G protein specificity

Cellular Signalling 14 (2002) 407 – 418 www.elsevier.com/locate/cellsig

Review article

G protein specificity: Traffic direction required Paul R. Albert*, Liliane Robillard Ottawa Health Research Institute, Neuroscience, University of Ottawa, 451 Smyth Road, Ottawa, ON, Canada K1H-8M5 Received 15 August 2001; accepted 2 October 2001

Abstract This review focuses on the coupling specificity of the Ga and Gbg subunits of pertussis toxin (PTX)-sensitive Gi/o proteins that mediate diverse signaling pathways, including regulation of ion channels and other effectors. Several lines of evidence indicate that specific combinations of G protein alpha, beta and gamma subunits are required for different receptors or receptor – effector networks, and that a higher degree of specificity for Ga and Gbg is observed in intact systems than reported in vitro. The structural determinants of receptor – G protein specificity remain incompletely understood, and involve receptor – G protein interaction domains, and perhaps other scaffolding processes. By identifying G protein specificity for individual receptor signaling pathways, ligands targeted to disrupt individual pathways of a given receptor could be developed. D 2002 Elsevier Science Inc. All rights reserved. Keywords: G protein; Receptor; Effector; Signaling; Adenylyl cyclase; Calcium; Potassium; Channel; Conductance; cAMP; Gb; Gg; RGS; Ga; Pertussis toxin

1. G protein specificity With the sequence of the human genome nearly complete, the number of heterotrimeric G protein subunits variants identified includes 27 G-alpha, 5 G-beta, and 14 G-gamma subunits. This leads to a theoretical diversity of 27
5
14 (1890) combinations of heterotrimers. Although some combinations do not form in vitro, this represents a huge diversity. Several questions arise: First, does G protein specificity exist; and if so is it important? Several levels of G protein specificity have been described. The first level of specificity is dictated by the G-alpha subunit, which determines G protein – effector specificity. For example, the Gaq/Ga11/Ga14/Ga16 family is coupled to a subclass of receptors and ubiquitously activates all PLC-b subtypes [1]. Gas and Gai recognize AC, but at

Abbreviations: Ga, Gb, Gg, G protein alpha, beta, gamma subunits; AC, adenylyl cyclase; PLC, phospholipase C; 5-HT, serotonin; SSTR, somatostatin receptor; PTX, pertussis toxin; PKC, protein kinase C; GIRK, G-protein-regulated potassium (channel); MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; RGS, regulators of G protein signaling; PI3K, phosphatidyl inositide 30-kinase; i2, i3, second and third receptor intracellular domains * Corresponding author. Tel.: +1-613-562-5461; fax: +1-613-5625403. E-mail address: [email protected] (P.R. Albert).

different sites to mediate activation or inhibition, respectively [2]. In general, Gaq, Gai and Gas recognize different families of receptors [3], although some receptors can cross these boundaries [4]. For example the thyrotropin receptor can activate all three families of G proteins, although with differing efficacies [5] and some receptors (e.g., a2-adrenergic) couple to both Gas and Gai [6]. Receptor – G protein specificity appears to be dictated by receptor structure such that homologous receptors display similar coupling. For example, the 5-HT1 receptor family is composed of five subtypes that all couple primarily to Gi/Go proteins to inhibit AC, whereas the 5-HT2 receptors are structurally divergent and couple to Gq to stimulate PLC. This suggests that receptor structure, particularly intracellular domains of the receptor that recognize G proteins, dictates the selection of G protein subunits by distinct receptor subtypes [7]. However, the primary sequences of intracellular domains for receptors that couple to specific G proteins (e.g., Gi/Go) are highly divergent, and no ‘‘consensus’’ G protein recognition sequence can be identified. Instead, it is suggested that amphipathic alpha helices form critical receptor– G protein interaction sites, which have similar secondary structures despite their divergent sequences [8]. The recent X-ray diffraction structure of rhodopsin reveals little regarding the structure of these domains [9], and cocrystallization of receptor and G protein may be necessary to stabilize coupling domain structures.

0898-6568/01/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 8 - 6 5 6 8 ( 0 1 ) 0 0 2 5 9 - 5

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Table 1 Gai/o specificity for receptors and effectors Gi1 Receptor – G protein 1. SSTR2/IP-western/CHO 2. Cannabinoid-CB1-CT/IP 3. Cannabinoid-CB1-i3/IP 4. 5-HT1A/1B-Sf9 5. Mel1a-HEK 6. m-Opioid/GTP labeling; SH-SY5Y 7. d-Opioid/GTP labeling; SH-SY5Y 8. d- or m-Opioid/fused to G protein 9. APP-coupling; IP Ca+ + channel (iCa) 10. SSTR/+ L-iCa; GH3 11. SSTR/+ L-iCa; RINm5F/GH3 12. SSTR/+ L-iCa/ovine somatotroph 13. Dopamine-D2/5-HT1A/SSTR/Ach-M4; + L-iCa; GH4 14. Ach-M4; + L-iCa; GH3 15. Galanin/Carbachol; + iCa; RINm5F/GH3 16. Ach-M4/a2aAR; + iCa; sympathetic neuron 17. SSTR + N-iCa; sympathetic neuron 18. a2aAR + N-iCa; sympathetic neuron 19. Ad.-A1, PGE2; + N-iCa; sympathetic neuron 20. Carbachol/+ L-iCa/cardiomyocytes K+ 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

channel (iK) SSTR/* iK-IR/GH adenoma SSTR/* iK/ovine somatotroph Carbachol; * K-IR; AtT-20 Carbachol; adenosine/* iK/cardiomyocyte PAMP-20/* iK/PC-12 Adenosine-A1/GIRK a2aAR/GIRK Dopamine-D2S/GIRK Ach-M4/GIRK GABAR1a/GIRK GABAR1b/GIRK

