------------------------------------------------------------------------------ receptor-heterotrimer specificity Galpha oA = o1, oB = o2 ------------------------------------------------------------------------------ adenosine A1R bovine coupling (GTPgammaS loading, agonist binding) ...in Sf9 membranes - with alphai1 or alphai2, beta1: gamma2 gamma1(S74L) > gamma1 gamma2(L71S) CAAX box X mutants change prenyl, L geranylgeranyl S farnesyl) - the four beta-gammas reconstitute into membrane equally well Yasuda et al. JBC 271(31):18588 (1996) ...in phospholipid vesicles - with alphai2, beta1: gamma2,3 (geranylgeranyl) > 1 (farnesyl) - beta1-gamma1 and beta1-gamma2 have similar affinity for alphai2 Figler et al. Biochem 36:16288 (1997) adenosine A1R and D2R couple to qi4,qi5,qz5,qo5 but not alphaq wt q E Y N L V i2 D C G L F o G C G L Y z Y I G L C Conklin et al. Nature 363:274 (1993) adenosine A2aR coupling with alphas, gamma2, eval by EC50 GTPgammaS loading beta4 (1.3 nM) > beta2 (5.7), beta3 (5.9) > beta1 (15.7) > beta5 (232) McIntire et al. JBC 276(19):15801 (2001) alpha1AR subtypes: A couples to alphaq,11 B couples to alphaq,11,14,16 C couples to alphaq,11,14 Wu et al. JBC 267(36):25798 (1992) alpha2AAR coupling Gi > Gq,Gs Chabre et al. JBC 269(8):5730 (1994) alpha2AAR high-agonist-affinity state promotion in Sf9 membranes - with alphai, beta1: gamma 2,3,4,7,11 > 5,10 > 1 yet beta1-gamma5,10,1 are competent to bind alphai (support ADP-rib) as much as beta1-gamma4,11 - beta3 can pair with gamma 4,11 (promotion ~beta1), 5 (>beta1) Richardson and Robishaw JBC 274(19):13525 (1999) alpha2AAR human expressed in CHO cells epinephrine and norepinephrine signal via this receptor to Gi and Gs (the latter at lower potency, evident after PTX treatment) whereas synephrine and octopamine (agonists above minus one ring OH) are less potent and signal only to Gi potency: (-)>(+), meta>para Airriess et al. British J Pharmacol 122:191 (1997) angiotensin AT1R couples to Gq/11, AT2R couples to Gi2,i3 Inagami J Am Soc Nephrol 10:S2-S7 (1999) beta2AR coupling with alphas, gamma2, eval by EC50 GTPgammaS loading beta4 (0.5 nM) > beta3 (0.9), beta1 (1.0) > beta2 (2.7) > beta5 (17.1) McIntire et al. JBC 276(19):15801 (2001) beta2AR-Galpha fusion proteins in Sf9 cells used to assess coupling; by t1/2 of isoproterenol-stim GTPgS binding: sL > sS > i,q by promotion of high-ag-affinity state: s > 16 > i3 (GTPgS-insens); not i2,q by coupling factor, (Bmax agonist-dep GTPgS binding)/(Bmax radiolab antag): sL,sS,i2,i3 > 16 > q ~1 0.53 0.08 Wenzel-Seifert and Seifert Mol Pharmacol 58:954 (2000) betaAR coupling to alphas in HEK293 cells involves beta1-gamma7 - ribozyme suppression of gamma7 reduces cell levels of beta1 but not other beta or alpha subunits Wang et al. JBC 274(24):17365 (1999) betaAR activation by Kir - tried beta1,5; gamma1,2,7,11 - beta1AR act greatest with beta5-gamma1, beta5-gamma11 - beta2AR act greatest with beta1-gamma7, beta1-gamma11, beta5-gamma2 Robillard et al. Cell Signal 12:673 (2000) beta2AR phosphorylation by PKA switches coupling from Gs to Gi; this switch does not occur in mutant w/o PKA sites (S->A at IC3 261,262 and Cterm 345,346) Daaka et al. Nature 390:88 (1997) (but see rebuttal to this in Clark et al. TIPS 20:279 (1999)) C5aR couples to G16 not G11; 11/16 chimeras tested for coupling 1-237/241-374 no activity 1-216/220-374 65% activity 207-359/1-209 40% activity 151-359/1-153 no activity thus specificity determinants in mult regions Lee et al. Mol Pharm 47:218 (1995) I:26,27 IC1 (4 res) II:0-4 CCKAR Gq,Gs low gastrin affinity IR NKRM RTVTN | | | CCKBR Gq high gastrin affinity GL SRRL RTVTN