----------------------------------------------------------------------------- receptor residues implicated in binding to G protein ----------------------------------------------------------------------------- * MUSCARINIC (1,3,5) receptors and Gq/11 Zhu et al. Mol Pharm 45:517 (1994) m1R III:25 R123N uncpled to low-aff state, severe decr PLC act; antag aff ~wt III:26 Y123F some decr coupling even after controlling for decr expr III:24 muts D-> in m1R and alpha2aAR decr ag potency but Emax ~same; however, severe decr coupling in B2AR Blin et al. JBC 170(30):17741 (1995) put m3 residues in m2, looked for coupling to Gq/11 (PI hydrolysis), thus showing role in specificity m3R "i2" S168 III:28 R171 III:31 R176 IC2 R183 IV:2 "Ni3" especially Y254 V:26 "Ci3" A488 VI:1 A489 VI:2 L492 VI:5 S493 VI:6 Burstein et al. JBC 271(6):2882 (1996) Hill-Eubanks et al. JBC 271(6):3058 (1996) Burstein et al. Biochem 37:4052 (1998) * summarizes the preceding works * and suggests general picture of hydrophobic coupling pocket with basic rim m5R Ni3 random mutagenesis, RSAT proliferation assay L212 V:21 conserved Y213 V:22 conserved I216 V:25 mostly hydrophobic - structural Y217 V:26 specific side chain important T220 V:29 mostly hydrophobic - structural R223 V:32 basic best m5R Ci3 random mutagenesis, RSAT proliferation assay K439 VI:0 basic best - involvement of charge in activation A440 VI:1 structural, coupling pocket A441 VI:2 affinity for G protein L444 VI:5 Lee et al. Mol Pharm 50:140 (1996) m1R BBXXB motifs, importance of the basic residues "B": KRTPR140 IV:(-3-0)-1 not important for coupling to AC or PLC KKAAR365 VI:-1-3 important for coupling, all three basic residues contribute but not additively; the first seems the most dispensible Moro et al. JBC 268(30):22273 (1993) bulky hydrophobic in IC2 [III:(33)] may be generally important for coupling m1R human L131 coupling to PLC m3R human L174 coupling to PLC B2aR F139 coupling to AC Hogger et al. JBC 270(13):7405 (1995) m1R human muts->Ala significantly decreased PI hydrolysis I211A V:25 Y212A V:26 K362A VI:0 Bluml et al. JBC 269(15):11537 (1994) m3R rat Gq coupling - bulky hydrophobic at V:26 Y254 (F,W) is important Wu et al. JBC 275(12):9026 (2000) m3R rat IC3 (~252-490) binds betagamma via (289-330), which is required for effective phosphorylation by GRK2 at the 331-3 and 348-351 motifs (IC3 also binds arrestin). Betagamma binds upon Galpha release/dissoc. GST-(289-330) betagamma binding: abolished by E324A/D327A/D329A, FWF312-4AAA, F312A, F314A decreased by Y292A, Y307A little changed by W313A, E324A, D327A, D329A increased by K317A m3R del(289-330) or F312A abolished betagamma binding and impaired agonist- induced internalization without changing ligand binding, Gq coupling; however, m3R del(274-496) has normal agonist-induced internalization. Suggests anti-internalization region (331-496) masked by betagamma binding and/or GRK2 phosphorylation. * MUSCARINIC (2,4) receptors and Gi/o Zhu et al. Mol Pharm 45:517 (1994) m2R III:25 R121N some decr coupling to Gi (AC inhib) Kostenis et al. Biochem 26:1487 (1997) put m3 residues in m2, looked for decreased ability to couple with qo5, thus showing residues important in specificity for Galpha Cterm Baldwin