Adenylyl cyclase (AC) 32. Dopamine-D2L/ /SSTR; + Gs-stimulated AC; GH4 33. 5-HT1A; + Gs-stimulated AC; GH4 34. Ach-M4 + Gs-stimulated AC; GH4 35. Dopamine-D2L; + F-stimulated AC; GH4 36. Dopamine-D2S; + F-stimulated AC; GH4 37. Dopamine-D2S; + F-stimulated AC; Ltk38. Dopamine-D2S; + Gs-stimulated AC; Ltk – 39. Dopamine-D2L; + F-stimulated AC; NS20Y 40. Galanin/+ F-stimulated AC/RINm5F Other 41. fMLP/* [Ca2 + ]i; HL-60 42. d-Opioid/* [Ca2 + ]i 43. Erythropoeitin; * iCa/erythroblasts 44. LPA/* c-fos/fibroblasts 45. G*-induced JNK 46. a2aAR-Ga/RGS4 47. Autophagic sequestration/HT-29 colon 48. Catecholamine uptake; PC-12

 ++ 

Gi2

Gi3

Go1

Go2

+  ++ + ++

++ ++  ++ ++ +





+ 



+ ++

± ++



     

     

     

  

++ + ++ ++

   

  

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++ ++  ++ ++ ++ +  ±  

++ ++ ++  ++ ++   

++  ++   ++  ++

 + +    

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++ ++ +



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P.R. Albert, L. Robillard / Cellular Signalling 14 (2002) 407–418

409

different receptors from the same cells are examined, they show distinct G protein specificity, arguing against this trivial explanation [10 – 12]. Nevertheless, the cell type (and possibly the G protein abundance in different cells) can influence the G protein specificity of a given receptor subtype. For example, in AtT-20 pituitary cells Gi3 mediated SSTR-induced potassium conductance, while in neurons of the locus coeruleus, Gi2 mediated the response [13]. However, differences in receptor or channel subunits could explain this result. How then can specificity be achieved between highly related G protein subunits? One answer may be that it is the heterotrimer that dictates specificity, not solely one subunit. As shown in Fig. 1, there is much greater sequence divergence between members of Gb and Gg families (30 –80% amino acid identity) than for the members of the Gai/o family ( > 80% amino acid identity). The diversity of Gbg subunits could explain how receptors might favor certain G protein combinations.

2. Receptor – G protein specificity: calcium channels

Fig. 1. Amino acid alignment of Gai/o, Gb and Gg protein sequences. The protein sequences obtained from Genbank for rat Gai/o (A), human Gb (B) and human Gg (C) are aligned by the Clustal method using the DNAStar program. Shown is the dendrogram plot for percent divergence in amino acid sequence (shown as different scales) versus protein identity. Note that Gai/o proteins have much higher percent identity (lower percent divergence) compared to Gb and Gg.

Table 1 provides a summary of some of the more recent findings regarding Ga specificity, focusing on studies that address receptor– G protein – effector specificity for multiple members of the Gai/o family. Structurally, Gai/o proteins are highly homologous particularly between Gai1– 3 (Fig. 1). While the data cited in Table 1 suggest that specificity does exist, one might argue that any receptor ‘‘specificity’’ between members of the G protein family could simply reflect the relative expression level of endogenous G proteins rather than any receptor selectivity. However, when

Following the cloning of multiple Ga and Gbg subtypes, clear evidence for additional levels of specificity beyond the Ga family came from electrophysiological studies of the Schultz lab. By nuclear microinjection of GH3 pituitary cells with antisense oligonucleotides against individual Ga, Gb and Gg subunit RNAs, they produced selective depletions of the desired subunits and examined the specificity of receptor coupling to inhibition of L-type calcium current. They demonstrated a remarkable specificity of endogenous somatostatin (SSTR2) and muscarinic-M4 receptors for Gao2b3g3 and Gao1b1g4, respectively [10,14,15] for inhibition of L-type calcium channels. In similar experiments in RINm5F insulinoma cells, the transfected SSTR2 coupled via the Gao2b1g3 combination [16] to inhibition of calcium channels, suggesting that the specificity observed in pituitary GH3 cells involves coupling to SSTR2 receptors. Importantly, subsequent studies in GH3 cells have shown that in PTX-treated cells (to inactivate endogenous Gi/o

Notes to Table 1: The relative importance of specific Gai/o proteins to the indicated receptor – G protein or G protein – effector coupling is indicated as strongest (++) to none (  ). 1. [54,108]. Note: SSTR2a and SSTR2b had opposite actions on cell proliferation; 2 – 3. [109]; 4. [110]; GTP-induced shift in agonist affinity; 5-HT1D/1E receptors showed weak shifts. 5. Brydon et al. [111]; coimmunoprecipitation in HEK cells. 6 – 7. [51]; receptor immunoprecipitation, G-protein labeling; 8. [112]; receptors were fused at C-terminal with Gai1 or Gao1. 9. [90]; GTPgS binding/GTPase activity; 10. [10]; Ga-antisense oligonucleotide injection; 11. [16]; Gai/o proteins added to PTX-treated membranes; 12. [21,22]; anti-Ga or Ga-antisense oligonucleotides. 13. [11,20]; stable expression of antisense Ga; * either or both Gao1 and Gao2 implicated. 14. [10]; Ga-antisense oligonucleotide injection. 15. [16]; Ga proteins added to PTX-treated membranes. 16. [25 – 27]; Ga-antibody or Ga-antisense oligonucleotides. 17 – 19. [33]; PTX-insensitive Ga + Gbg combinations. 20. [113]; cardiomyocytes from Gai2  /  and Gai3  /  were compared to +/+. 21. [13]; anti-Ga antibodies. 22. [21,22]; anti-Ga antibodies and Ga-antisense oligonucleotides. 23. [43]; Cys/Ser PTXinsensitive Ga. 24. [42]; cardiomyocytes from Gai2  /  , Gai3  /  , and Gao  /  mice were compared to +/+. 25. [41]; anti-Ga antibodies. 26 – 31. [44]; HEK cells were stably transfected with Kir3.1/3.2; transiently with receptors and PTX-insensitive C351G Gai/o. 32. [11]; stable expression of Ga-antisense cDNA. 33 – 34. [20]; stable expression of Ga-antisense cDNA. 35 – 36. [49]; transfection of PTX-insensitive Ga mutants. 37 – 38. [47]; transfection of PTXinsensitive Ga mutants. 39. [48]; transfection of PTX-insensitive Ga mutants. 40. [18]; anti-Ga antibodies; Ga-antisense oligonucleotides. 41. [89]; transient expression of Ga-antisense constructs. 42. [52]; Ga-antisense oligonucleotides; ND8 – 47 neuroblastoma cells. 43. [114]; microinjection of Ga protein or antiGa antibody. 44. [115]; anti-Ga antibody; PTX-insensitive Ga mutants. 45. [116]; constitutively active Ga mutant coupling to JNK. 46. [96]; a2aAR-Ga fusion constructs; GTPase activity ± RGS4 in Cos7 cells. 47. [117]; Ga-antisense oligonucleotides, constitutively active Gai3 mutant. 48. [118]; anti-Gao, addition of Ga proteins in vitro.

