Non-receptor activators of heterotrimeric G-protein signaling (AGS proteins)

Seminars in Cell & Developmental Biology 17 (2006) 334–344

Review

Non-receptor activators of heterotrimeric G-protein signaling (AGS proteins) Mary J. Cismowski ∗ Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, Rootstown, OH, United States Available online 16 March 2006

Abstract G-protein coupled receptor (GPCR) signaling represents one of the most conserved and ubiquitous means in mammalian cells for transferring information across the plasma membrane to the intracellular environment. Heterotrimeric G-protein subunits play key roles in transducing these signals, and intracellular regulators influencing the activation state and interaction of these subunits regulate the extent and duration of GPCR signaling. One class of intracellular regulator, the non-receptor activators of G-protein signaling (or AGS proteins), are the major focus of this review. AGS proteins provide a basis for understanding the function of heterotrimeric G-proteins in both GPCR-driven and GPCR independent cellular signaling pathways. © 2006 Elsevier Ltd. All rights reserved. Keywords: Heterotrimeric G-protein; AGS protein; Signaling; Receptor-independent; G-protein coupled receptor

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional screening for AGS proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AGS1—a Ras-related direct G␣i/o activator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular functions of AGS1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other direct heterotrimeric G␣ activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AGS3-6—members of a family of G␣i/G␣o-specific GDIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular functions of GPR domain proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AGS2, AGS7, AGS8—a new family of G␤␥ interacting proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In canonical G-protein coupled receptor (GPCR) signaling, extracellular stimuli are transduced inside the cell via binding of specific ligands to their cognate seven-transmembrane receptors. Receptor conformational changes elicited by ligand binding are transferred to a heterotrimeric G-protein, triggering exchange of GDP for GTP on the G␣ subunit followed by heterotrimer dissociation and activation of GTP–G␣ and G␤␥ specific effectors

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and their downstream signaling pathways. Upon GTP hydrolysis, the heterotrimer can then reform and be re-activated by receptor. Tight regulation of each of these events within the cell insures optimization of signal efficiency, duration and specificity, as well as appropriate integration of cellular responses. One key area for signal regulation is that of the activation state of the heterotrimer. Much effort over the last decade has gone into understanding how GPCRs function as guanine nucleotide exchange factors (GEF) for G␣, thereby activating the heterotrimer, and many excellent reviews are available on this topic [1–6]. The discovery and characterization of a family of proteins that enhance the intrinsic GTPase activity of G␣ (the regulators of G-protein signaling or RGS proteins) has also greatly

M.J. Cismowski / Seminars in Cell & Developmental Biology 17 (2006) 334–344

increased our understanding of the regulation of G-protein signal duration once receptor activation has occurred (reviewed in Refs. [7–11]). In the large body of work characterizing the heterotrimeric G-protein activation/deactivation cycle, several observations have been made suggesting that G-protein activation can occur independently of a GPCR, thus lending credence to the idea that heterotrimeric G-proteins function in multiple signal pathways within the cell, some of which may not require GPCR input. Both small molecule cationic amphiphiles and short cationic peptides, including the family of wasp venom mastoparans and derivatives of Rab3 effector domains, are capable of directly activating heterotrimeric G-proteins by facilitating GTP/GDP exchange on the G␣ without altering basal GTP hydrolysis rates (reviewed in Refs. [12,13]). The activation of G␣i/o by cationic peptides is sensitive to G␣ ADP-ribosylation by pertussis toxin, and each activating peptide is capable of folding into a cationic amphiphilic helix, suggesting a mechanism of action similar to that of the third intracellular loops of GPCRs ([14–16]; however, see also Ref. [17]) as well as the existence of proteins with similar domains that may also function as direct G␣ activators. The identification by numerous laboratories of G-protein complexes localized to intracellular compartments involved in vesicular trafficking, exocytosis, autophagy and cell division, each apparently lacking GPCR association, further suggested alternative means for signal input to heterotrimers [18–29]. Additional evidence for intracellular regulators of heterotrimeric G-proteins derived from observations of differences in the efficiency and specificity of receptor-G-protein coupling in different cell backgrounds, as well as observations of differences in the kinetic properties of purified G-protein signaling components versus their cellular counterparts [30–35]. Finally, the elucidation of intracellular signaling networks in which heterotrimeric G-proteins cross-talk with signaling proteins from non-GPCR pathways (reviewed in Refs. [36–39]) strongly suggests the presence of additional cellular regulators of G-protein function.

