to be determined.

THE Gß₅ SUBUNIT
Gß₅: A Divergent, Brain-Enriched Subunit
The Gß₅ subunit is an outlier in the Gß protein family (Figure 4), with only 50% identity to the four other family members, which among themselves share greater than 80% sequence identity (33, 34). The gene for Gß₅ was originally cloned in 1994 by Watson et al. from a mouse brain library; northern blots of various mouse tissues showed robust mRNA expression in the brain and considerably lower expression in the kidney, heart, lung, and skeletal muscle (33, 34). Shortly after Gß₅ was cloned, a retinal-specific, longer splice variant, Gß_5L, was discovered (35). This longer form contains a 42-residue N-terminal extension. Although the shorter Gß₅ subunit (Gß_5S) is also expressed in the retina, only the longer Gß_5L is expressed in the rhodopsin-rich rod outer segment membranes, suggesting an involvement of Gß_5L in phototransduction (see below) (35). The human Gß₅ gene is highly similar to the mouse form (99.4% protein sequence identity), and is also expressed primarily in brain and retina (Figure 4) (34, 36–38). In the brain, the Gß₅ subunit is associated with the membrane; however, a significant amount (30%–50%) has also been found in the soluble fraction (35, 38, 39), which is in contrast to other Gß subunits which are generally only found associated with the membrane (14, 45, 38).

View larger version (56K): [in this window] [in a new window]   Figure 4. The novel nature of the Gß5 subunit. A. An unrooted dendrogram depicting the relationship among human Gß subunit protein sequences is superimposed over a space-filling model structure of the Gß1 subunit (cyan; PDB id 1TBG) and a space-filling model of the Gß5 subunit (yellow; model coordinates generously provided by Dr. John E. Sondek, UNC-Chapel Hill). B. Northern blot analyses of human Gß5 transcript expression patterns. Blots of 20 mg total RNA (left) or 2 µg poly(A+) RNA from various human tissues were serially hybridized with a mouse Gß5 cDNA probe and, as a control for RNA loading and quality, a human glyceraldehyde-3-phosphate dehydrogenase probe. Data adapted from Snow et al. (1998) Proc. Natl Acad. Sci. USA, 95, 13307–13312. Copyright ©1998 by The National Academy of Sciences of the United States of America.

RGS6, RGS7, RGS9, and RGS11 are members of the R7 subfamily of proteins containing an RGS domain that accelerates the intrinsic GTPase activity of G subunits, thereby promoting the formation of inactive G protein heterotrimers and terminating signaling from G subunits and Gß dimers (42–44). In addition to the RGS domain, R7 proteins contain a Dishevelled/Egl-10/Pleckstrin (DEP) homology domain that mediates membrane association (45, 46) and a G protein gamma-like (GGL) domain that is responsible for dimerization with Gß₅ (36, 47, 48).

Gß₅/R7 Dimerization
The highly specific association of Gß₅ with the GGL domain of R7 family subunits is required for the stability of both members of the heterodimer. Deletion of the GGL domain from RGS7 eradicates the ability to bind Gß₅ (48); conversely, truncated mutant proteins that only contain the GGL and RGS domains of RGS6, RGS7, or RGS11 competently associate with Gß₅ (36, 47). The interaction with GGLcontaining RGS proteins is specific to the Gß₅ subunit; Gß₁, Gß₂, Gß₃ and Gß₄ are unable to associate with RGS6, RGS7, or RGS11 (36, 47, 49). However, exchanging the GGL domain of RGS7 with a portion of the sequence from G₁ allows for association with Gß₁ (48). Because of the considerable amino acid sequence homology between GGL domains and G subunits (Figure 2), it is likely that Gß₅ interacts with RGS proteins in a manner structurally analogous to a Gß–G interaction (50). We have shown that mutation of residues in the GGL domain analogous to residues that are involved in Gß₁–G₁ binding diminish or eradicate Gß₅–RGS protein association (47). It is thus unlikely that Gß₅ could simultaneously associate with both an RGS protein and a G subunit (see also Box 1).