numb. m2 m3 VI:1 V A VI:2 T A VI:5 I L (VI:6 not important) L S mutated residues in Cterm of Gqalpha, looked for coupling to wt m2R Gq E Y N L V | | | each of these 3 single mutants could couple to m2R Gi1,2 D C G L F to some extent Gi3 E C G L Y Kostenis et al. JBC 272(38):23675 (1997) Gs Q Y E L L Gq/11 E Y N L V mutated residues in Cterm of Gsalpha, looked for coupling to Gq/11-coupled receptors: m3R V1a vasopressin gastrin-releasing peptide Gs wt n n n Gs -5 Q->E y y n Gs -3 E->N y y y Liu et al. PNAS USA 92:11642 (1995) m2 and m3 VI:1,2,5,6 interact w/ 5 last residues of Galpha m2 VTIL -> Gi/o m3 AALS -> Gq SUMMARY (UNION) OF PUTATIVE muscR COUPLING SPECIFICITY-DETERMINING RESIDUES 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) 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 Wu et al. JBC 273(10):7197 (1998) upon activation, betagamma binds IC3 and acts as an adaptor for kinase (used m2R and m3R sequences) Wu et al. JBC 275(12):9026 (2000) m3R IC3 interaction with betagamma inhib by alpha (thus postulated to occur upon activation); important for phosphorylation by GRK2 and internalization but not ligand binding or signaling; important residues F312, F314, acidic stretch E324-D329 ----------------------------------------------------------------------------- * RHODOPSIN-TRANSDUCIN Klein-Seetharaman et al. Biochem 38(25):7938 (1999) rhodopsin single Cys mutants transducin activation <40% wt at 67,68 (IC1) Yang et al. Biochem 35(38):12464 (1996) 226 (V:25), 229,230,233,234,242 (IC3), 243,244 (VI:-6,VI:-5) Cai et al. Biochem 38(25):7925 (1999) VII:21,VII:22,VII:24,VII:25 and residues 313,317 (Cterm) Acharya et al. JBC 271(41):25406 (1996) ERY R but not E needed to stimulate GDP release by transducin; E apparently regulatory; neither needed to bind transducin tail peptide III:24 E->F,L like wt; S,I,Q constitutively active III:25 R->G,Q inactive; E134Q/R135Q double mutant also inactive Franke et al. JBC 267(21):14767 (1992) E,R III:24,25 reversal or neutralization abolished transducin activation; significant decr in activation caused by IC3 S240A VI:-6 T243V VI:-1 K248L Min et al. JBC 268(13):9400 (1993) mutants found in autosomal dominant retinitis pigmentosa spectrally normal but deficient in activating transducin: I:21 T58R III:25 R135L,W Ernst et al. JBC 270(18):10580 (1995) bound Gt (light-dependently) but were impaired in stimulation of GDP release: IC2 replacement of 140-152 CKPMSNFRFGENH -> GTEGPNFYVPFTS (from N-terminal sequence) IC3 deletion 237-249 (245-249 is VI:-4-0) impaired in binding Gt: II:1,2 deletion 70,71 (low activity detected) III:24,25 ER->RE IC2 deletion 143-150 Shi et al. JBC 270(5):2112 (1995) bovine rhodopsin IC3 mutations; some of them (marked x) decr Gt coupling V:30-32 VI:-4-3 wt KEA AAQQQESATTQ KAEKEVTR AA | || | G GG || | x V:32/(IC3) 233-235 N NN || | x V:32/(IC3) 233-235 AAA || | AA || | AA | x (IC3) 242,243 A A | AAA | AAA x VI:1/VI:2/VI:3 250-252 Hamm et al. JBC 262(22):10831 (1987) antibodies recognizing rhodopsin middle of VII -> Cterm region block interaction with transducin Konig et al. PNAS USA 86:6878 (1989) rhodopsin peptides inhibit Gt stabilization of MII rhod? IC1 peptide (61-75; I:24-27, 4 residues, II:0-6) no IC2 peptide (141-153; III:31,32, 8 residues, IV:1-3) yes IC3 peptide (230-252; V:29-32, 11 residues, IV:-4-3) yes IC4 peptide (310-321; VII:25,26 and 10 residues) yes tail peptides (323-334) (327-338) or (337-348) no Ernst et al. JBC 175(3):1937 (2000) transducin (IC50 3 microM) stabilizes MII; stabilization lost for rhodopsin mutant 310-312 NKQ -> SPD (as in B2AR) in helix VIII transducin alpha peptide (340-350) (IC50 50 microM) stabilizes MII but (340-350)K341R/L349A does not; stabilization lost for rhodopsin mutant 310-312 NKQ -> SPD (as in B2AR) transducin gamma peptides (50-71)-far (IC50 285 microM) and (60-71)-far stabilize MII but (60-71) and (60-71)F64A/L67A-far do not; stabilization lost for rhodopsin mutant 310-312 NKQ -> SPD (as in B2AR) but enhanced for rhodopsin mutant C322S/C323S (not palmitoylated) Marin et al. JBC 275(3):1930 (2000) rhodopsin peptides inhibit Gt act by rhod? IC1 peptide (61-75; I:24-27, 4 residues, II:0-6) no IC2 peptide (132-144; III:22-32 and 2 residues) yes IC3 peptide (240-252; 5 residues and IV:-4-3) yes IC4 peptide (310-321; VII:25,26 and 10 residues) yes and binds Gt and free alphat rhodopsin mutant 310-312 NKQ -> SPD (as in B2AR) does not activate Gt, but mutations in 315-320 and C322S/C323S (not palmitoylated) do not significantly impair activation Acharya et al. JBC 272(10):6519 (1997) transducin tail peptide (340-350) stabilizes MII state of rhodopsin via required stretches 136-139 III:26-29 and 247-251 VI:-2-2 (note ER of ERY not essential for binding) Phillips and Cerione Biochem J 299:351 (1994) rhodopsin interacts with alphat at least partly via the 325-338 region (activation-blocking peptide) Weiss et al. Biochem 33(24):7587 (1994) rhodopsin truncated after 315 (middle of VIII) still couples normally to Gt Abdulaev et al. JBC 275(50):39354 (2000) rhodopsin loops alone or in series separated by GP linkers inserted into a loop of the globular protein thioredoxin ("1" means IC1, etc.): Gt act: (400-600nM) 2-3 > 1-2-3-4 no Gt act: (up to 1 microM) 1,2,3,4,1-2,1-3,1-4,1-2-3 inhibition of Gt-MII interaction (<400nM) 1-2-3 Terakita et al. JBC 277(1):40 (2002) bovine rhodopsin (Gt-coupled) chimeras with scallop rhodopsin (Go-coupled) (cannot compare *between* Gt, Go, Gi due to different conditions used) IC2 (134-152) replacement decr Gt act (mostly rescued by III:24 C->E); decr Go act; Gi act ~wt IC3 (231-252) replacement decr Gt act; Gi act ~wt; incr Go act 4x relative act of G alpha chimeras with wt bovine rhodopsin and IC3 replacement Gi(wt) 1 1 i/o5 1 1 i/q5 0 0 i/t11 1 1 i/o11 0.25 1 i/q11 0 0 suggesting direct contact of IC3 with Galpha residues -6 and -10 in o R A in t,i K K ----------------------------------------------------------------------------- * OTHER Okamoto and Nishimoto JBC 267 (12):8342 (1992) Ikezu et al. FEBS Lett 311:29 (1992) general characteristics of G protein activating sequences, although not sufficient or strictly necessary: B=basic Ar=aromatic 10-26 residue stretch ending with BBXXB/Ar or BBXB/Ar and containing at least two other B EC50 (micromolar): ~1 ~10 ~100 ~1000 VI:-4 3 B2AR human 259-273 peptide RRSSKFCLKEHKALK Gs m4R 130-147 (IC2) peptide Gi,o Gs m4R 382-400 (IC3) peptide Gi,o Gs alpha2aAR 131-148 (IC2) doesn't have Nterm