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A variety of methods have been used to identify and characterize intracellular regulators of heterotrimeric G-protein activity. Protein interaction screens, such as the yeast 2-hybrid screen, have proven successful in isolating proteins binding to heterotrimeric subunits (for examples, see Refs. [40–44]), and yeast 2-hybrid, expression cloning and differential expression techniques have all proved useful in identifying novel GPCR interacting proteins (for examples, see Refs. [45–48]). In addition, several laboratories have used biochemical assays to characterize a group of proteins that appear to activate G-proteins in the absence of receptor, including GAP-43 [49], Ric-8 [50], phosphatidylethanolamine binding protein (PBP) [51], presenilin [52], cysteine string protein [53] and a partially purified protein from NG108-15 cells [54,55] (see Section 5). Though these different methods have proven successful in isolating signal regulators, each is hampered by either a lack of throughput or a lack of directly available functional information. Therefore, a screening system was designed in the yeast Saccharomyces cerevisiae that combined high throughput with a direct functional readout in an effort to further identify receptor independent activators of heterotrimeric G-proteins. 2. Functional screening for AGS proteins High throughput screens were designed in haploid yeast to take advantage of its endogenous GPCR-based mating pathway. This pathway, which is driven by the release of free G␤␥ following receptor activation by pheromone, was modified to allow screening of mammalian cDNA expression libraries to isolate those expressed cDNAs that activate a pheromone-responsive reporter [56,57]. These modifications included deletion of the endogenous yeast GPCR and the introduction of a pheromoneresponsive reporter that allows for selection of cDNA-derived activators via a simple growth assay [56]. Reasoning that mammalian proteins would be more likely to functionally interact with mammalian G-protein subunits than with their yeast

Table 1 AGS proteins derived from yeast functional screensa Classification

Alternate names

Library derivation

G-protein selectivity

Properties

Group 1 (GEF) AGS1

Dexras1, RASD1b

Human liver

G␣i2, G␣i3

Ras family member

Group 2 (GDI) AGS3 AGS4 AGS5 AGS6

GPSM1b GPSM3b LGN, GPSM2b RGS12b

NG108-15 cell line Prostate leiomyosarcoma Prostate leiomyosarcoma Prostate leiomyosarcoma

G␣i2, G␣i3 G␣i2, G␣i3 G␣i3c G␣i3c

Four GPR domains Three GPR domains Four GPR domains One GPR domain

Group 3 (G␤␥) AGS2 AGS7 AGS8

Tctex-1, DYNLT1b TRIP13b KIAA1866, FNDC1b

NG108-15 cell line Prostate leiomyosarcoma Ischemic rat heart

G␣i2, G␣i3, G␣s, G␣16 G␣i2, G␣i3, G␣s, G␣16 G␣i2, G␣i3, G␣s, G␣16

Binds G␤␥ Binds G␤␥ Binds G␤␥

a AGS proteins were isolated via yeast functional screen of the cDNA library indicated and are numbered according to their order of isolation. G-protein selectivity, unless otherwise stated, was determined using yeast strains individually expressing human G␣i2, human G␣i3, rat G␣s or human G␣16, each of which was made to couple to yeast G␤␥ [56]. Abbreviations used: GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide dissociation inhibitor; AGS, activator of Gprotein signaling; RASD1, dexamethasone-inducible Ras; GPSM, G-protein signal modulator; RGS, regulator of G-protein signaling; DYNLT, dynein, light chain, Tctex-type; TRIP, thyroid hormone interacting protein; FNDC, fibronectin type III domain containing. b Designation given to the human ortholog by the Human Genome Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature/). c Not tested in yeast strains expressing human G␣i2.

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counterparts, the endogenous yeast G␣ was also replaced with a human or rat G␣ sequence that efficiently couples to yeast G␤␥ (e.g., G␣s, G␣i2, G␣i3, G␣16). Additional similarly modified yeast strains, with alterations that interrupt the yeast mating pathway at different points, were used in epistasis tests to identify those cDNA-derived activators that functioned at the heterotrimeric G-protein itself, and heterotrimeric subunit interactions confirmed in pull-down assays using purified mammalian G␣ and G␤␥ [56,58–60]. This subset of direct heterotrimeric activators were deemed AGS proteins (for activators of Gprotein signaling). AGS proteins appear to fall into three distinct functional classes: those that function as direct G␣ activators, those that modulate G␣–G␤␥ interaction by binding to G␣, and those that modulate G␣–G␤␥ interaction by binding to G␤␥ (Table 1 and Fig. 1). As predicted, the AGS proteins that directly interact with G␣ displayed selectivity for the mammalian G␣ family used in the screen (in this case G␣i/o) and not for other mammalian G␣ proteins or the yeast G␣ [56,58,59]. Class 2 AGS proteins all