Box 1: The Case For a Gß5 Dimer? A conundrum currently exists in the Gß5 field. It is well established that Gß5–RGS protein complexes exist in vivo (39, 41, 51); however, there is currently no direct evidence for the in vivo existence of Gß5 dimers. In fact, some have speculated that Gß5 only exists in a complex with GGL domain–containing RGS proteins and not with G subunits (50, 51, 85). Does Gß5 function in a traditional Gß sense? Two general functions characterize all Gß dimers: 1) the ability to support ligand-induced, receptor-catalyzed nucleotide exchange on G subunits; and 2) the ability to directly regulate effectors (14). Numerous laboratories have examined the ability of Gß5 to regulate known Gß effectors. For example, when Gß5 was cotransfected with a G subunit (e.g., G2) and PLC-ß2 into COS-7 cells, a significant increase in IP3 was observed (33, 35). In comparing Gß1 and Gß5 functions in cotransfection assays, Zhang et al. demonstrated that Gß52 could activate PLC-ß2 similarly to Gß12; however, unlike Gß12, Gß52 had no effect on downstream activation of MAPK or c-Jun N-terminal kinase pathways (86). Using purified proteins reconstituted in lipid vesicles, Lindorfer et al. showed that Gß52 could directly stimulate turkey erythrocyte PLC-ß (87). In similar in vitro studies, Maier et al. and Yoshikawa et al. confirmed that the PLC-ß2 isoform could be directly activated by Gß52 (88, 89). Phosphatidylinositol-3-kinase (PI3K) is another effector known to be regulated by Gß dimers (90, 91, 92). Using purified proteins in an in vitro assay system, Maier et al. discovered that Gß52 has no measurable effect on the enzymatic activity of either PI3Kß or PI3K (88). Activation of PI3K is known to result in downstream activation of MAPK and c-Jun N-terminal kinase pathways (93–95). The inability of Gß52 to activate PI3K may be the reason why co-transfection of Gß52 is unable to activate MAPK and c-Jun N-terminal kinase (86, 88). On the other hand, Gß5 dimers can regulate the function of two brain-enriched effectors. Zhou et al. investigated the ability of different Gß dimers to regulate N-type Ca2+ channels by transfecting specific Gß and G subunits into HEK293 cells stably expressing N-type Ca2+ channels; Ca2+ currents were measured by a whole-cell patch technique (54). The Gß52 dimer inhibited the channels similarly to other G2- containing dimmers. Most Gß dimers that have been examined activate G protein-activated inwardly rectifying K+ channels (96). Gß5-containing Gß dimers, however, inhibit both basal and receptor-activated currents generated by these channels (96). Binding studies demonstrated that Gß52 can bind to the same channel domains as other Gß dimers (96); results from competition binding assays and activity assays suggest that the mechanism of GIRK channel inhibition is competition between Gß5 dimers and other Gß dimers for GIRK channel interactions (96, 97). Gß5 dimers can also bind G subunits and couple to receptors. Fletcher et al. demonstrated that purified Gß52 could bind Gq but not Gi1, Gi2, Go, or Gs (20). Gq, and possibly related subunits such as G11, can be selectively purified from bovine brain membrane extracts using Gß52 immobilized on an agarose column (20). Purified Gq and purified Gß52 are able to couple to the M1 muscarinic receptor and the endothelin B (ETB) receptor and participate in ligandinduced nucleotide exchange on the G subunit (87). Thus, it appears that the Gß5 subunit may have two independent binding partners (i.e., G subunits and GGL-containing RGS proteins), and different functions have been described for each complex. Gß52 can participate in functions that typify Gß dimers: functional interactions with a G subunit and receptor as well as the ability to regulate known effectors. Unfortunately, there is a dearth of direct evidence to support the in vivo existence of Gß5 dimers, which has led some to doubt the relevance of Gß5 dimers (50, 85). Indeed, a number of technical considerations have hampered the experimental investigation of Gß5 dimer function. Gß5 clearly does not engage in the typical Gß–G dimer interaction. Certain non-denaturing buffer conditions (e.g., ionic detergents such as cholate) appear to perturb the Gß5–G association (20, 47, 89, 98). Even coimmunoprecipitation procedures have been reported to dissociate Gß5 from G from lysates known to contain active dimer (99), although the Gß5–RGS interaction is maintained under similar conditions. Therefore, investigation of Gß5 dimers must give due regard to the sensitivity of the Gß5– interaction to experimental conditions.