B's Gs alpha2aAR 218-228 (IC3-N) peptide Gi,o,s alpha2aAR 301-313 (IC3-M) peptide (inactive) alpha2aAR 356-372 (IC3-C) ends BBXXF Gi,o Gs Chabre et al. JBC 269(8):5730 (1994) alpha2aAR coupling Gi > Gq,Gs Eason and Liggett JBC 271(22):12826 (1996) alpha2aAR coupling to Gi requires IC2, IC3 Nterm OR IC3 Cterm alpha2aAR coupling to Gs requires IC2, IC3 Nterm AND IC3 Cterm Taylor et al. JBC 269(44):27618 (1994) Taylor et al. JBC 271(7):3336 (1996) Wade et al. Mol Pharm 50:351 (1996) alpha2aAR IC3 Nterm peptide "P" CRIYQIAKRRTRV, Cys + V:24-32 + 3 residues -> N- and C-term (stim partly blocked by PTX) of Galphao/i proposed role: coupling specificity (along with IC2) and IC3 Cterm peptide "Q" RWRGRQNREKRFTC, 6 residues + VI:-4-2 + Cys -> N-term of Galphao/i (xlinker at end of Q labels 1-17), Cterm of Gbeta (xlinker at end of Q labels 281-341) proposed role: coupling effector (membrane-distal), modulator (mem-prox) Wade et al. Mol Pharm 56:1005 (1999) alpha2A AR Ala mutations of basic residues in IC3 region IC3 R361A/R363A/R365A decr Gs act; Gi act ~wt VI:-3,-1,0 R368A/K370A/R371A some decr Gs act; decr Gi act but not binding VI:-1,0 K370A/R371A Gs act ~wt; decr Gi act but not binding Delos Santos et al. JBC 2006 Feb 24 Epub beta1 AR human, residues in helix VIII: D382L decr coupling, R384Q/E incr coupling and constit act Inagami J Am Soc Nephrol 10:S2-S7 (1999) AT1R couples to Gq/11 AT2R couples to Gi2,i3 Daviet et al. Life Sci 69:509 (2001) AT1R stimulation -> growth, AT2R stimulation -> growth inhib, apoptosis; specificity due to IC3 because: - chimera AT1R/IC3-AT2R has binding like AT1R, signaling like AT2R - AT2R IC3 peptide can cause downstream effects like AT2R - AT2R del middle of IC3 (240-244) destroys signaling, del at either end of IC3 did not Shirai et al. Hypertension 25:726 (1995) AT1R couples to Gq,i,o; peptides effect at <100 microM P1, IC2 Nterm III:21-32 + 2 residues P2, IC2 Cterm 8 residues + IV:1-6 act G? (purif Gq not tried) P3, IC3 Nterm V:23-32 + 5 residues act Gi1,i2,o P4, IC3 Cterm 3 residues + VI:-4-4 P5, Ctail VII:24-26 + 13 residues act Gi1,i2,o Franzoni et al. JBC 274(1):227 (1999) angiotensin II rat AT1aR IC3 based on NMR structures of peptides Ni3 (V:20-32 + 6 residues) and Ci3 (6 residues + VI:-4-5), postulate the Nterm part of IC3 is needed for coupling, while the Cterm part is regulatory (switch, selectivity); activation involves increased amphipathic helix structure in IC3 and Cterm tail, decreased TM3-TM7 interaction, increased TM6-TM7 interaction Hunyady et al. JBC 270(17):9702 (1995) AT1aR rat V:22 Y215F decreases coupling Hunyady et al. PNAS USA 92:10040 (1996) AT1aR rat V:29 L222 nonpolar residue important for coupling Zhang et al. JBC 275(21):15782 (2000) AT1aR rat VI:1,2 nonpolar residues important for coupling to Gq/11 VI:-5-3 Ala scan little effect; VI:1 I238 del,D,K,S decr coupling; VI:2 F239 L,Y little effect, del,N,S,D decr coupling, K,R incr coupling; none of these caused constitutive activation Wang et al. JBC 270(28):16677 (1995) AT1R human -> Gq via IC3 Nterm V:24-32 (217-225) and Cterm VI:-3-0 (234-237) based on chimeras with AT2R (not Gq-coupled) Jung et al. FEBSA Lett 358:133 (1995) betaAR turkey IC3 Cterm peptide can couple to Gs; NMR