share a similar structural motif of ∼20 amino acids, which has been termed the G-protein regulatory (GPR) [58] or GoLoco [61] motif. There is, however, no obvious consensus motif in the G␤␥ interacting AGS proteins. It should be stressed that the classification of AGS proteins as activators of heterotrimeric signaling is based on their functional activity in the yeast screens, and many of these proteins were previously identified based on non-AGS properties (Table 1). Because the transcriptional readout used to identify AGS proteins in the yeast screens is driven by G␤␥, screening for specific effects of expressed cDNAs on G␣-mediated signaling pathways (e.g., activation of adenylyl cyclase or phospholipase C) is not possible. Within the context of mammalian cells carrying multiple GPCR- and non-GPCR signaling pathways, AGS proteins exhibit complex behaviors (see below) and a full understanding of their cellular functions requires additional study. Nevertheless, the yeast functional screen continues to be a valuable tool for the identification of new and unexpected heterotrimeric Gprotein signal regulators.

Fig. 1. Potential modulation of heterotrimeric G-protein signaling by AGS Groups 1–3 proteins. AGS proteins may compete at the plasma membrane with GPCRs for limiting pools of heterotrimeric G-protein (mechanism I) or may interact with a distinct pool of heterotrimer either at the plasma membrane or intracellular membranes (mechanism II). Group 1 proteins (A), as exemplified by AGS1, can function as direct guanine nucleotide exchange factors (GEFs) on heterotrimeric G␣i/o, though AGS1 has also been reported to interact with G␤␥ [63]. Group 2 proteins (B), as exemplified by AGS3, function as guanine nucleotide dissociation inhibitors for G␣i/o, perhaps releasing upon activation of the heterotrimeric G␣ by one or more GEFs. Group 3 proteins (C), as exemplified by AGS8, bind to G␤␥ and may either dissociate the inactive heterotrimer or ‘capture’ G␤␥ released upon heterotrimer activation.

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3. AGS1—a Ras-related direct G␣i/o activator Full length AGS1 (RASD1, DexRas1; GenBank accession # NM 016084), a Ras-related protein with all of the conserved Ras-like motifs required for nucleotide binding/hydrolysis, membrane localization and effector recognition, was isolated from a human liver cDNA library [56] and is the only GEF isolated thus far using the yeast functional screen (see Section 5). AGS1 potently activates G␣i heterotrimers expressed in yeast, but not G␣s, G␣16 or native yeast heterotrimers. AGS1 activation of G␣i signaling in yeast can be eliminated in the presence of a G␣i2 mutant incapable of stably binding GTP (G␣i2–G204A) and is antagonized by co-expression of RGS proteins that act on G␣i (RGS4 and RGS5), consistent with AGS1 directly facilitating GTP binding on G␣ [56]. Purified AGS1 does indeed function as a GEF for G␣i/o, activating both free and heterotrimeric G␣ subunits [62]. Interestingly, while purified AGS1 interacts in vitro with His6 -tagged G␣i2 but not yeast G␤␥ [56], in both yeast 2-hybrid assays and co-immunoprecipitation assays in mammalian cells AGS1 interact specifically with G␤1 but not G␣i2 [63]. Though its actions in yeast and in vitro are only consistent with a direct G␣ activation function, an interaction of AGS1 in vivo with both G␣ and G␤␥ may help explain the multiple roles AGS1 appears to play in regulating signaling in mammalian cells (see Section 4). Indeed, AGS1 has also been shown to interact with adaptor proteins not directly linked to GPCR signaling pathways, such as CAPON [64] and Nck-2 [65], suggesting it may function in vivo either independently of heterotrimer signaling or to facilitate cross-signaling between G-protein heterotrimer and non-GPCR signaling pathways. Aside from its Ras homology AGS1 contains unique N- and C-terminal extensions not seen in the majority of Ras family members, each of which contains numerous basic amino acids. It is within the C-terminal extension (amino acids 194–250) that AGS1 appears to recognize G␤␥ [63]. AGS1 function likely requires guanine nucleotide binding, as mutation of glycine residues (G31V or G36V) required for stable P-loop formation [66] renders AGS1 inactive in both yeast and mammalian cells [56,62,67,68]. AGS1 contains several sequence differences in comparison to Ras within its nucleotide binding/hydrolysis domains, and mutation of these residues in Ras leads to constitutive activity [69]. It remains unclear, however, how these sequence differences affect AGS1 activity. Though AGS1 is active in yeast-based assays without any apparent stimuli and AGS1 purified from yeast extracts is GTP bound [62], AGS1 expressed in mammalian cells appears to be mainly GDP bound [70,71] suggesting the presence of mammalian regulators of AGS1 nucleotide binding and/or hydrolysis. Mutation of alanine-178 to valine in AGS1 reportedly causes constitutive activity [70], though this mutation does not appear to greatly alter the in vivo GDP/GTP binding ratio of AGS1. The post-translational S-nitrosylation of cysteine-11 in AGS1 also appears to be required for full activity [71,72]. Clearly there is a need for the further identification of key residues in AGS1 required for its activity in both yeast and mammalian systems.