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proteins) Gao2 could reconstitute SSTR coupling, while Gao1 reconstituted M4 response to inhibit L-type channels [12]. While this level of specificity has rarely been reported, this group has also documented the G protein specificity of galanin receptor coupling to N-type calcium channels in rat insulinoma RINm5F cells for Gao1b2g2 > Gao1b3g4 [17]. By contrast, Gai3 was essential for galanin-induced inhibition of forskolin-stimulated AC in RINm5F cells, but not Gao or Gai1/2 [18]. It is important to note that the antisense strategy, which depletes endogenous G protein subunits, often reveals a higher level of specificity than biochemical approaches. For example, when GH3 or RINm5F cell membranes were prepared, each of these receptors (M4, SSTR, galanin) demonstrated coupling to both Gao1 and Gao2 as detected by [a-32P]GTP azidoanilide labeling [12]. The observation that G protein specificity is greater in intact cells than in membrane preparations suggests that preformed receptor– G protein complexes may exist in intact cells and are disrupted upon cell lysis [8,19]. The electrophysiological experiments also pointed out the importance of Go in receptor coupling to calcium channels. Using stable transfection of antisense Gai/o constructs, we found a predominant role of Go in coupling SSTR, M4, dopamine-D2 (short (D2S) and long (D2L) variants) and 5-HT1A receptors to dihydropyridine-sensitive (L-type) calcium channels in pituitary GH4C1 cells [11,20]. In ovine pituitary cells, Gao2 mediates SSTR coupling to calcium channels [21,22]. Furthermore, in dorsal root ganglia (DRG) neurons the Gao1 subtype mediates coupling of GABA-B receptors to inhibition of the L-type calcium channels [23,24]. In sympathetic neurons, M4 muscarinic and a2-adrenergic receptor regulation of N-type calcium channels has been shown by anti-Ga antibody or Ga-antisense experiments to require Gao1 [25 – 27], as observed for M4 actions on L-type calcium channels in GH3 cells. Similarly in NG108-15 cells, d-opioid and a2adrenergic coupling to omega-conotoxin-sensitive currents were reconstituted with PTX-insensitive Gao1, but not SSTR-mediated action [28]. Despite the crucial role for the Gao subunit to connect receptor and N-type calcium channel, it is the Gbg dimer that interacts directly with the N-type channel (a1B) subunit to inhibit channel opening [29 – 31]. The mechanism by which Go regulates L-type channels is unclear since both channel a1C and a1D subunits fail to bind Gbg [32]. Perhaps a heteromeric L-type channel comprising at least one a1B subunit would be sensitive to Gbg. In summary, Go plays a crucial role in coupling multiple receptors to both L- and N-type calcium channels, although it appears to be the Gbg subunits that actually transduce the receptor signal. Recently, Jeong and Ikeda have expressed multiple combinations of PTX-insensitive Gai/o and Gbg subunits in sympathetic neurons to examine receptor coupling to Ntype calcium channels [33]. Upon pretreatment with PTX to inactivate endogenous G proteins, PTX-resistant Gi1 and Gi3 failed to reconstitute channel coupling, whereas both

Gi2 and Go were active. The order of efficiency was receptor-dependent: Go2 = Gi2 > Go1 for SSTR (similar to the Go2 selectivity of antisense experiments) and Go > Gi2 for a2-adrenergic receptor. These studies further confirm results from antisense studies that although Gbg subunits mediate coupling to N-type channels, the Ga subtype plays a determining role in coupling to the channels. In contrast to the antisense experiments, no dependence on particular Gbg combinations was identified and all Gb1 –4/Gg combinations tested coupled. The observed lack of specificity for Gbg could reflect elevated expression of Gbg upon cotransfection with Ga. For example, the selectivity in vitro of Gbg combinations that couple Gi1 to the adenosine-A1 receptor can be overcome at high enough Gbg concentrations [34]. On the other hand, no receptor coupling to channels was observed upon transfection of Ga alone without Gbg [33]. Conversely, Gai1 inhibited a2-adrenergic coupling to the channel and cotransfection of a stoichiometric amount of Gbg subunits was necessary to permit PTX-insensitive coupling. Thus, the exogenous PTX-insensitive Ga proteins did not appear to exchange and combine with endogenous Gbg –receptor complex to render it PTX-insensitive, except after receptor activation. Similarly, transfections of Gbg subunits did not exchange with endogenous receptor– Gbg complexes in this system [35]. This lack of exchange of transfected Gbg with endogenous Gbg – receptor complexes could explain the reduced receptor – Gbg selectivity compared to antisense depletion. Thus, although multiple Gbg have the potential to couple to the N-type channels, the endogenous receptor– G protein-channel coupling is somehow restricted to a preferred combination of Gabg, as observed in antisense experiments.