structure in the presence of micelles shows Nterm-flexible region...helical region-Cterm Mason et al. JBC 274(18):12670 (1999) beta1AR human polymorphism at position 389 in Cterm VII:(33) ~26% Gly, 74% Arg; the latter has sl. higher basal and much higher agonist-stim activity Cheung et al. FEBS Lett 279:277 (1991) beta2AR hamster 15-mer IC3 Nterm and Cterm peptides activate Gs not Gi/o; phospholipid vesicles required Okamoto et al. Cell 67:723 (1991) beta2AR peptide R259-K273 (IC3 5 residues and VI:-6-3) activates Gs > Gi; phosphorylation by PKA decr Gs stim but incr Gi stim Daaka et al. Nature 390:88 (1997) 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) O'Dowd et al. JBC 263(31):15985 (1988) beta2AR coupling to AC (via Gs) involves IC3 Cterm, Ctail Nterm; IC2 and conserved Cys in Ctail may also contribute Kolakowski et al. JBC 270:18077 (1995) C5aR IC3 implicated in coupling to G protein VI:-4 T235A and VI:-1 T238R disrupted coupling to PLC activation Mukhopadhyay et al. Biochem 38:3447 (1999) CB1R tail peptide (401-417, VII:24-26 + 14 residues) Gi-act EC50 32 microM RSKDLRHAFRSMFPSSE * acetylation decr max inhib AC (efficacy) # * x# x palm-C in wt R, # removal decr affinity/efficacy Mukhopadhyay et al. Mol Pharm 57:262 (2000) CB1R cannabinoid receptor Ctail region involved in coupling to Gi/o; CB1R co-IP's with Gi1,i2,i3 and two isoforms of Go but not Gs,q,z CB1R tail peptide (401-417) competes with CB1R for Go,i3 not i1,i2; IC3 peptides, CB2 tail peptide, etc. do not compete Wu et al. JBC 272(14):9037 (1997) CCKAR IC1 implicated in coupling to Gs (compare to TSHR IC2, B2AR IC3) Wu et al. Mol Pharm 55:795 (1999) 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) Pommier et al. J Neurochem 73:281 (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) Wang J Neurochem 68:1728 (1997) CCKBR rat coupling to Gq requires basic cluster 333-335 VI:-2-0 (IC3 Cterm) KKR MTL individually or collectively decrease Gq coupling (esp. latter two) RRK maintains Gq coupling Doi et al. Biochem 38:3090 (1999) 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 Grasso et al. Mol Cell Endo 110:35 (1995) FSHR rat peptide amide (533-555) V:24,25 + 10 residues + VI:-6-4 at concentrations of 10-250 microM - stim GTPgammaS binding to rat testis membrane - inhib FSHR-Gs coupling Miettinen et al. JBC 274(39):27934 (1999) FPR human couples to Gi proteins; blocking peptides implicate IC1,IC2,Ctail, regions in V,VI mutations that significantly decrease coupling; formyl peptide affinity ~wt II:6 S63W II:14 D71N,A D71N complemented by VII:17 N297D III:25 R123A,G III:26 C124S/III:28 C126S VII:15 C295S VII:21 Y301A mutations with coupling and formyl peptide affinity ~wt II:0 H57S II:16 C73S IV:4 K143A V:21 S220A V:22 Y221A V:24 L223A/VI:2 L240A V:25 I224A VI:3 R241A VI:5 L243A VI:8 V246A VII:17 N297A Amatruda et al. JBC 270(47):28010 (1995) FPR human IC1 involved in coupling to Galpha16 IC1 II:0 fMLF-stimulated coupling to Galpha16: residues 54-62: RMT HTV TTI (IC1) AAA attenuated II:0/II:1/II:2 AAA nearly wt II:3/II:4/II:5 AAA abolished Benya et al. Mol Pharm 46:495 (1994) GRPR mouse couples to Gq III:25 R139G abolished PLC act and GPP(NH)P shift VI:1 A263E abolished PLC act but not GPP(NH)P shift (suggests