the exchanges marked by "|" cause a gain in Gs coupling and decr gastrin affinity in CCKBR background, a loss of Gs coupling in CCKAR background (and R->L increases gastrin affinity) Wu et al. Mol Pharm 55:795 (1999) CCKBR rat expressed in CHO cells couples to PLC via PTX-insensitive G protein; to PLA2 via PTX-sensitive G protein; not to Gi (no AC inhib) Pommier et al. J Neurochem 73:281 (1999) CRHR1 human (SecR family) alpha isoform activates alpha s > q,o > i; d isoform (deletion of VII:14-26 + 1 res by Lichtarge alignment) couples weakly with s,o and not with q,i Grammatopoulos et al. Mol Endo 13:2189 (1999) D1R couples to Gs, Go; D5R couples to Gs, Gz Sidhu Mol Neurobiol 16:125 (1998) D2SR couples to alphai3 to inhibit Gs-stim AC, alphai2 to inhibit forskolin- stim AC in transfected fibroblast cells Ghahremani et al. JBC 274(14):9238 (1999) D2LR rat in Sf21 reconstitution system with beta1-gamma2 couples to alpha o>i2 - higher affinity in high-aff state for some agonists - one agonist only shows a high-aff state with alpha o, not alpha i2 - generally higher agonist potency in stimulating GTPgS binding to alpha Cordeaux et al. JBC 276(31):28667 (2001) D4R rat can couple to mouse Gt2 to inhibit forskolin-stim AC; does not couple to mouse alphai2,oA,oB Yamaguchi et al. JBC 272(26):16599 (1997) D4R human variants with 2,4,7 imperfect hexadecapeptide repeats in IC3 and construct w/o any repeats all couple well to alpha i1, i2, i3 (at about equal levels); also couple to o,t Kazmi et al. Biochem 39:3734 (2000) endothelin receptors ETAR, ETBR in CHO cells, A => Gs not Gi; chimera with IC3 of B => Gi B => Gi not Gs purified/reconstituted Gs not activated by either Gq Emax A>B EC50's ~equal Gi Emax ~equal EC50's A>B Go Emax ~equal nonpalmitoylated mutant ETAR decr aff Gq, decr stim Gq,i but not Go Doi et al. Biochem 38:3090 (1999) FPR human readily couples to Galphai1,i2,i3 Wenzel-Seifert et al. JBC 274(47):33259 (1999) FPR human, fluorescent agonist assay in detergent-solubilized system with bovine brain beta-gamma: coupling alphai3 (Kd ~1microM) > i2 (~3 microM) > i1 (>3 microM) >> o (>10 microM) Bennett et al. JBC 276(25):22453 (2001) frizzled (rat) stim of primitive endoderm formation by F9 teratocarcinoma cells agonist Gprot (by antisense) also involves frizzled-1 Wnt-8 alphao,q MAPK,PKC NOT: -5a alphat1,t2,i2,i3,s,11 PI3K, PD beta1,2,4 frizzled-2 Wnt-5a alphao,t2; beta2 PI3K NOT: -8 alphat1,i1,i3,s,q,12,13 MAPK,PKC,PD beta1,3,4 Liu et al. JBC 274(47):33539 (1999) Liu et al. PNAS 96(25):14383 (1999) galanin-receptor-mediated Ca++ current inhibition in rat pituitary and B cell lines mediated mainly by alphao1-beta2-gamma2, less by alphao1-beta3-gamma4 (prevention of effect by antisense; not prevented by antisense to alphai,q, 11,14,15; beta1,4; gamma1,3,5,7) Kalkbrenner et al. Embo J 14:4728 (1995) mGluR6 human (retinal) can activate rat Go > bovine Gt in reconst systems Weng et al. JBC 272(52):33100 (1997) muscarinic receptors summary of determinants III:28 31 (36) IV:2 V:24 26 28 29 VI:1 2 5 6 m2/4->Gi/o C K P M H S A S V T I L m1/3/5->Gq/11 S R R R R Y E T A A L S Hill-Eubanks et al. JBC 271(6):3058 (1996) Kostenis et al. Biochem 26:1487 (1997) Burstein et al. Biochem 37:4052 (1998) IC3 m5R RSAT summary Blin et al. JBC 170(30):17741 (1995) coupling to Gq/11->PI hydrolysis Liu et al. PNAS USA 92:11642 (1995) m1R-mediated Ca++ incr (PLCbeta act) in a rat basophilic leukemia cell line inhibited by antisense to alphaq,11 not alphao1,14 " " " " beta1,4 not beta2,3 " " " " gamma4 not gamma1,2,3,5,7 Dippel et al. PNAS USA 93:1391 (1996) m2R-mediated activation of Gi (GTPgS binding) in reconstituted