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Mammalian AGS1 orthologs have been identified both as a dexamethasone inducible mRNA (Dexras1) in mouse AtT-20 corticotroph cells [73], as a blood-loss inducible mRNA from rat kidney [74] and as a cycling mRNA during circadian rhythm in the mouse suprachiasmatic nuclei [75]. AGS1 mRNA is widely distributed in human and rat tissues, but appears to be downregulated in many cell lines and in some tumors [64,65,68,76]. AGS1 has a closely related human homolog termed Rhes (Ras homolog enriched in striatum; RASD2; Tumor Endothelial Marker 2, TEM2; GenBank Accession # NM 014310), which was identified as a thyroid hormone inducible gene [77–79]. Though AGS1 and Rhes appear to form a distinct branch within the Ras superfamily of small G-proteins, Rhes does not function as a G␣i activator in yeast (MJC, unpublished data). 4. Cellular functions of AGS1 Studies of AGS1 function both in cell culture and in animals suggest AGS1 regulates multiple cell signaling events. Transiently transfected AGS1 activates the ERK1/2 signaling pathway in mammalian cells, and either pretreatment with pertussis toxin or sequestering of free G␤␥ blocks this activation [62,80]. AGS1 expression also blocks ADP-ribosylation of G␣i by pertussis toxin and inhibits cAMP accumulation in response to forskolin or a constitutively active G␣s [80,81]. In AtT-20 cells, mutation of AGS1 at alanine-178 leads to a decrease in both basal and cAMP-dependent peptide hormone secretion, an effect that appears to depend on AGS1 plasma membrane association [70]. Each of these experiments is consistent with AGS1 functioning as a direct G␣ activator in a manner similar to that of a G␣i-coupled GPCR. However, when transiently cotransfected with a G␣i-coupled receptor AGS1 actually blocks G␤␥-mediated signaling following agonist activation of that GPCR [67,80], and AGS1 blocks receptor mediated heterologous sensitization of adenylyl cyclase [82]. Similar results have been reported for Rhes, which disrupts the cAMP/PKA pathway by apparently uncoupling G␣s from its GPCR [83]. The seemingly contradictory effects of AGS1 on G␣i signaling may result from AGS1 and G␣i-coupled receptors competing for a limited pool of heterotrimeric G-proteins, from AGS1 altering GPCR/G-protein coupling in a manner similar to Rhes, or from AGS1 interacting with more than one signaling component in vivo. AGS1 expression is dramatically upregulated in both cultured cells in response to both glucocorticoid treatment [73] and in whole animals following severe blood loss [74], strongly suggesting AGS1 plays a role in mediating stress response. A glucocorticoid response element has been identified in the 3 flanking region of AGS1 [84]. Though no canonical hypoxiainducible factor binding site is apparent in either the 5 or 3 untranslated region (UTR) of AGS1, two sequences with significant homology to human erythropoietin promoter elements essential for hypoxic induction were identified in the 5 UTR [74]. Interestingly, AGS1 expression is downregulated in some tumors [76], suggesting it may have a growth inhibitory function in at least some tissues. More experiments are clearly needed to