3. Receptor – G protein specificity: potassium channels Positive regulation of G protein-activated inwardly rectifying potassium channels (GIRK) occurs via direct interaction of Gbg subunits with channel subunits [36,37]. A variety of experimental approaches in various systems have indicated that G protein specificity for coupling of multiple receptors to GIRK channels often involves Gi3 (Table 1). Antisense or antibody experiments in pituitary cells indicate that both dopamine-D2 and SSTR couple to Gi3 to increase potassium conductance [13,22,38 –40]. Injection of anti-Ga antibodies to block coupling indicates that proadrenomedullin N-terminal 20 peptide (PANP-20) mediates potassium conductance in PC-12 pheocytochroma cells via Gi3 [41]. Using cardiomyocytes derived from Gai2  /  , Gai3  / or Gao  /  mice, it was found that muscarinic regulation of potassium conductance utilizes both Gi2 and Gi3, but not Go [42]. In AtT-20 corticotroph cells, muscarinic activation of GIRK was reconstituted with PTX-insensitive Gi2 only, while the SSTR response was not reconstituted with PTXinsensitive Gi proteins [43]. More recently, the coupling of multiple receptors to PTX-insensitive Ga proteins was

P.R. Albert, L. Robillard / Cellular Signalling 14 (2002) 407–418

analyzed in PTX-treated HEK-293 cells transfected with GIRK subunits (Kir3.1 + 3.2A) [44]. Cotransfection of Gbg was not necessary to observe coupling to GIRK, in contrast to receptor coupling to N-type calcium channels in neurons [33]. This suggests that the transfected Ga subunits could exchange with endogenous Gbg subunits in HEK-293 cells to render coupling to GIRK insensitive to PTX. Some receptors were Gi selective (e.g., dopamine-D2S, GABAB) but others (e.g., adenosine-A1, a2-adrenergic) displayed little Gi selectivity (see Table 1). While Gai2, Gai3 and Gao were active, Gai1 was the weakest for coupling to GIRK. This is consistent with the reported inhibition of Gbg – GIRK coupling in GIRK1-expressing Xenopus oocytes or cardiomyocyte membranes by exogenous GTPgS-activated Gai1 but not Gai2 or Gai3 [45]. In GIRK-transfected HEK293 cells, the dopamine-D2S receptor coupled exclusively via Go1, in contrast to Gi3 in pituitary cells. Discrepancies between these results may be explained by expression of different GIRK subunits in pituitary cells compared to the defined channels expressed in transfected HEK-293 cells. However, as observed for N-type calcium channels [33], the identity of the Ga subunit appears to dictate Gbg coupling to GIRK channels.

4. Receptor – G protein specificity: adenylyl cyclase Other studies have addressed the specificity of receptor – G protein coupling to specific effectors using antisense approaches (discussed above) or the introduction of PTXinsensitive G protein mutants. As summarized in Table 1, these data argue for specific roles of particular Ga subunits in the coupling of diverse receptors to specific effectors. For example, Go couples multiple receptors to inhibition of calcium channels. In cases where multiple Gai subtypes couple receptors to a response (e.g., inhibition of AC), each receptor appears to have a different pattern of preferences for Gai1, Gai2, or Gai3. For inhibition of Gs-stimulated AC in GH4 cells, the muscarinic-M4, dopamine-D2L and somatostatin receptors utilize Gai2 primarily, while dopamineD2S receptor utilized a Gai2-independent pathway [11,20]. However, a precise G protein specificity was difficult to define for coupling to AC. For example, antisense to more than one Gai inhibited coupling to AC for muscarinic-M4 and 5-HT1A receptors [20], suggesting that coordinate inhibition of a diversity of AC subtypes by different Gi proteins is required to block Gs-coupled cAMP responses. Defining G protein specificity in coupling to AC is complicated by the presence of multiple subtypes of AC [46,47]. Different AC subtypes may display distinct preferences for G proteins, and this could lead to differences in apparent receptor– G protein – AC specificity depending on the AC subtypes present. For example, in GH4 cells the dopamine-D2L receptor utilized Gai2 to inhibit basal and Gs-stimulated cAMP; on the other hand, Go couples the receptor to inhibition of calcium channel activation but not

411

to regulation of cAMP levels. In cortical neurons and NS20Y neuroblastoma cells the D2L receptor inhibited forskolin-stimulated adenylyl cyclase via Go [48]. Such differences in G protein specificity in various cell types could reflect expression of different AC subtypes with distinct G protein recognition. Differences in Gai selectivity may also result from inhibition of basal, Gs- or forskolinstimulated AC by distinct Gai proteins. Using PTX-insensitive Gi proteins, Senogles found that the D2S receptor utilized Gai2, while the D2L receptor used Gai3 to couple to inhibition of forskolin-stimulated AC in GH4 pituitary cells [49], whereas we found that Gai2 was not involved in D2S inhibition of Gs-stimulated AC. In Ltk-fibroblasts, the D2S receptor utilized Gai2 to inhibit forskolin-stimulated AC, but Gai3 to inhibit Gs-stimulated AC [47]. The different Gi specificity in coupling to Gs- or forskolininduced AC in the same cell type could reflect statedependent regulation of AC or distinct activation of AC subtypes with different Gi selectivity. An additional complicating factor is Gbg-mediated stimulation of ACII and ACIV for Gi-induced increase in cAMP formation. In GH4 pituitary cells, antisense depletion of Gai1 resulted in a paradoxical stimulation of AC by 5-HT1A activation, whereas depletion of Gai3 resulted in stimulation of AC by M4-muscarinic receptors [20]. Gi-induced stimulation of AC was presumably mediated by ACII that is expressed in these cells and is activated by the 5-HT1A receptor [46]. In summary, the diversity of AC subtypes coexpressed in cells provides a diversity of regulation that is highly receptor- and cell-type-dependent.

5. Receptor – G protein association/activation studies Several studies have demonstrated that receptors and G protein subunits remain associated following receptor activation, such as the association of b2-adrenergic or muscarinic-M2 receptors with Ga subunits [50]. Two Gi/ocoupled receptor subtypes that have been extensively studied with regard to receptor– G protein association are the opioid and somatostatin receptors. 5.1. Opioid receptors Specific recognition of G proteins by receptors has been measured by reconstitution of receptors with G proteins (coupling assessed by measuring agonist-induced GTPgS binding or GTPase activity). In human SHSY5Y neuroblastoma cell membranes incubated with [a-32P]GTP azidoanilide, d- versus m-opioid agonists preferentially labeled Gai1 or Gai3, respectively, but not Gai2 [51]. Although Gai2 associated more weakly with the d-opioid receptor [51], Gi2 is utilized for d-opioid-mediated calcium mobilization in ND8 –47 neuroblastoma
dorsal root ganglion cells, which have undetectable levels of Gai1 [52]. Thus, the G proteins that associate biochemically with a receptor