 

Gß₅ and R7 proteins are obligate binding partners; expression and solubility of either is limited by stoichiometric expression of the binding partner (39, 40, 51). Data from Gß₅ knockout mice clearly show that RGS6, RGS7, RGS9, and RGS11 protein stability in vivo is also dependent on Gß₅ expression (52). Protein expression of all four GGL-containing RGS proteins is eliminated or greatly reduced in the retina and striatum in the absence of Gß₅. Likewise, in the RGS9 knockout mice, expression of the retinal specific Gß_5L isoform is lost (53). Expression of the shorter Gß₅ isoform is maintained in striatum of RGS9-deficient mice, possibly reflecting an association of Gß₅ with other R7 family members.

GGL/Gß₅ Dimers as Conventional Gß Pairs
Because GGL-containing RGS proteins associate with Gß subunits in a manner analogous to the G–Gß association, one might speculate that Gß₅–RGS complexes play a role in cellular signaling analogous to Gß dimers. If Gß₅–R7 dimers promote either receptor–G protein coupling or effector activation (in addition to their GAP function), they would be positioned to regulate G protein activity at multiple levels (Figure 5). To date, no such Gß₅–R7 function has been demonstrated biochemically. In fact, Posner et al. report that recombinant Gß₅–RGS6 and Gß₅–RGS7 dimers do not form heterotrimeric complexes with G_o•GDP or G_i•GDP, nor were they capable of directly regulating effectors such as ACI, ACII, ACV, PLC-ß1 or PLC- ß2 (49). In addition, Gß₅–RGS11 was found to be unable to regulate N-type Ca²⁺ channels (54).

View larger version (52K): [in this window] [in a new window]   Figure 5. A model depicting a possible G–GDP–Gß5–R7 heterotrimer coupled to a GPCR. GDP-bound G would undergo ligand-induced nucleotide exchange when coupled with a Gß5–R7 dimer. The Gß5–R7 complex would be tethered to the membrane via binding of R9AP (or an R9AP-like molecule) to the DEP domain of the RGS protein. Upon activation, the RGS domain of the Gß5–R7 complex would be conveniently localized to accelerate the hydrolysis of GTP to GDP.

R7 GAP Activity
Several laboratories, including our own, have found the GAP activity of R7 proteins to be specific for members of the G_i/o family, with the highest GAP activity exhibited toward the brain-enriched isoform G_o. Shuey et al. demonstrated that the RGS domain in vitro can increase the GTPase activity of soluble purified G_i1 and G_o but not of G_s (60). We have shown that Gß₅–RGS11 dimers in vitro can increase the GTPase activity of soluble G_o without significantly affecting G_i1, G_s, G_q, G_z, G₁₂, or G₁₃ (36). Posner et al. similarly observed G_o-specific GAP activity in Gß₅–RGS6 and Gß₅–RGS7 dimers (61).