system inhibited by geranylgeranyl-gamma5 peptide (14 residues); m4R-mediated Ca++ current inhibition in SCG neurons but not that mediated by SSR, alpha2AR, or m1R inhibited by geranylgeranyl-gamma5 peptide (14 residues) but not nonprenylated gamma5 peptide or prenylated gamma7 or gamma12 peptide Azpiazu et al. JBC 274(50):35305 (1999) m2R-mediated activation (+ carbachol) of alpha GTPase rate in reconst system, reflecting the rate of nucleotide exchange (note this assay is more sensitive than GTPgS binding and allows a 1:1 ratio of Galpha:R to be studied): alphao-beta1-gamma7 > (~2x) alphao-beta1-gamma5 > (~2-3x) alphai2-beta1-gamma7, alphai2-beta1-gamma5 although the four types of heterotrimer formed equally well Hou et al. JBC 275(50):38961 (2000) m2R-mediated activation (+ carbachol) of alpha GTPase rate in reconst system, reflecting the rate of nucleotide exchange (note this assay is more sensitive than GTPgS binding and allows a 1:1 ratio of Galpha:R to be studied): alphao-beta4-gamma2 > alphao-beta1-gamma2 although heterotrimers formed equally well Hou et al. JBC 276(23):19982 (2001) m2R-mediated activation (+ carbachol) of alphao GTPase with beta1-gamma5(gg) faster with gamma5scr (scrambling in 13 residues before final C) than gamma5wt; differences NOT evident in GTPgS assay; beta1gamma5wt and beta1gamma5scr bind alphao and activate PLCbeta3 about equally well; however, m2R-alphao-beta1-gamma5 coupling is blocked by gamma5wt peptide, but not gamma5scr peptide (each with geranylgeranyl and 14 residues) Azpiazu and Gautam JBC 276(45):41742 (2001) ORL1R (nociceptinR) can couple to alphaz and alpha16 (similar to opioid R's) Chan et al. J Neurochem 71:2203 (1998) rhodopsin bovine coupling - with alphat, beta1: gamma1 > 2,3 - by IP, the three beta-gammas bind alphat equally well Kisselev and Gautam, JBC 268(33):24519 (1993) - gamma peptide specific Cterm residues and farnesyl important for stab MII Kisselev et al. JBC 269(34):21399 (1994) - gamma1 Cterm 5-22-residue peptides max MII stab attained with 12-14 res and longer; farnesyl > geranylgeranyl > geranyl Kisselev et al. JBC 270(43):25356 (1995) 5HT1AR human high-agonist-affinity state promotion (E. coli membr, recomb prots) - with betagamma from bovine brain Gi/o (shown to bind all alphas tried here): potency alphai3 > i2 > i1 >> o not s Bertin et al. JBC 267(12):8200 (1992) 5HT1AR human high-agonist-affinity state promotion in Sf9 membranes - with beta1-gamma2: alpha i1,i2,i3,o1,z (not s,q) - with alphai1, beta1: gamma 2,3,5,7 > 1 smaller affinity enhancement with just alpha or just beta-gamma Butkerait et al. JBC 270(31):18691 (1995) 5HT1AR activates Na+/H+ exchange in CHO-K1 cells via alphai2, alphai3 Garnovskaya et al. JBC 272(12):7770 (1997) 5HT1BR couples to Gi/o not Gt although Cterms are very similar; chimeras used to identify determinants i1 t (in Galpha helix 4) Q304 K300 E308 L304 Bae et al. JBC 274(21):14963 (1999) 5HT1BR coupling efficiency Gi3, Gi1 > Go > Gi2 Clawges et al. Biochem 36:12930 (1997) 5HT1DR human with beta1gamma2 couples to alpha i1,i2,i3,o not q in Sf9 cells Brys et al. Mol Pharm 57:1132 (2000) 5HT2cR in rat choroid plexus epithelial cells activates PLC via alphaq, not via release of betagamma from alphai Chang et al. JBC 275:7021 (2000) 5HT4R couples to G13 and 5HT7R couples to G12 in mouse hippocampal neurons Kvachnina et al. J Neurosci 25:7821 (2005) somatostatinR SSTR1 can couple with alphai3 SSTR2A can associate with alphai1,i2,i3,o2 SSTR3 can interact with alphai1,i2,i4,i6 Patel Frontiers Neuroendocrinol 20:157 (1999) TXA2R alpha isoform couples to Gq and Gh, beta to Gq but not Gh even though it can be