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may not reflect the G protein specificity of coupling to specific effectors. One possible explanation for discrepancy between the G proteins associated with receptor and those required for coupling is a ‘‘reshuffling’’ of the G proteins associated with a receptor upon activation. Using receptor immunoprecipitation followed by Western blot for G protein subunits, the solubilized d-opioid receptor was shown to associate with Gai1, Gai3, Gao and Gb1/2 in the inactive state, but with Gai2, Gai3 and Gb in the presence of added agonist [53]. Thus, Gai2 association was detected only for the activated receptor, while Gao was associated only with the inactive receptor. 5.2. Somatostatin receptors The SSTR –G protein association was studied in Chinese hamster ovary (CHO) cells using an antibody specific for SSTR2A to immunoprecipitate the somatostatin-bound receptor. In the presence of agonist, G proteins remained associated with the receptor and Gai1– 3 were detected, but not Gao [54]. The G proteins that remained associated with the somatostatin receptor did not match the G protein abundance in CHO cells (Gai3 and Gao are most abundant [55]). Similarly, affinity purification of SSTR from GH4C1 cells resulted in copurification of Gai2 and Gai3, with no detectable Gai1 or Gao [56,57], despite the fact that Gai2 and Gao are the most abundant subtypes in these cells [20,57]. Furthermore, SSTR-induced inhibition of L-type calcium channels is dependent on Go in these cells [11] and in the parental line, GH3 [10]. Using anti-Ga antibodies to disrupt SSTR coupling in CHO cell membranes [55], SSTR2 was shown to couple to Go activation, whereas Gao protein was not detected in immunopurified receptor [54]. These studies indicate that the SSTR recognizes G proteins based not on their abundance but on agonist-induced receptor– G protein interactions. They also suggest that some interactions were not detected following agonist treatment and/or receptor purification, such as the receptor –Gao interaction that mediates functional coupling of SSTR to calcium channels. As observed for SSTR, upon reconstitution with the adenosine-A1 receptor, Gai1 –3 associated with the receptor but not Gao [58] even though Go couples this receptor to calcium channels [33]. It remains unclear why A1 receptor interaction with Gao was undetectable, but agonist was present in the assay and Gao may be released under this condition as observed for the d-opioid receptor [53]. The mechanism by which G protein association with the receptor is regulated and altered in intact cells is unclear. Upon purification, there may be disruption of scaffolding interactions that stabilize the receptor – Gao interaction; alternately, ligand binding may recruit scaffolding proteins that displace Gao. Who might these mystery traffic directors be? This question remains unanswered but candidates do exist. For example, RGS proteins that function not only as GTPase activators may also direct G protein specificity in the kinetics of coupling to calcium channels [59]. Unknown

proteins may be identified that mediate this function [58]. A variety of scaffolding (PSD-95, InaD), actin-binding (filamin, spinophillin), calcium-binding (calmodulin) and chaperone (14-3-3) proteins interact with G-protein-coupled receptors in a G-protein-independent manner [60 –62], but may also contribute to G protein specificity in intact cells as discussed in the conclusion.

6. GB;– effector specificity: calcium channels The Gbg subunit has been implicated in coupling to a large diversity of effectors including ACII and ACIV, PLCb2/b3, and by direct interaction with potassium and calcium channels [63]. Overlapping domains of the Gb subunit that mediate coupling to these effectors have been extensively mapped using point mutagenesis [64]. The specificity of Gbg combinations for effector coupling has been examined most intensively for coupling to calcium channels and divergent specificity has been obtained depending on how the experiment was done (Table 2). Using nuclear injections of rat superior cervical ganglion cells with various combinations of Gb1– 5g3 cDNAs, a Gb1/2g3 preference was identified for coupling to N-type calcium current to enhance prepulse facilitation following a2-adrenergic receptor stimulation, while Gb3– 5g3 were weakly coupled [65]. Furthermore, it was shown by yeast two hybrid that Gb3 – 5 interacted poorly with the crucial I– II domain of the Ntype channel a1b subunit [65]. However, using the same microinjection approach, Ruiz-Velasco and Ikeda showed that all Gb1 – 4/g1– 3 combinations could inhibit N-type calcium currents [35]. In both cases, Gb5 was inactive, presumably reflecting specific coupling of Gb5 to Gaq subtype [66]. In these experiments, the level of Gbg expression relative to endogenous levels was not assessed. Thus, differences in specificity could be explained by lowlevel expression that could lead to insufficient Gbg for coupling. The transfected Gbg subunits did not appear to displace endogenous Gbg subunits to mediate activation. This was tested using Gb mutants that could not couple GIRK channels but did couple to calcium channels. This effector specificity was retained upon transfection [35], suggesting a lack of exchange between exogenous Gb mutants and fully active endogenous Gb subunits. Thus, the observed lack of Gbg –calcium channel coupling specificity is not due to exchange/mobilization of random combinations of endogenous Gbg. The specificity (or lack thereof) observed for Gbg coupling to N-type calcium channels in SCG neurons could reflect solely Gbg –effector specificity, rather than receptor– Ga/Gbg –effector specificity, which may be of higher order. Analogous experiments have been done in HEK-293 cells stably transfected with specific combinations of calcium channel subunits to examine direct Gbg modulation of a ‘‘pure’’ calcium channel type [67]. The C2D7 clone stably expressing a1b, a2d and b1 – 3 channel subunits was

