----------------------------------------------------------------------------- GPCR residues suspected of mechanistic importance - mutation disrupts coupling although not thought to interact directly with agonist or G protein, or produce gross structural disruption some overlap with ligs, coupling, and efficacy files; hard to separate ----------------------------------------------------------------------------- II:14 D many references, some conflicting; affinity same or decr; regulation by Na+ Bihoreau et al. PNAS USA 90:5133 (1993) AT1aR rat D->N,E uncoupled although agonist affinity is maintained Ceresa et al. JBC 269(47):29557 (1994) summary table alpha2AR D->E ~wt N,Q loss of allosteric regulation by G protein and Na+, but coupling maintained as assessed by agonist-stim GTPase activity Donnelly et al. Biochem J 339:55 (1999) NK2R human D79A,N but not E impaired Du et al. Prot Engr 10:109 (1997) NPY1R D Fanelli et al. Mol Pharm 56:214 (1999) oxytocinR human D85A no coupling Fraser et al. Mol Pharm 36:840 (1989) m1R rat D->N Fraser et al. PNAS USA 85:5478 (1988) B2AR human D79N single low-aff state Gilbert et al. J Receptor Sig Trans 19:327 (1999) fMLFR human D71A Jagerschmidt et al. Mol Pharm 48:783 (1995) CCKBR D->N decr coupling Ji and Ji JBC 268(23)14953 (1991) LHR rat D383N decr coupling Kong et al. JBC 268(31):23055 (1993) delta opR D Na+ effect II:14 D->N decreases affinity for d-selective peptide & nonpeptide ags affinity ~same for d-selective antags, nonselective ags site of Na+ regulation (uncoupling to low-agonist-affinity state) Kong et al. Mol Pharm 44:380 (1993) SSTR2 D89N Na+ insensitive but coupled Kong et al. JBC 269(32):20548 (1994) mu opR D Kolakowski et al. JBC 270:18077 (1995) C5aR human D82N Marie et al. Mol Pharm 55:92 (1999) bradykinin B2R human D76N,A Martin et al. Mol Pharm 55:210 (1999) in NTR reqd for Na+ mod but not couplg Miettinen et al. JBC 274(39):27934 (1999) FPR human D71N,A signif decr couplg Nie et al. J Neurosci 21:8758 (2001) CB1R D164N decr basal not ag-stim act Parent et al. BBRC 219:968 (1996) PAFR human D63 Quintana et al. Mol Endo 7:767 (1993) LHR D->N Raffetseder et al. Eur J Biochem 235:82 (1996) C5aR D Roche et al. Mol Pharm 56:611(1999) CB1R rat D164N MAPK act, Ca++ current inhib, and AC inhib ~wt, but decr Kir act and internalization Rose et al. FEBS Lett 362:243 (1995) ETBR human D147A no coupling to PLC Strader et al. Ann Rev Biochem 63:101 (1994) betaAR D Strader et al. Ann Rev Biochem 63:101 (1994) NK1R E Surratt et al. JBC 269(32):20548 (1994) mu opR D->A,N,E comparison of II:14 D->N effects among receptors: - decreased agonist but not antagonist affinity: betaAR, deltaOR, LHR - affinities ~same: somatostatin R, ATR, muscarinic R, alpha2AR Tao and Abood JPET 285:651 (1998) human CB1R and CB2R D->E,N Vincent et al. TIPS 20:303 (1999) neurotensin NTS1R D->A (NTS2R has A or G) Vogel et al. Arch Biochem Biophys 361:283 (1999) m2R D69N decr coupling but effects differ wrt diff agonists and wrt AC versus PLC coupling Yang et al. Biochemistry 39:14900 (2000) MC4R human D90A Wang et al. Mol Pharm 40:168 (1991) alpha2A AR human D79N Wang et al Mol Pharm 43:931 (1993) 5HT2R D->N decr coupling Webb et al. Biochem 35:2548 (1996) ETAR human D126A no coupling to PLC Wess TiPS 14:308 (1993) muscR D Wilson et al. Mol Pharm 59:929 (2001) alpha2A AR D79N,Q decr coupl, E ~wt *counterexample Sun et al. Prot Sci 8:2304 (1999) C3aR human D68A ~wt II:17 Webb et al. Biochem 35:2548 (1996) ETAR human Y129A decr Emax 50-75% II:21 ligand-binding AND transduction? Hibert et al. J Receptor Sig Trans Res 19:589 (1999) VP1aR Q104A III:7 ligand-binding AND transduction? Vogel et al. Arch Biochem Biophys 361:283 (1999) m2R D103N decr coupling Lu and Hulme JBC 274(11):7309 (1999) m1R rat D105A Hulme et al. Life Sci 56:891 (1995) m1R, m2R D->E,N decr high-aff proportion III:8 Lu and Hulme JBC 274(11):7309 (1999) m1R rat Y106A III:10 ligand-binding AND