More recently, we published a comprehensive study examining the ability of purified, recombinant Gß₅–R7 dimers to increase the steady-state GTPase activity of G_i1, G_i2, G_i3, G_o, G_q, and G₁₁ in the context of receptor-coupled heterotrimers reconstituted (with conventional Gß dimers) in proteoliposomes (62). We found that dimers of Gß₅ and RGS6, RGS7, RGS9, or RGS11 exhibited GAP activity against G_i proteins coupled to M2 muscarinic acetylcholine receptors but not against G_q or G₁₁ proteins coupled to M1 receptors (Figure 6). Further, there were differences in the potencies and efficacies of the various Gß₅–R7 dimers in their abilities to accelerate the GTPase activity within the G_i family. G_i subunits achieved two- to fourfold higher maximal GTPase rates in the presence of RGS11 or RGS6 as compared to RGS9 or RGS7 (62). Additionally, Gß₅–RGS9 and Gß₅–RGS11 were more potent (EC₅₀ = 25–80 nM) than Gß₅–RGS6 and Gß₅–RGS7 (EC₅₀ = 150–350 nM) for G_i1, G_i2, and G_i3, while all four Gß₅–R7 dimers exhibited similar potency for G_o (EC₅₀ = 16–47 nM).

View larger version (31K): [in this window] [in a new window]   Figure 6. The effects of purified Gß5–R7 dimers on the GTPase activity of Gi and Gq family G subunits. A. Recombinant epitope-tagged Gß5–R7 dimers were expressed in insect cells and purified; purified products are shown on a coomassie blue-stained SDS-PAGE gel. B. Relative steady-state GTPase activities of various purified G subunits reconstituted with receptors in proteoliposomes were determined in the presence of Gß5–R7 dimers. Go, Gi1, Gi2, and Gi3 (with Gß12) were reconstituted in phospholipid vesicles with the M2 muscarinic receptor, and Gq and G11 (with Gß12) were reconstituted with the M1 muscarinic receptor (62).

BIOLOGY OF Gß₅–R7 DIMERS
RGS9-1 Regulation of Phototransduction
The most well characterized physiological function of R7 subfamily RGS proteins is the role of RGS9-1, a retinal-specific splice variant of RGS9, in phototransduction in rod and cone cells. Phototransduction is mediated by the photon-activated receptor rhodopsin, which activates the Gi family G subunit transducin (G_t) and, in turn, the G_t effector cGMP-phosphodiesterase. GTP turnover by G_t is much too slow (tens of seconds) to account for the fast (<1 s) physiologic inactivation (66, 67). He et al. demonstrated that the recombinant RGS domain of RGS9 (66) or the recombinant Gß_5L–RGS9 complex (68) significantly increases the GTPase activity of G_t in vitro, suggesting that RGS9-1 is the GAP responsible for the fast inactivation of G_t. Furthermore, immunodepletion of RGS9 from rod outer segment detergent extracts removes most of the G_tmodulating GAP activity from the extract (67). Confirming these biochemical data, rod cells from RGS9 knockout mice exhibit slower recovery to light as compared to rod cells from wild-type mice (53).

Two laboratories have recently discovered a small, retinal-specific integral membrane protein that serves as a RGS9 anchor protein (R9AP) (45, 69). R9AP co-purifies with Gß_5L/RGS9-1 from rod outer segment membranes, and the interaction appears to be mediated by the DEP domain of RGS9-1 (46). As the name implies, R9AP functions by anchoring RGS9-1 to the membrane (Figure 5), thereby increasing the effective concentration of RGS9-1 at the membrane of outer segments. This localization enhances the ability of RGS9-1 to increase the GTPase activity of G_t in the retina (45, 46, 69, 70, 71). In the absence of R9AP, the stability of the Gß_5L/RGS9-1 complex in photoreceptors is severely compromised (72). Studies have yet to show if there are similar anchoring proteins for the other R7 family members or if R9AP similarly anchors other R7 family members. Interestingly, Snapin, a cytosolic homolog of R9AP implicated in SNARE-mediated membrane fusion, binds the DEP region of RGS7 (73) although, in the absence of a membrane targeting function, the significance of this interaction is unknown.