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identify additional AGS1 gene sequences required for its transcriptional repression and activation. AGS1 forms a complex with both CAPON and neuronal nitric oxide synthase (nNOS) in rat brain that facilitates its Snitrosylation, a modification which increases guanine nucleotide exchange on AGS1 and presumably enhances its activity [71,72]. As CAPON also serves as an adapter linking N-methyld-aspartate (NMDA) receptor-mediated calcium influx to nNOS activation [85] and, as NMDA-mediated activation of Erk1/2 in primary cortical neurons is pertussis toxin sensitive [86], it is tempting to speculate that AGS1 functions as a regulated intermediary between NMDA and G-protein signaling pathways in neurons. This speculation appears borne out by recent studies of the mammalian circadian clock. NMDA-mediated signaling is one component of this clock and the discovery of AGS1 as an mRNA in suprachiasmatic nuclei that cycles with the circadian rhythm [75] suggests a potential link between the NMDACAPON-nNOS-AGS1 complex and G-protein mediated circadian rhythms. A targeted disruption of AGS1 in mice does not affect viability or reproduction, but alters photic and nonphotic responses of the circadian clock involving signals arising from both glutamate and neuropeptide Y receptors [87]. Thus AGS1 may be mediating signal cross-talk between NMDA and neuropeptide Y receptors leading to activation of G␣i/o and Erk1/2. Further studies using conditionally null AGS1 mice will prove useful in confirming this exciting possibility. Other cellular functions of AGS1 may involve effector systems analogous to those regulated by other Ras-related proteins. Prolonged AGS1 expression in some cell types causes growth inhibition, and AGS1 expression prevents the development of tumors in athymic nude mice [68]. As this growth inhibition is unaffected by pertussis toxin, heterotrimeric G␣i/o signaling is not likely involved. Though AGS1 under these conditions may be interacting with G␣ proteins not affected by pertussis toxin or with G␤␥, cellular regulation of proliferation, senescence, apoptosis, and/or terminal differentiation is often through Ras-family protein interactions with effectors through their effector binding domains [88–91]. AGS1 shares homology with Ras in its effector domain, and the identification of putative AGS1 effectors should greatly aid our understanding of the role AGS1 plays in regulating cell growth. 5. Other direct heterotrimeric G␣ activators As mentioned in the introduction, many other direct activators of heterotrimeric G␣ proteins have been identified. Though not the focus of this review, they certainly bear mentioning. GAP43, an abundant protein in the neuronal growth cone, facilitates guanosine5-3-O-(thio)triphosphate (GTP S) binding to purified G␣o [49]. In vivo, GAP-43 amplifies GPCR signaling cascades in response to environmental stimuli via a palmitoylation- and phosphorylation-regulated mechanism, perhaps thereby serving to coordinate signal transduction complexes at the axonal growth cone [92]. Likewise PBP, a plasma membrane associated protein, facilitates binding of GTP␥S to purified G␣i1, and appears to augment G␣i/o coupled receptor signaling in vivo [51]. The C-

terminus of presenilin, an integral membrane protein involved in Notch signaling and implicated as a familial Alzheimer’s disease marker, interacts with and enhances both GTP␥S binding and GTP hydrolysis rates of G␣o [52]. Another G␣i/o specific activator has been identified in NG108-15 cells and has been partially purified [54,55]. Although not fully characterized, this NG10815 activator increases GTP␥S binding to G␣i/o in a pertussis toxin insensitive manner, distinguishing it from both AGS1 and GPCRs in terms of its mechanism of action [55]. Ric-8 (synembryn), a regulator of neuronal transmitter release and asymmetric cell division [93,94], plays a key role in GPCR dependent and independent signaling, at least in lower organisms [95]. Two mammalian orthologs of Caenorhabditis elegans Ric-8, termed Ric-8A and Ric-8B, were isolated from yeast 2-hybrid screens using G␣ proteins as bait [50]. Ric-8A showed specificity for G␣q and well as G␣i/o in 2-hybrid and pull-down experiments, and functioned as a specific GEF in vitro for this subset of G␣ proteins. Ric-8B, though not fully characterized, showed specificity for G␣s, G␣q and G␣olf in 2-hybrid tests and potentiates G␣olf cAMP accumulation in transfected cells [50,96]. An apparently G␣s-specific activator for both monomeric and heterotrimeric G␣s, cysteine string protein (CSP) has also been identified [53]. The guanine nucleotide exchange function of full length CSP, an abundant protein in neuronal secretory vesicles with homology to the DnaJ/Hsp40 family of co-chaperones, is regulated by two other proteins, Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein). As G␣ proteins, but not receptors, have been identified on various secretory vesicles (see Ref. [53] and references therein), CSP may be substituting for receptor in these vesicles to regulate secretion. 6. AGS3-6—members of a family of G␣i/G␣o-specific GDIs AGS3 (GPSM1; GenBank accession # NM 144745), isolated from the neuroblastoma-glioma hybrid cell line NG108, was the first member of a family of heterotrimeric G-protein GDIs to be identified via the yeast functional screen [56,58]. Like AGS1, AGS3 functioned at the level of the G␣i2 heterotrimer and is specific for G␣i proteins. However, AGS3 was distinguished in its mechanism of action from AGS1 by its ability to function in the presence of a mutant G␣i2 (glycine-204 to alanine) unable to stably bind GTP and by its ability to function in cells coexpressing RGS4 [58]. This suggested that AGS3 specifically recognizes the GDP-bound form of G␣i2, something confirmed in pull-down assays using purified proteins [58]. Full length AGS3 is a mosaic protein with seven N-terminal repeats of a tetratricopeptide (TPR) motif and four C-terminal GPR repeats. As originally isolated, AGS3 was N-terminally truncated and contained only a single GPR domain. An AGS3 construct incorporating all four GPR repeats also activated G␣i signaling in yeast but full length AGS3 did not (MJC, unpublished observations). Subsequent yeast screens using cDNA libraries derived from prostate leiomyosarcoma and human heart identified other