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413

Table 2 Gbg specificity in coupling to receptors and effectors b1

b2

b3

b4

b5

++ ++

++ ++

 ++

 ++

 

g1

g2

g3

g4

*

* *

*

g5

g6

g7

g8

g9

g10

g11

g12

++

Ca channels 1. RatSCG/(N)-VDCC 2. RatSCG/(N)-VDCC 3. SCG/M4/VDCC 4. SCG/SS/a2AR/VDCC 5. HEK-a1BVDCC (N) 6. HEK-a1AVDCC-PQ 7. HEK-a1BVDCC (N) 8. HEK-a1BVDCC (N) 9. HEK-a1BVDCC (N) * 10. DRG-(L)-VDCC K + channels 11. GIRK—in vitro 12. Xenopus/Kir3.2-b1AR 13. Xenopus/Kir3.2-b2AR PLC/P130K 14. PLC-b1/b2—in vitro 15. PLC-b3—in vitro 16. DRG-PLCb-Gai2 17. Cos7-MAPK/JNK 18. P13K-p85—in vitro 19. Cos7-P13K/Akt Receptors 20. Sf9-a2A/Gai1 21. Sf9-a2A/Gai1 22. HEK-b2AR-as 23. b1AR/A2a 24. Stimulated ACII/inhibited ACI 25. M2/Gao 26. M2/Gao 27. V6421 APP-apoptosis 28. GRK2-CT/in vitro 29. F-actin interaction

++  ++ ± ++

± ± ++

++ + ++

+ ++ ++

  ++

++

++  ++

++ ++ ++ ++ ++

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++

++

++ 



++

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++ 

++ 

++

*  +

* * ++ * * *

  



*

++

 + ++

++ ++ *

+± +±

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* ++ ++

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++

++ *

++

* ++ +± ++  *  ++



++

++   ++ ++ ++

* * ++ ++ *



++ ++

*

+ ++

++

+

++ ++

* * * * * * 

+

++





 ++

The relative importance of specific Gbg combinations to the indicated receptor – G protein or G protein – effector coupling are indicated as strongest (++) to none (  ). Opposite or negative regulation is indicated as ( ). An asterisk (*) is placed where the indicated subunit was used in multiple combinations. 1. [65]. 2. [33,35]. 3. [71]; Gg5-ger-ger peptide inhibition of M4 receptor coupling to inhibit Ca channel. 4. [71]; Gg5-ger-ger peptide on somatostatin or a2-adrenergic receptor coupling to inhibit Ca channel; 5 – 6. [68]; HEK-tsa201 clone (stably transfected with channel a1A/B/a2d/b1b) transfected with Gbg. 7 – 8. [67]; HEKC2D7 clone (stably transfected with channel a1b/a2d/b1 – 3) transfected with Gbg. 9. [69,119]; * PKC sensitivity; phosphorylation by PKC of aa-422 in the channel a1b I – II linker specifically uncoupled Gb1 from the channel; syntaxin recruits Gbg. 10. [83]. 11. [73]; * combined with the indicated Gb. 12. [74]; microinjection of Xenopus oocytes with receptor, Kir3.2, and Gas cDNAs. 13. [74]. Note: most active combinations were Gb1g7, Gb1g11 and Gb5g2. 14 – 15. [78]; in vitro. 16. [83]. 17. [79]; * combined with indicated Gb. 18. [78]; in vitro. 19. [81]; cotransfection of Gbg; inhibited by Gaq-Q209L 20 – 21. [84]; GTPgS-sensitive binding. 22. [105]; antisense depletion using anti-Gg7-ribozyme. 23. [75]; receptor reconstitution with Gs. 24. [75]; AC reconstitution with Gbg; [87] in vitro reconstitution of M2 with Gao/Gbg. 25 – 26. [86]; in vitro reconstitution of M2 with Gao/Gbg. 27. [91]; Gb2g2 transfected with V6421 App induced apoptosis in Cos7 cells. 28. [120]; GRK3-CT bound Gb1, Gb2 and Gb3. 29. [93].

used, and direct prepulse-sensitive inhibition of the channels by Gb1– 5g1 –3 combinations was examined. All Gbg combinations were effective except Gb5g2, which did inhibit the channel. The Gg subtype influenced inhibitory coupling to calcium channels, as Gg1 was the weakest. Northern blot analysis demonstrated that Gb subunit RNA was greatly overexpressed compared to endogenous RNA. In HEK cells transiently cotransfected with Gb1 – 5g2 combinations and calcium channel subunits for either Ntype (a1b, a2-d, b1b) or P/Q-type (a1a, a2-d, b1b) channels, Arnot et al. found a greater G protein specificity [68]. In

this case, Gb1g2 and Gb3g2 exhibited the greatest prepulse facilitation of N-type channels, but Gb4g2 was most effective at P/Q-type channels. This higher specificity may be due to the use of a single (b1b) channel b-subunit rather than multiple subunits (b1 –3). The channel b-subunit competes with Gbg binding at the I –II domain of the channel a1 subunit. Interactions between the channel bsubunit, PKC, and Gbg at the channel a1 subunit I– II domain may be crucial to determine Gbg – effector specificity. For example, PKC phosphorylation of the I – II domain inhibits coupling of Gb1g2 to N-type channels

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transfected in HEK-293 cells, but does not affect coupling of Gb2 – 4g2 [69]. In addition, syntaxin-1A enhances Gbg signaling to N-type calcium channels, and could also be a determinant of Gbg –effector specificity [70]. Another approach to determine receptor –Gbg –calcium channel specificity in vivo has been the use of Ggspecific blocking peptides [71] in SCG neurons. Peptides for Gg5 specifically block muscarinic M4 coupling to Ntype currents, but not somatostatin or a2-adrenergic receptor coupling. In addition, Gg7 and Gg12 peptides were poor inhibitors of M4 coupling to calcium currents. This supports the specificity identified in antisense experiments [15], and suggests that the highest level of specificity is observed when intact receptor– Gbg– effector systems are examined.