still bound to G protein, perhaps not Gq; wt does not activate Gs) Arora et al. JBC 273(40):25581 (1998) GnRHR IC1 implicated in coupling cAMP IP I:23 L58A max decr 80% ~wt II:0 L73R max decr 80% ~wt II:1 S74E abolished max decr, EC50 incr ~10x II:7 L80A abolished max decr, EC50 incr ~10x Arora et al. Mol Endo 11:1203 (1997) GnRHR IC2 mutations, coupling to Gq/11 (IP generation) III:24 D138N,E incr coupling but poor expression III:25 R139Q signif decr coupling III:26 S140Y,A coupling ~wt Arora et al. JBC 270(30):22820 (1995) GnRHR IC2 mutations, coupling to Gq/11 (IP generation) III:26 S140Y coupling ~wt, incr agonist affinity IC2 (III:33) L147A,D decr coupling Chung et al. JBC 274(50):35756 (1999) GnRHR mouse IC3 nonpolar residue important in coupling to Gq/11 V:29 L237 Gilchrist et al. JBC 271(32):19283 (1996) LHR rat couples to Gs and Gq, and these pathways are separable (point mutations can knock out one pathway while preserving the other) Min and Ascoli Mol Endo 14:1797 (2000) LHR human V:22 Y546F impaired hCG-dependent Gs coupling (expression ~wt) Sagan et al. JBC 274(34):23770 (1999) NK1R human couples to Gs->AC (major population) and Gq/11->PLC (minor component); also known to couple to PLA2 and PLD Vincent et al. TIPS 20:303 (1999) neurotensin receptors NTS1R, NTS2R NTS1R coupling to PLC (Gq) involves IC3 Hoare et al. JBC 274(40):28682 (1999) oxytocinR rat couples to Gq/11 and Gi/o proteins; Ctail truncation after 343 (del51) prevents coupling to Gq/11, decreases OT affinity ~5x, decreases expression ~90%; truncation after 355 (del39), palmitoylation mutant C351S/C352S essentially ~wt Zhong et al. Am J Physiol Endocrinol Metab 2006 Epub Dec 5 oxytocinR human IC3 Cterm important for coupling; in Ala scan of RVSSVKL, mut R prevented coupling; muts S,S,V reduced couping; mut L (VI:-7) was constit act; others had little effect Carlson et al. Mol Pharm 53:451 (1998) PAFR IC3 implicated in coupling to Gq Parent et al. JBC 271(14):7949 (1996) PAFR human A230E VI:1 abolished coupling, A230Q had decreased PAF affinity but could couple Verrall et al. JBC 272(11):6898 (1997) PAR1 thrombinR IC2 implicated in coupling to Gq Seibert et al. Eur J Biochem 266:911 (1999) PAR1 human known to couple to Gi,q,12; can also couple to Gt purified from bovine retinae, reconstituted in Sf9 membranes Lembo et al. Mol Pharm 52:164 (1997) 5HT1AR IC2 implicated in coupling to Gq, and phosphorylation by PKC of T149 decreases Ca++ mobilization. T149A mildly decreases Gi alpha-mediated decr in cAMP, but knocks out Ca++ mobilization (Gi2 betagamma-mediated) and inhibition of Ca++ channel activation (Go betagamma-mediated). T149 may interact more with Go and Gi2 than other Gi's. Highly conserved in GPCR's, including those that couple to Gq and Gs. Kushwaha et al. Mol Pharm 2006 Jan 12 Epub 5HT1AR most point mutations of >60 in IC2 impair coupling to Gi alpha and/or betagamma Clawges et al. Biochem 36:12930 (1997) 5HT1BR coupling efficiency Gi3, Gi1 > Go > Gi2 Niswender et al. JBC 274(14):9472 (1999) 5HT2cR human wt vs. RNA-edited forms III:29,31,(33) constit activity INI (wt) ~ VSI,INV,VNV,ISV > VSV >> VGV basal IP's, EC50 shift, presence of high-affinity state; thus III:31 N promotes coupling more than G Hirata