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proteins with GPR domains and properties similar to AGS3. AGS4 (GPSM3; G18.1b; GenBank accession # NM 022107), a protein encoded by the major histocompatibility complex class III region and containing three GPR consensus motifs [58,61], associates with G␣i/o and, like AGS3, inhibits GTP␥S binding to the heterotrimeric G␣ [59]. AGS5 (GPSM2; LGN; GenBank accession # NM 013296), previously identified in a yeast 2hybrid assay as a binding partner for G␣i [42], shares the greatest homology with AGS3, including the presence of seven TPR and four GPR motifs. AGS6 (RGS12; GenBank accession # NM 198229) encodes the C-terminal region of RGS12 [60]. Aside from AGS4 each isolated cDNA encoded an N-terminally truncated product, though each expressed protein contained at least one intact GPR motif. In addition to their discovery via AGS screens, proteins with GPR domains have been identified through their ability to interact with G␣i, G␣o and/or G␣z or via homology searches [42,44,61,97,98]. In mammals these include RGS14, Rap1GAP (GTPase activating protein), and Purkinje cell protein-2 (PCP2). Many GPR proteins have additional domains that serve important regulatory or secondary functions, such as PDZ (PSD95/Discs Large/ZO-1) and phosphotyrosine binding domains (seen in AGS6/RGS12), Ras binding domains (seen in AGS6/RGS12 and RGS14) and GTPase activating domains (seen in AGS6/RGS12, RGS14 and Rap-GAP). Of particular interest, the combination of GTPase activating domains and GPR domains in AGS6/RGS12 and RGS14 presents an intriguing scenario for the dual regulation of G␣i subunits via enhancing GTPase activity and stabilizing GDP–G␣i [99–105]. The TPR domains in AGS3 and AGS5 are also likely to serve a regulatory function, as TPR domains in other proteins are key sites for protein–protein interactions (reviewed in Refs. [106,107]). Indeed, the TPR domains of AGS3 and AGS5 interact with potentially important regulatory proteins and may function as determinants of subcellular localization (see Section 7). It is possible that the lack of function of full length AGS3 in the yeast assay arises from TPR domain interaction with a conserved eukaryotic protein that sequesters AGS3 from the heterotrimeric G-protein. This possibility requires further investigation. Of note a splice variant of AGS3 lacking TPR sequences but containing three GPR domains has been identified as the major expressed form of AGS3 in the heart [108], suggesting tissue specific differences in regulation of AGS3 function. Though GPR domain proteins were initially postulated to function as GEFs [44], additional studies of intact proteins as well as isolated GPR domain sequences have determined that GPR domains instead function as GDIs, inhibiting GDP release from G␣i/G␣o [102,109–114]. Residues required for GDI function have been identified [109,113,115–118] and, though most data indicates a selectivity for G␣i versus G␣o, sequences outside of the core GPR motif may influence G-protein selectivity in interacting with the helical domain of G␣ proteins [104,113]. AGS3 GPR domains can dissociate mammalian G-protein heterotrimers [114,115], making it likely that AGS3-6 were identified in the yeast assay via their ability to directly dissociate yeast G␤␥ from GDP-bound G␣i2.