RGS9 and its membrane anchor R9AP have recently been implicated in hereditary abnormalities in photoresponse recovery (70). Patients with recessive mutations that affect either RGS9 or R9AP and result in severely diminished G_t-directed GAP activity are unable to see moving objects accurately, especially under lowcontrast conditions, and have difficulty adjusting to changes in light intensity. This is the first known human pathology associated with a defect in an RGS protein (70).

RGS9-2 Regulation of Dopaminergic and Opioid Signaling
A second role for the Gß₅–RGS9 dimer in regulating signal transduction is emerging. A longer splice form of the RGS9 gene, RGS9-2, contains a proline-rich 205 amino acid C-terminal extension in addition to the DEP, GGL, and RGS domains. The physiological function of this C-terminal region remains uncharacterized, but recent studies suggest a possible role in substrate selectivity (74) or nuclear localization (75). RGS9-2 expression is highly enriched in striatum, a brain region rich in dopamine and dopaminergic innervation that has been implicated in mediating the reward effects of both dopamine and opioid receptor activation. In addition to its predominant expression in striatum, RGS9-2 is also expressed in the superficial laminae of the dorsal horn, a site implicated in opiate analgesic effects.

Recent in vitro and in vivo work implicates Gß₅–RGS9-2 dimers in the regulation of inhibitory signaling downstream of D2 dopamine receptors (D2Rs) and µ-opioid receptors (µORs), presumably by increasing the GTPase activity of the G_i subunit. Overexpression of RGS9-2 attenuates D2R-mediated inhibition of adenylyl cyclase activity in HEK293 cells (76) and accelerates the kinetics of D2R-mediated GIRK channel activation in CHO cells (76) and Xenopus oocytes (77). Further, in a melanophore dispersion assay, expression of RGS9-2, but not RGS9-1, lowers maximal responses to µOR agonists (78). Additional studies are necessary to determine if there is specificity for, or direct interaction with, these receptors or if RGS9-2 interacts solely with the activated G subunit.

Studies of mice that either overexpress or lack RGS9-2 in striatum support a physiologic role for RGS9-2 in the regulation of dopaminergic and opioid signaling. Viral-mediated overexpression of RGS9-2 specifically in the nucleus accumbens reduces the locomotor response to cocaine and D2R agonists (77). Further, RGS9 knockout mice display heightened locomotor and reward responses to cocaine, and striatal extracts of RGS9 knockout mice show 50% more D2R-mediated inhibition of forskolin-stimulated cyclase activity as compared to wild-type extracts (77). Likewise, RGS9 knockout mice show tenfold higher sensitivity to the reward effect of the opioid receptor agonist morphine as measured in place-preference conditioning assays. This phenotype is reversed by viral-mediated expression of RGS9-2 specifically in the nucleus accumbens of the knockout animals (79). RGS9-deficient mice also show heightened sensitivity to morphine in behavioral assays of analgesic effects (79, 80). These results suggest that inhibition of RGS9-2 may be a valuable adjuvant to opiate-mediated analgesia.

The steady-state level of RGS9-2 protein expression in striatum appears to be regulated by opioid and dopaminergic receptor activation. RGS9-2 protein levels (but not mRNA levels) are significantly increased following chronic cocaine exposure, consistent with a role for RGS9-2 in the development of tolerance (77). The effects of morphine on RGS9-2 expression are more complex. Acute morphine treatment results in an increase in RGS9-2 expression, but chronic treatment decreases expression. Additionally, RGS9-deficient mice show delayed or reduced development of morphine tolerance and enhanced withdrawal symptoms (79, 80). Dysregulation of RGS9-2 (and Gß₅) expression and/or stability represents a potential mechanism for the development of tolerance and addiction to opioid and dopaminergic agents. Additional studies are needed to determine the significance and therapeutic value of this mechanism.