transduction? Miura et al. JBC 274(11):7102 (1999) AT1R rat N111 <-> ATII Tyr4 amino-arom III:12 Cavalli et al. FEBS Lett 399:9 (1996) alpha1B AR hamster T130A *counterexample Ji and Ji JBC 268(23)14953 (1991) LHR rat E429Q (consrvative) III:14 S Cavalli et al. FEBS Lett 399:9 (1996) alpha1B AR hamster S132A Lu and Hulme JBC 274(11):7309 (1999) m1R rat S112A Conchon et al. J Am Soc Nephrol 10:S8 (1999) AT1R S115 III:21 Lu and Hulme JBC 274(11):7309 (1999) m1R rat I119A III:24 D/E direct interaction with G protein? (see also coupling file) Fraser et al. Mol Pharm 36:840 (1989) m1R rat D->N Fraser et al. PNAS USA 85:5478 (1988) B2AR human D130N high-affinity state sensitive to GTP but no agonist-dep Gs act (maybe CA? not addressed) Wang et al. Mol Pharm 40:168 (1991) alpha2A AR human D130N Ho et al. JBC 276(2):1376 (2001) KSHV-GPCR R143K,Q,A Franke et al. JBC 267(21):14767 (1992) rhodopsin Vogel et al. JMB Epub 2008 May 17 rhodopsin *counterexample Arora et al. Mol Endo 11:1203 (1997) GnRHR D->N,E increased signaling when normalized to Bmax *counterexample Scheer et al. Mol Pharm 57:219 (2000) alpha1bAR D142A is CA *counterexample Chen et al. BBRC 284:596 (2001) adenosine A3R D107N,K,R ~wt *counterexample LHR rat E441D,Q ~wt binding of hCG and coupling to Gs (cAMP Emax ~wt, EC50 incr 3-4x) Wang et al. Mol Endo 7:85 (1993) [note these are fairly conservative mutations, however] *counterexample Ghanouni et al. JBC 275:3121 (2000) B2AR-Gs fusion D130A ~wt [note this is a fusion with Galpha, however] *see cams file for more counterexamples *III:24,25 mutation constitutively activates some receptors (mechanism role predominates), not others (direct coupling role predominates) Rovati et al. Mol Pharmacol Epub 2006 Dec 27 III:25 R direct interaction with G protein? (see also coupling file) (note Kudo et al. Mol Endo 14:272 (2000) C. elegans GPCR has EMS not DRY) Arora et al. Mol Endo 11:1203 (1997) GnRHR R->Q decr IP generation ~50% Hulme et al. Life Sci 56:891 (1995) m1R R->other decr high-aff proportion (ternary cplx) and G prot act; E>L,Q>A>K (K incr affinity of some agonists) Miettinen et al. JBC 274(39):27934 (1999) FPR human R123A,G signif decr cplg Alewijnse et al. Mol Pharm 57:890 (2000) histamine H2R rat R116A,N Franke et al. JBC 267(21):14767 (1992) rhodopsin Feng and Song Biochem Pharmacol 65(7):1077 (2003) CB2R R->A Min et al. JBC 268(13):9400 (1993) rhodopsin R135L,W Vogel et al. JMB Epub 2008 May 17 rhodopsin Scheer et al. Mol Pharm 57:219 (2000) alpha1bAR R143H,D,A,N,I,E not K low fxn *counterexample B2AR human R131H,N,L...but also several examples in Seibold et al. Receptors and Channels 5:375 (1998) *counterexample Chini et al. Adv Exp Med Biol 449:367 (1998) oxytocinR CAM *counterexample Barak et al. PNAS USA 98:93 (2001) V2R human R137H *counterexample Chen et al. BBRC 284:596 (2001) adenosine A3R R108N,E,H ~wt *counterexample Shi et al. Biochem 37:4869 (1998) rhodopsin R135K,L,Q,A rate of Gt activation >= 50% wt in membranes, less in detergent *more counterexamples Schulz et al. Mol Endo 13:181 (1999) LHR R464A ~wt, H decr max cAMP; m1R R->K modest impairmt *counterexample Rosenkilde et al. Mol Pharmacol 68(1):11 (2005) ORF74-EHV2 not R, mut->R decr constit but maintained lig-dep activity *see cams file for more counterexamples Wilbanks et al. Biochem 41:11981 (2002) mutations in V2R, AT1aR, alpha1aR, alpha1bR result in constitutively desensitized receptors (uncoupled from G proteins, associated with beta-arrestins, found in endocytic vesicles) *III:24,25 mutation constitutively activates some receptors (mechanism role predominates), not others (direct coupling role predominates) Rovati et al. Mol Pharmacol Epub 2006 Dec 27 III:29 I/V direct interaction with G protein? (see also coupling file) Ballesteros et al. JBC 273(17):10445 (1998) GnRHR I143A but not V,L incr IP EC50 ~30x IV:7 Kolakowski et al. JBC 270:18077 (1995) C5aR human C157S IV:17 Liggett Am J Resp Crit Care Med 