et al. J Clin Invest 97:949 (1996) 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 Zhou et al. BBRC 264:171 (1999) TXA2R human alpha isoform IC2 (III:33) F138D,Y coupling to PLC signif impaired; antag binding ~wt Capra et al. Mol Pharmacol (2004) epub Jun 30 TXA2R alpha III:25 R130V coupling to PLC signif impaired D'Angelo et al. JBC 271:6233 (1996) TXA2R human platelet mutants with normal lig binding but decr Ca++ signaling (downstream of PLC): V:30 T221M decr signaling V:32 C223A decr signaling; C223S no signaling mutants with normal ligand binding and Ca++ signaling: IC3 (V:33) H224R, (V:36) H227R Spurney and Coffman JPET 283:207 (1997) TXA2R mouse Ctail trunc (del 22 residues) decr coupling to PLC; lig aff ~wt Vezza et al. JBC 274(18):12774 (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 Biebermann et al. FASEB J 12:1461 (1998) TSHR human V:22 Y601A,F,H,S,W and V:26 Y605A decr. basal cAMP and destroy IP coupling Buck et al. Endocrinology 141:3717 (2000) TRHR type 1 mouse IC3 "ends" important in coupling to Gq (by alignment to rhodopsin 1f88, V:25 I214 and VI:-6 T257 are the extents of helicity) oocyte assay fxn in HEK293 cells wt 100% del 226-260 89 up to VI:-3 ~wt del 226-262 61 up to VI:-1 del 226-263 25 up to VI:0 del 223-260 75 del 222-260 42 del 226-260/R261A 93 VI:-2 del 226-260/R261E 0 del 226-260/K262A 58 VI:-1 del 226-260/K262E 15 del 226-260/Q263E 86 VI:0 del 226-260/Q263R 96 R261Q/K262Q VI:-2/VI:-1 decr del 226-260/R261Q ~wt del 226-260/K262Q ~wt del 226-260/R261Q/K262Q decr Liu and Wess, JBC 271(15):8772 (1996) Erlenbach and Wess, JBC 273(41):26549 (1998) 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) Rosenthal et al. JBC 268(18):13030 (1993) V2R III:25 R137H normal agonist affinity but no incr cAMP (Gs coupling) Erlenbach et al. JBC 276(31):29382 (2001) V2R human in yeast system; random saturation mutagenesis of IC2 (138-160) couples to: yeast alpha alpha-s5 alpha-q5 wt no yes no M145L,W (III:33) no yes yes M145A,G (III:33) no yes no M145del yes yes yes (note equivalent to M145L/L146del) M145del/H150R (IV:-3) no yes yes ----------------------------------------------------------------------------- family 2 ----------------------------------------------------------------------------- Cypess et al. JBC 274(28):19455 (1999) GLR rat inferred by replacing IC regions with residues from D4R IC1 coupling to Gs IC2, IC3 processing/surface expression IC1 Cterm region, Ctail Nterm region Mathi et al. Mol Endo 11:424 (1997) GLP1R rat IC loop residues found by Ala muts to be important in coupling: II:3 R176 V:25,26 V327, I328 V:29 V331 V:32-34 K334, L335, K336 Iida-Klein et al. JBC 272(11):6882 (1997) PTHR human? coupling to AC (Gs) and PLC (Gq) K319 IC2 (IV:1) ->E decr IP signaling but not cAMP signaling ->A both types of signaling modestly decreased ->R ~wt mutations of E317 or K318 have some effects, mutations of Y320 have none Cterm tail truncation incr cAMP signaling but not IP signaling Huang et al. Endocrinology 140:1294 (1999) PTHR opossum coupling to Gs and Gq, effect of IC3 mutations V:29 L379A ~wt ~30% V:31 T381A 60-80% 60-80% V:32 K382A <10% <10% ----------------------------------------------------------------------------- family 3 ----------------------------------------------------------------------------- Gomeza et al. JBC 271(4):2199 (1996) chimeras with mGluR3 (not PLC-coupled) suggest mGluR1 coupling to PLC requires IC2 and at least one other IC domain; most important (but not sufficient) are the 16 C-term residues of IC2 Mary et al. JBC 273(1):425 (1998) mGluR1 coupling to PLC in the absence of agonist inhibited by Ctail RRKK (877-890, "36 residues" after TM7) but longer-tail isoforms overcome this inhibition and are consitutively active El Far et al. JBC 276(33):30662 (2001) mGluR7 Ctail (approx 851-915) contains overlapping sites for betagamma and Ca++/CaM; Ca++/CaM displaces betagamma and promotes betagamma signaling. Delta(864-876) mGluR7 binds betagamma but not Ca++/CaM, and has normal agonist-dependent Galpha but not Gbetagamma signaling. Peptide (851-875) binds betagamma and Ca++/CaM as strongly as does the full tail peptide (851-915); K860E or S862E but not Q857A significantly decrease betagamma binding. Ca++/CaM also binds mGluR 4A and 8 but not 4B or 6. ----------------------------------------------------------------------------- other ----------------------------------------------------------------------------- Clark et al. JBC 269(12):8831 (1994) STE2 S. cerevisiae IC3 (231-243) implicated in coupling to G protein n + (need to be uncharged and positive, respectively) -RSRRFLGLKQFDS- no Pro tolerated; weak constit act G237A, Q240A, F241A Dosil et al. Mol Cell Biol 20:5321 (2000) STE2 S. cerevisiae Ctail approx residues 297-431 G protein binding evaluated by: dom neg R inhib signaling from wt R; unoccupied wt R inhib signaling from constit act R; rescue of lethal phenotype due to mutant Galpha IC3 L236H impairs activation but not G protein binding Ctail truncs (326,345,360,391) impair G protein binding but not activation Duran-Avelar et al . FEMS Microbiol Lett 197:65 (2001) STE2 S. cerevisiae Ctail N388S decr signaling (mating efficiency, Fus1 induction ~50% wt) but is well-expressed and alpha-factor Kd ~wt By 2-hybrid assay, Ctail peptide N388S decreased binding of Galpha (GPA1) ~50% Gbeta (STE4) ~50% Caterina et al. JBC 269(2):1523 (1994) cAMP receptor of Dicty mutations in Ni3 (V:29-32, 4 residues in IC3) disrupt coupling to G protein and several G-protein-independent responses ----------------------------------------------------------------------------- NONCLASSICAL (NON-SERPENTINE) GPCR's ----------------------------------------------------------------------------- Murthy and Makhlouf, JBC 274(25):17587 (1999) Takahashi et al. PNAS USA 90:11772 (1993) Parnell et al. BBRC 251:625 (1998) single-TM natriuretic peptide clearance receptor NPR-C 17-residue peptide 469-485 RRtqqeesnlgKHRelR activates Gi2,i1 not Gi3,s,q/11 single-TM IGFII/mannose-6-phosphate receptor 14-residue peptide 2410-2423 RvglvRgeKaRKgK activates Gi2 > i1=i3 > o multi-TM polycystin-1 receptor Cterm 20-residue peptide RRlRlwmgfsKvKefRHKvR activates brain Gi/o (EC50 0.2 microM) but min binding domain defined by truncation/IP includes flanking regions for a total of 74 residues Sun et al. JBC 272(9)L5413 (1997) single-TM EGFR juxtamembrane peptide 645-657 activates Gs -----------------------------------------------------------------------------