7. GB; – effector specificity: GIRK The interaction of Gb1 with Gg2 is crucial for activation of GIRK-1/2 in transfected HEK-293 cells [72], suggesting that Gbg specificity may exist for signaling to GIRK. The Gbg specificity of coupling to GIRK channels has been studied in HEK-293 cells stably transfected with GIRK1 and GIRK4 expression plasmids [73]. All Gb1 –4g2 or g11 combinations were effective to activate the GIRK-1/4 channel, and were also effective at GIRK-1/2 (neuronal channel). Interestingly, Gb5g2 or g11 combinations inhibited basal, Gb1g2 and 5-HT1A receptor-mediated activation of GIRK-1/4. It was suggested that Gb5 mediates actions of Gq-coupled receptors to inhibit the GIRK current, since Gb5 is associated with Gq [66]. In transfected HEK cells, there was little discrimination between Gb1 –4 in the regulation of GIRK channels, as observed above for Gbg –calcium channel specificity. A higher level of Gbg –channel specificity was observed for receptor-mediated coupling to Kir3.2. Gbg-mediated coupling of the b1- and b2-adrenergic to GIRK (Kir3.2) was examined in Xenopus oocytes injected with RNA encoding b-adrenergic receptor, Gas, Kir3.2 channel and various Gbg combinations [74]. For the b1-adrenergic receptor, Gb5/g1 or g11 were the most effective combinations while Gb1/g combinations were inactive. The lack of activity of Gb1g2 was not because of inability of this Gbg dimer to interact with the b1-adrenergic receptor [75]. For the b2-adrenergic receptor it was Gb1/g7 or g11, and Gb5/ g2. This specificity was not observed for Gbg binding to GST-Kir1 in vitro [74]. Interestingly, the Gb1/g7 combination appears to be a preferred endogenous combination of the b2-adrenergic receptor in HEK-293 cells. Ribozymemediated depletion of Gg7 results in a specific loss of only Gb1, and blocks b2-adrenergic (but not prostaglandin E1) coupling to adenylyl cyclase [76,77]. These experiments in Xenopus oocytes and HEK-293 cells suggest that the same Gb1g7 dimer is preferentially utilized in conjunction by the b2-adrenergic receptor to activate potassium channels or

AC. This high level of specificity appears to reflect receptor – Gbg – effector specificity, which is greater than the Gbg– effector specificity examined in the above experiments addressing Gbg –effector coupling only.

8. GB;– effector specificity: enzymes It has been difficult to demonstrate Gbg specificity in vitro for a variety of enzymes, such as PLC-b2, PLC-b3, or AC. Recently, some specificity has been detected in vitro, particularly for the Gb5 subunit. For example, Gb5g2 can activate PLC-b1 or -b2, but not PLC-b3, PI3K [78] or MAPK/JNK [79], whereas Gb1g2 was effective for all pathways. Interestingly, distinct structural determinants at the C-terminal of Gb1 are required for it to activate MAPK versus JNK pathways [80]. In transfection experiments in Cos7 cells, Gb5g2 failed to induce PI3K, whereas Gb1/2g2 pairs were effective [81]. The Gb5g2 pair inhibited ACII, whereas the Gb1g2 and Gbg subunits purified from tissue stimulated ACII [82]. Recently, Gb1– 5g2 specificity in coupling to ACI or ACII in Sf9 insect cells was examined. All combinations activated ACII and inhibited ACI (Gb3g2 was weaker) except Gb5g2 [75]. Thus, the Gb5 subunit, compared to other Gb subunits, has an atypical signaling phenotype that can be detected in vitro. This may reflect the relatively low structural homology (50% amino acid identity) of this subunit compared to Gb1– 4. Recent work from Diverse-Pierluissi and collaborators is consistent with a greater level of Gbg– effector specificity in vivo. Injection of Gbg subunits in sensory neurons inhibits calcium channels indirectly via a PLC-b/PKC pathway [83]. Recombinant Gb1g2, b3g2, b5g2 but not Gb2-containing dimers activated PLC-b in injected neurons, but in vitro both Gb1g2 and Gb2g2 were equally effective to activate PLC-b, although at a 30-fold higher concentration than in vivo. These experiments indicate that a higher order of Gbg – effector specificity is present in intact cells, but that at high concentrations of Gbg this specificity may be lost.

9. GB; specificity: receptors The level of receptor– Gbg specificity has recently been examined in vitro for a2a-adrenergic/Gai1 complex in Sf9 insect cells transfected with various Gbg combinations [84]. Differences between Gb1 and Gb3 in combination with various Gg subunits to promote high-affinity binding were noted, but most combinations were at least partially effective except Gb1g1 and Gb5 dimers. Upon expression in Sf9 cells of mammalian 5-HT1A receptor/Gai1 and Gbg combinations, Gb1g1 was also weak to promote high-affinity binding, whereas other Gb1g combinations were effective [85]. Further evidence of specific recognition of Gbg dimers by receptors has been obtained in vitro [86,87].

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Muscarinic-M2 receptor/Gao interacted with Gb4g2 but not Gb1g2, whereas Gb1g5 and especially Gb1g7 interacted effectively with this receptor/Gao combination. In these experiments, no preference was observed in coupling of Gbg dimers to ACII, PLC-b2, or PLC-b3 as described above. Thus, there is evidence in vitro of preferential receptor– Gbg recognition.

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associates with F-actin and mediates action of PKC on cellular motility via phosphorylation of an N-terminal Ser2 [93]. Other Gg subunits lack interaction with F-actin and did not influence cellular motility.

11. Conclusion: traffic direction needed 11.1. Receptors as traffic directors

10. G protein specificity: biological readouts Most studies of G protein specificity have focused on immediate coupling of receptors to effectors such as ion channels or AC. In some cases, a high level of receptor– Ga– effector specificity in signaling to downstream biological responses has been identified in intact preparations using either antisense approaches or transfection of PTXinsensitive G proteins. Activation of the dopamine-D2S receptor expressed in Balb/c-3T3 fibroblasts stimulates p42/44 MAPK phosphorylation, cell proliferation, and cellular transformation, actions that are all blocked by PTX pretreatment [88]. Using cells stably transfected with PTX-insensitive point mutants of Gai1– 3 or Gao1, we showed that while Gai2 mediates MAPK phosphorylation and DNA synthesis, only Gai3 mediated foci formation but not MAPK activation or DNA synthesis. This suggests that in intact cells G protein specificity can be transmitted from receptor to effector and ultimately affect the biological outcome of receptor signaling. By stable transfection of antisense Gao, fMLP and leukotrieneB4-induced calcium mobilization was blocked by 70% in HL-60 leukocytes, whereas the combination of antisense Gai1 – 3 had no effect [89]. In Gai2 antisense HL-60 clones, agonistinduced inhibition of forskolin-stimulated cAMP was blocked, but not affected in antisense Gao clones. In antisense Gao clones, downstream events such as secretion of b-glucuronidase and chemotaxis were also reduced, but not completely blocked. Thus, G protein specificity at the level of second messengers can confer specificity in biological readouts. There is also evidence for Gbg specificity in coupling to downstream events such as apoptosis. Okamoto and colleagues have identified coupling between V642I APP and Gao2 to mediate cell death in NK1-Cos cells [90]. They have further demonstrated that while constitutively active Gao2 did not induce apoptosis, block of Gbg signaling reduced cell death suggesting a role for Gbg [91]. They demonstrated that Gb2g2 was the only combination that when cotransfected induced cell death, whereas other Gb1 – 3g2 –3 combinations did not. In normal Cos7 cells, Gb2g2 activates JNK, but does not result in cell death [92]. The signaling pathway leading to Gb2g2-induced apoptosis in NK1-Cos cells remains to be elucidated. Nevertheless, these results suggest a role for specific Gbg combinations in mediating specific biological responses. Another example of Gbg specificity is provided by Gg12, a subunit that