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7. Cellular functions of GPR domain proteins The identification a class of heterotrimeric G-protein GDIs suggests cellular functions both in the context of classical GPCR signaling and in receptor-independent G-protein signaling. In mammalian cells as in yeast, GPR proteins may bind to Gprotein heterotrimers and actively promote subunit dissociation while maintaining G␣ in its GDP-bound state. In the context of GPCR signaling this loss of available heterotrimer for receptor coupling might be expected to dampen agonist induced signals. Alternatively as G-protein heterotrimers undergo either basal or agonist-stimulated cycling through activate/inactive states, GPR proteins may “capture” GDP-bound G␣ before it can rebind to G␤␥. In this case the activity of G␤␥-regulated effectors would likely be enhanced and signaling via G␣ pathways decreased, and regeneration of a functional heterotrimer would require prior dissociation of the GPR protein from GDP–G␣. These mechanisms of G-protein regulation might influence receptor regulation and/or cellular adaptations to prolonged activation of G␣i-coupled GPCRs [103,119]. Another intriguing function of GPR proteins may be in regulating G␣ independent of heterotrimer and classic GPCR coupling. In this scenario GPR proteins may function in a manner akin to G␤␥, maintaining G␣ in an inactive state until a nucleotide exchange factor disrupts the GPR–G␣ complex. Or the GPR–G␣i complex might itself function in a novel signaling pathway. The additional motifs present on many of GPR proteins might then serve as binding scaffolds for G␣ complexes that include either nucleotide exchange factors or other regulatory proteins. Several recent studies have provided intriguing clues that members of the GPR family do indeed serve to organize intracellular signaling complexes. The TPR domains of AGS3 anchor it along with GDP-bound G␣i3 on the endoplasmic reticulum (ER) of the intestinal cell line HT-29 [120,121], and these TPR domains expressed independently of AGS3 GPR domains localized to intracellular membranes [108,120]. GDP–G␣i3 free of G␤␥ was previously shown to control macroautophagy in these cells [23,122], a process accelerated by the presence of the RGS protein GAIP [123]. Later studies showed that an ER-anchored AGS3/GDP–G␣i2 complex positively stimulates an early phase of this autophagic response to nutrient starvation [120]. This stimulation requires full length AGS3 and is inhibited by expression of either the GPR or TPR domains alone, presumably due to competition between full length AGS3 and the isolated GPR and TPR domains for binding of free GDP–G␣i3 or an ER-derived protein, respectively. A second example of scaffolding in GPR proteins, initially discovered in Drosophila melanogaster and C. elegans, is that of the regulation of asymmetric cell division via G␣ without GPCR input [124–130]. The identification in C. elegans of a complex containing a GPR protein, G␣i/G␣o subunits, a GEF (Ric-8) and a GTPase activating protein (RGS7) confirmed a coordinated receptor-independent signaling cascade that differentially control pulling forces on the mitotic spindle allowing the generation of daughter cells of different sizes [131]. Current efforts in this field are addressing the nature of upstream signal input

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into this complex and the mechanism whereby spindle-pulling forces are controlled. An analogous system appears to be in play in mammalian cells. AGS5/LGN localizes to the spindle poles and the midbody in non-asymmetrically dividing cells via interaction of its TPR domains with the nuclear mitotic apparatus protein [132–134]. A similar process likely controls asymmetric cell division during neurogenic development, where AGS3 appears to regulate the activity of G␤␥ presumably by sequestering GDP–G␣i/o proteins [135]. LKB1, a serine/threonine specific kinase involved in the regulation of cell cycle progression and cell polarity [136], may play a role in regulating AGS3/AGS5 function in cell division. LKB1 was isolated as a binding partner of AGS3 TPR domains and phosphorylates AGS3 GPR domains in vitro, disrupting the ability of AGS3 to inhibit GTP␥S binding to G␣i [137]. The demonstration of phosphorylation sites at or near GPR domains in AGS3, AGS4, AGS5 and RGS14 suggests a common mechanism for regulation of GDI activity in this family of proteins [103,137,138]. The presence of multiple GPR motifs in AGS3, AGS4 and AGS5/LGN and the demonstration that up to four GDP–G␣i proteins can simultaneously complex with AGS3 [115,117] also suggests the possibility that GPR proteins may function as scaffolds for signaling complexes. The significance of these putative complexes in regulating G␣i function in the cell as well as their structural arrangement remains to be determined. As mentioned above, expression of a distinct splice variant of AGS3 lacking TPR domains is enriched in the heart, while full-length AGS3 is preferentially expressed in the brain [58,108]. The expression of this short cytosolic form of AGS3 is developmentally regulated, but little else is known of its function in the heart. The high levels of expression of AGS3 in the adult brain and the potential for GPR proteins to control adaptive responses to chronic heterotrimeric G-protein stimulation [139] suggests a possible function for GPR proteins in regulating behavior adaptations in the central nervous system. Indeed, AGS3 expression is upregulated in the prefrontal cortex during withdrawal from chronic cocaine administration, and studies with both a membrane permeable consensus GPR peptide and AGS3 antisense oligonucleotides indicate that AGS3 plays a key role in regulating the reinstatement of drug-seeking behavior and locomotor sensitization following cocaine withdrawal [140]. Similarly, AGS3 expressed in the nucleus accumbens positively regulates heroin seeking behavior through enhancing G␤␥ signaling [141]. Though much work remains in elucidating the role of AGS3 and other GPR proteins in these adaptive responses, in cell division and in additional GPCR-dependent and independent cellular functions, it is clear that this family of heterotrimeric GDIs plays a key role in signal regulation. 8. AGS2, AGS7, AGS8—a new family of G␤␥ interacting proteins The functional screens in yeast were designed with a bias towards the identification of G␣ activators. Nevertheless, several cDNAs have been isolated that encode proteins that appear to function in yeast and in mammalian cells by directly interacting