Dopamine, Parkinson’s Disease, and RGS9-2
Parkinson’s disease is a neurodegenerative disease that manifests motor disturbances as a result of deficient striatal dopaminergic signaling. The primary symptoms of the disease are related to dysregulated motor function, including bradykinesia, muscular rigidity, resting tremor, and abnormal posture. The events leading to the onset of Parkinson’s disease remain a mystery, but the role of dopaminergic signaling in the progression of the disease has been recognized for nearly half a century. In 1960, it was shown that levels of striatal dopamine in patients with Parkinson’s disease were reduced more than ninety percent (81). This deficiency reflects a loss of dopaminergic neurons in the nigrostriatal tract that have cell bodies in the substantia nigra and axonal projections onto cholinergic interneurons in the striatum (Figure 7). Dopaminergic stimulation in the striatum leads to two outflow pathways: a direct pathway mediated by excitatory D1 receptor–expressing neurons and an indirect pathway mediated by inhibitory D2 receptor–expressing neurons that converge in the substantia nigra pars reticulate and medial globus pallidus and provide feedback to the cerebral cortex. Both pathways are dysregulated in Parkinson’s disease and contribute to reduced excitatory input to the cortex.

View larger version (19K): [in this window] [in a new window]   Figure 7. Dopaminergic transmission between the substantia nigra and striatum. (A) Normally, neurons originating from the substantia nigra and terminating in the striatum release dopamine to propagate signals to cholinergic interneurons and GABAergic neurons, leading to inhibition of output from the GABAergic neurons. (B) In Parkinson’s disease, these nigrostriatal dopaminergic neurons are progressively lost (gray), causing an imbalance in the normal control of posture and fine motor movement owing to increased GABAergic output from this circuit. L-DOPA, the immediate precursor of dopamine, is used in anti-Parkinson’s pharmacotherapy to restore dopamine receptor-mediated inhibition of this neuronal circuit. Adding an RGS9-2 inhibitor as an adjuvant to L-DOPA should potentiate dopamine receptor signaling by blunting negative regulation via RGS domain–mediated GAP activity.

	CONCLUSION

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
The Gß dimer, formerly considered as a mere bystander to the G- mediated coupling of GPCRs to effectors, is now known to have its own rich set of downstream signaling targets. Recent evidence has now highlighted that some of these targets are selectively responsive to particular subsets of Gß dimers. Moreover, the diversity of potential Gß–G pairings, of specific expression patterns, and of potential new functions has been complemented by the discovery that Gß₅, by binding to the GGL domains of R7 RGS proteins, is functionally distinct from other Gß isoforms. Significantly, the emergent properties of the Gß dimer should suggest new avenues—be they gene-therapy or small-molecule approaches—to the therapeutic modulation of signaling by GPCRs.

Shelley B. Hooks, PhD, was a senior postdoctoral research associate in the Harden laboratory at UNC during the preparation of this review article, but is currently on the faculty of the College of Pharmacy at the University of Georgia in Athens.

David P. Siderovski, PhD, is an Associate Professor in the Department of Pharmacology at UNC-Chapel Hill and is a member of the UNC Neuroscience Center and the Lineberger Comprehensive Cancer Center.

Miller B. Jones, PhD, is a senior postdoctoral research associate in the Siderovski laboratory. Address correspondence to SBH. E-mail SHooks{at}rx.uga.edu; fax 706-542-5358.

	ACKNOWLEDGMENTS

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 
The authors would like to thank Dr. T. Kendall Harden for his critical role in developing and supporting the experiments and hypotheses discussed regarding R7 family RGS proteins. SBH is supported by the National Institutes of Health (F32 GM66561). MBJ is a postdoctoral fellow of the Pharmaceutical Research And Manufacturers of America (PhRMA) Foundation. Research on the R7 subfamily of RGS proteins in the Harden and Siderovski labs is supported by NIH grant P01 GM065533.

	References

TOP Summary Introduction Gß{gamma} Signaling:... Gß{gamma} specificity... Gß{gamma} Specificity... Gß{gamma} Signaling:... Conclusion References

 

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