A large number of studies have addressed receptor –G protein or G protein –effector specificity in vitro. In some cases, specific receptor –G protein associations have been identified. However, results in vitro do not always correspond to the functional coupling observed in vivo. Thus, Go mediates coupling of multiple receptors to calcium channels, yet Go did not copurify with affinity- or immunopurified receptors (e.g., somatostatin receptors). A greater receptor – G protein – effector specificity is often observed in vivo than can be detected by in vitro assays. A general conclusion from the above examples is that although studies in vitro display some G protein specificity, there is a higher level of G protein specificity in vivo. This suggests the presence of an organized network of receptor– G protein – effector interactions, at least in cases where a high level of specificity is observed. This could be mediated by specific networks of receptor– G protein – effector, as has been suggested to explain the rapidity and specificity of receptor coupling to ion channels [19]. The formation of receptor dimers may favor specific associations between G proteins in the receptor –G protein complex [94,95]. 11.2. G protein regulators as traffic directors Cofactors may determine in part receptor – G protein specificity. For example, the Gg-like (GGL)-domain RGS proteins interacts specifically with Gb5, and blocked coupling of the Gb5g2 dimer to N-type calcium channels, illustrating a potential role for RGS proteins in modifying Gbg specificity [67]. RGS4 selectively activated GTPase activity of a2A-Gao1 fusion over a2A-Gai2 fusion protein [96]. Although no difference in intrinsic specificity of RGS4 for Gao versus Gai2 was observed, RGS7 shows a marked intrinsic preference for GTPase activation of Gao over Gai2 [97]. In chick dorsal root ganglion neurons, GAIP and RGS4 regulate a2-adrenergic receptor coupling via Go and Gi, respectively, to N-type calcium channels [59]. These results suggest that RGS proteins may also modulate receptor – Ga activation specificity. The finding that syntaxin-1A can enhance Gbg coupling to N-type calcium channels, suggests that it could also influence Gbg selectivity [70]. Finally, novel as yet unidentified proteins may dictate G protein specificity. For example, a cofactor that confers specific G protein association to the A1-adenosine receptor has been suggested [58]. These examples serve to illustrate

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that some traffic direction appears to be required to generate G protein specificity. 11.3. Scaffolding proteins as traffic directors The stability and specificity of a receptor– G protein – effector complex could be enhanced by the presence of a scaffolding protein. The demonstration that such a scaffolding protein can be indispensable for proper signaling is well illustrated by the InaD protein, a polypeptide containing five PDZ domains that regulates visual signaling of Drosophila photoreceptors [98]. Through specific PDZ domains InaD assembles a calcium channel (TRP), PLC-b (the effector for rhodopsin/Gq) and PKC (inhibitory regulator) into a ‘‘transductosome’’ protein complex. This permits rapid activation of TRP by PLC activation by rhodopsin, and deactivation of TRP through phosphorylation by PKC to terminate signaling. Disruption of InaD results in abnormal signaling presumably due to abnormal subcellular localization of TRP [98,99]. In addition, TRP has a GTPase activating function that is crucial to inactivate Gq, leading to rapid termination of the light response [100]. Conceivably, the assembly of receptor, G protein and effectors by scaffolding proteins could lead to specificity of G protein signaling. Recently, several G-protein-coupled receptors have been shown to directly interact with scaffolding or signaling proteins as illustrated figuratively in Fig. 2. For example, the C-terminal end of the b1-adrenergic receptor interacts with PSD-95, a PDZ-domain protein that binds to NMDA receptors [101]. In some cases, interactions with scaffolding proteins enhance signaling of the receptor. The interaction of the actin-binding protein filamin with the D2-dopamine receptor i3 domain is crucial to direct membrane localization of the receptor, and functional coupling to inhibition of AC [102,103]. Interestingly, the filamin-D2 interaction is inhib-

Fig. 2. Traffic jam at the receptor. A variety of proteins that have been shown to interact directly with G-protein-coupled receptors (GPCR) are shown figuratively. Note that not all of these proteins interact at once with the same receptor, and several proteins such as calmodulin (CAM) may compete with G proteins.

ited by PKC activation [103]. Filamin interacts with the C-terminal domain of the calcium-sensing receptor, another Gi-coupled receptor, to direct signaling to MAPK activation [104]. Oppositely, interactions with scaffolding proteins may inhibit receptor signaling. Calmodulin interacts with the i3 domains of the m-opioid [105] and dopamine-D2 [106] receptors and inhibits their coupling to G protein activation. The specificity of this action for Ga or Gbg subunits is not known but could provide a mechanism to select for preferred (high affinity) receptor –G protein interactions. Some interactions are modified by agonist stimulation, such as the interaction of spinophilin with the a2-adrenergic receptor [107], and might explain the shuffling of receptor-associated G protein subunits that occurs upon agonist activation. In short, much more remains to be understood regarding the protein interactions that may mediate efficient and receptor selective signaling in intact cells.

Acknowledgments PAR is Novartis/CIHR Michael Smith Chair in Neuroscience. This study was supported by grants from the Canadian Institutes of Health Research, Ontario Mental Health Foundation, Parkinson Society of Canada and National Cancer Institute of Canada.

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