co-IP'ed with the latter; Gh is a high-MW G protein that (like Gq) activates a PLC, but in the non-GTP-bound form apparently acts as a specific transglutaminase Vezza et al. JBC 274(18):12774 (1999) TXA2R alpha isoform couples to Gq and Gs, beta (longer Cterm) Gq and Gi II:0 R60L impairs: alpha coupling to both, beta just Gq coupling Hirata et al. J Clin Invest 97:949 (1996 TXA2 human in platelets copurifies with alpha13 and alphaq Djellas et al. JBC 274(20):14325 (1999) vasopressin receptors determinants, coupling specificity (chimera analysis) V1aR couples to Gq/11 IC2 V2R couples to Gs Nterm part of IC3 (esp V:Q22,E28), shorter (specific aa's not importnt) IC3 middle, Nterm part of Ctail made chimeras coupled to both Gq/11 and Gs but not Gi (thus not promiscuous) Liu and Wess, JBC 271(15):8772 (1996) Erlenbach and Wess, JBC 273(41):26549 (1998) ----------------------------- arranged by function below ----------------------------- GTP-gammaS loading in Sf9 membranes with beta1-gamma2 after activating... beta1AR: alphas 5HT1AR: alphaz (not 12,13,s) neurokinin1R (substance P): alphaq,12,13 > z thrombinR: alphaq,12,13 > z Barr et al. JBC 272(4):2223 (1997) inhibition of voltage-sensitive L-type Ca++ channels... ...by activated m4R involves alphao1-beta3-gamma4 ...by activated somatostatinR involves alphao2-beta1-gamma3 Kleuss et al. Science 259:832 (1993) (GH3 cells) Degtiar et al. FEBS Lett 380:137 (1996) (SST2R, rat insulinoma RINmF5 cells) voltage-dependent inhibition of N-type Ca++ channels in SCG neurons is betagamma-mediated; no involvement of alphai1,i3 found although they can couple to alpha2AR; with alpha2AR, beta1-gamma2, beta1-gamma3, beta2-gamma2, beta3-gamma4, beta4-gamma4 approx equivalent, but not beta5-gamma2 which is known to be alphaq-selective; with beta1-gamma2, the inhibition... ...by activated alpha2AR involves alphaoA,oB > i2 ...by activated adenosineR or PGE2R involves alphai2 > oA,oB ...by activated somatostatinR involves alphai2,oB > oA Jeong and Ikeda PNAS USA 97(2):907 (2000) specificity of inhibition by beta, by injection into cultured rat neurons: 1,2 > 5 > 3,4 Garcia et al. J Neurosci 18:9163 (1998) by antisense method, Ca++ influx in rat portal vein myocytes after activating... ...alpha1AR mediated by alphaq,11; beta1,3; gamma2,3 Macrez-Lepretre et al. JBC 272(8):5261 (1997) ...AT1R mediated by alpha13-beta1-gamma3; alpha13 Cterm interacts with R Macrez-Lepretre et al. JBC 272(15):10095 (1997) ...endothelin A receptor (ETAR) mediated by alpha11-beta3-gamma5 Macrez et al. Mol Pharm 55:684 (1999) by antisense method, AC stim in rat pituitary GH3 cells after activating... ...VIPR mediated by alphas-beta2-gamma2 ...TRHR mediated by alphas-beta2-gamma2 and AC inhib in rat pituitary GH3 cells after activating... ...somatostatinR mediated by alphai2-beta1-gamma3 ...D2R mediated by alphai2-beta1-gamma3 and PLC act in rat pituitary GH3 cells after activating.. ...TRHR mediated by alphaq/11-beta4-gamma2 and PLC inhib in rat pituitary GH3 cells after act ...D2R mediated by alphao-beta3-gamma4 Johansen et al. Cellular Signalling 13:251 (2001) by antisense method, inhib of basal AC activity in rat pituitary GH4C1 cells is mediated by all three alphai subunits; inhib of VIPR-activated AC by... ...5HT1AR mediated by alphai2, alphai3 ...M4R mediated by alphai1, alphai2 Liu et al. JBC 274(23):16444 (1999) ------------------------------------------------------------------------------ G protein selectivity, however, may depend on agonist... D2R human, long form Gazi et al. Br J Pharmacol 138:5):775 (2003) ------------------------------------------------------------------------------