with G␤␥ [56,58,60] and thus form a third class of AGS proteins (Table 1). AGS2, isolated from NG108 cells, is identical to mouse Tctex-1 (DYNLT1; GenBank accession # NM 009342), a light chain component of both the cytoplasmic motor protein dynein and ciliary dynein [142]. Though the role AGS2–G␤␥ complexes may play in regulating dynein function remains unclear, AGS2/Tctex-1 has been demonstrated by multiple laboratories to interact with a variety of signaling molecules including GPCRs [143–147] suggesting dynein may organize signaling complexes in the cell to regulate events such as organelle movement. AGS7 (also called thyroid hormone interacting protein-13 or TRIP13; GenBank accession # NP 004228) was isolated from a prostate leiomyosarcoma cDNA library as a partial clone containing a putative ATP binding domain (MJC, unpublished data). TRIP13 has also been identified via yeast 2-hybrid assays as a protein interacting specifically with the non-liganded nuclear thyroid hormone ␤1 receptor [148] and a human papillomavirus type 16 E1 variant [149], though the nature of these interactions in relation to heterotrimeric G-protein signaling, if any, remains unclear. Further characterization of AGS7 as a G␤␥ binding protein will be published elsewhere (MJC, manuscript in preparation). AGS8 (GenBank accession # DQ256268) was isolated from a rat heart model of repetitive transient ischemia [60]. AGS8, as isolated from the yeast screen, encodes the C-terminal 372 amino acids of a large (1 730 amino acids), and largely uncharacterized, gene product termed KIAA1866 (in humans, FNDC1). The KIAA1866 protein sequence is conserved within mammals and encodes four fibronectin domains, three at the N-terminus and one C-terminal. The C-terminal fibronectin domain is present in the isolated AGS8 sequence and is the only obvious protein motif. Intriguingly, AGS8 expression is induced in adult ventricular cardiomyocytes specifically by hypoxia and not by other cardiac insult, and AGS8 expression is not induced by hypoxia in cardiac fibroblasts [60]. This highly restricted expression suggests a role for heterotrimeric G-protein signal modulation in myocyte-specific adaptation to hypoxic stress. Further experiments in transfected cells are consistent with AGS8 binding to G␤␥ and excluding G␣ binding without affecting the ability of G␤␥ to interact with its downstream effector phospholipase C-␤2 [60]. Though the characterization of this newly identified class of G␤␥-associated AGS proteins as signal regulators is still in its infancy, it is intriguing to note that at least some of these proteins appear capable of affecting G␣ association with G␤␥ but not effector association with G␤␥ (which is likely the reason for their isolation in the yeast screen), and that these AGS proteins appear to recognize a G␤␥ interface common to both yeast and higher eukaryotes. The former observation suggests these proteins either have G␤␥ binding sites distinct from known effector binding sites, or that these proteins facilitate transfer of G␤␥ from GDP-bound G␣ to an effector. Alternatively these proteins may function themselves as direct effectors of heterotrimeric G␤␥ or may function as binding partners for a population of G␤␥ existing independent of G␣ or GPCRs. The identification

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of novel G␤␥-specific binding partners along with the recent identification of G␤␥ ‘hot spots’ as potential coordinating sites for binding of multiple targets and regulation of multiple downstream effector signaling pathways [150–154] should dramatically improve our understanding of G␤␥-mediated signaling. The ability to effectively isolate specific G␣ and G␤␥ signal regulators from a relatively simple yeast functional screen provides a ready opportunity for further identification of important mammalian heterotrimeric G-protein signal regulators, including those that are likely disease specific [60]. The further expansion of these yeast functional screens to include other disease state-based libraries and possibly other mammalian signal targets (e.g., other mammalian G␣ or G␤␥ constructs) will greatly aid in this quest.

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Acknowledgements The author gratefully acknowledges the continuing collaboration and fruitful discussions with Drs. Stephen M. Lanier, Motohiko Sato, Joe Blumer and Amanda Struckhoff and other members of the Lanier laboratory at the LSU Health Sciences Center, the ongoing contributions of members of the author’s laboratory, and the helpful input of Dr. Emir Duzic at Cephalon, Inc. Work in the author’s laboratory has been supported in part by funding from the Ohio State Board of Regents and a subcontract from the National Institutes of Health (R01 GM074247-01A1; P.I., Stephen M. Lanier).

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