156:5156 (1997) beta2AR human T164I IV:18 Lu et al. JBC 276(36):34098 (2001) m1R rat W->A V:6 ligand-binding AND transduction? Wess TiPS 14:308 (1993) muscR T DeMartino et al. JBC 270(27):15966 (1995) C5aR R Raffetseder et al. Eur J Biochem 235:82 (1996) C5aR R Wiens et al. Mol Pharm 54:435 (1998) D2sR S->A Wang et al. Mol Pharm 40:168 (1991) alpha2A AR human S->A V:7 ligand-binding AND transduction? Wiens et al. Mol Pharm 54:435 (1998) D2sR S->A Ambrosio et al. Mol Pharm 57:198 (2000) B2AR S->A but only double mut V:7,10 V:10 ligand-binding AND transduction? Wang et al. Mol Pharm 40:168 (1991) alpha2A AR human Holst et al. Mol Pharm 53:166 (1998) NK1R T201H/I204H decr Emax but T201H Emax ~wt; agonist affinities not drastically changed Ambrosio et al. Mol Pharm 57:198 (2000) B2AR S->A but only double mut V:7,10 Conway et al. BBRC 239:418 (1997) mel1aR ovine H211F,L aff decr 6-8x;no coupl V:22,26,29 direct interaction with G protein? - see coupling file VI:1 direct interaction with G protein? - see coupling file VI:8 Lin et al. JPET 294:1053 (2000) GRPR mouse F270A aff ~wt, decr Gq cplg VI:12 Jagerschmidt et al. Mol Pharm 53:878 (1998) CCKBR F347A decr coupl, bind ~wt Chen et al. EMBO J 19:4265 (2000) alpha1BAR F303G,N no coupl but incr ag aff VI:15 Fraser JBC 264(16):9266 (1989) B2AR C285S normal binding but decr AC stim VI:16 Chauvin et al. Mol Pharm 57:625 (2000) GnRHR rat W279S Kd incr 3x, no signlg VI:17 Han et al. Mol Endo 12:810 (1998) AT1AR rat V->A decr max IP response without major change in agonist affinity Cho et al. J Neurochem 65:2105 (1995) D2R human L->A VI:18 Puett et al. Mol Cell Endo 125:55 (1996) LHR P562L ~52% Emax VI:19 ligand-binding AND transduction? Wess TiPS 14:308 (1993) muscR Y Ward et al. Mol Pharm 56:1031 (1999) m1R rat Y381A 50x decr est Ach efficacy Han et al. Mol Endo 12:810 (1998) AT1R Miura et al. JBC 274(11):7102 (1999) AT1R rat H256 <-> ATII Phe8 stacking AT1AR rat H->A decr max IP response without major change in agonist affinity Lin et al. JPET 294:1053 (2000) GRPR mouse N281A aff ~wt, decr Gq cplg VI:20 ligand-binding AND transduction? Granas et al. J Receptor & Signal Trans Res 18:225 (1998) 5HT1BR human F->A VI:22 Han et al. Mol Endo 12:810 (1998) AT1R AT1AR rat F->Y decr max IP response without major change in agonist affinity VI:23 ligand-binding AND transduction? Wieland et al. PNAS USA 93:9276 (1996) beta2AR N Hannawacker et al. Mol Pharm 62:1431 (2002) beta2AR human N293D decr agonist act but not CA Gros et al. Eur J Biochem 251:590 (1998) beta3AR N Holst et al. Mol Pharm 53:166 (1998) NK1R F268A,H not W decr Emax; agonist affinities ~wt except substance P affinity decr 5-11x VI:24 Ward et al. Mol Pharm 56:1031 (1999) m1R rat L386A 20x decr est Ach efficacy VI:26 Mouledous et al. Mol Pharm 57:495 (2000) ORL1R human Q286A bind ~wt, no coupl Yang et al. Biochemistry 39:14900 (2000) MC4R human Y268A VI:27 Toledo et al. J Clin Endo Metab 81:3850 (1996) LHR human A593P no Gs couplg VII:3 Raffetseder et al. Eur J Biochem 235:82 (1996) C5aR D282A but not N decr aff Fernandez and Puett JBC 271(2):925 (1996) LHR rat K583E,Q,R,P Ryu et al. Mol Cell Endo 125:93 (1996) LHR rat K583R,D,E,A,deletion Gilchrist et al. JBC 271(32):19283 (1996) LHR rat K->R,E,L ~no coupling; A no incr cAMP but incr IP at least as much as wt with decr EC50; del no incr cAMP but some incr IP with decr EC50 (incr potency!) VII:6 Cho et al. J Neurochem 65:2105 (1995) D2R human F->A VII:9 Chauvin et al. Biochem Pharm 62:329 (2001) GnRHR rat F310L Kd incr 4x no sig VII:11 Marie et al. JBC 269(33):20815 (1994) AT1AR rat Y->F decr coupling although ag/antag affinity is maintained Puett et al. Mol Cell Endo 125:55 (1996) LHR rat P591L ~23% Emax Conchon et al. J Am Soc Nephrol 10:S8 (1999) AT1R Y292 *weak counterexample Marie et al. Mol Pharm 55:92 (1999) bradykinin B2R human short form NOT affected by Y->F at this position VII:13 N/S Hunyady et al. Mol Pharm 54:427 (1998) AT1AR rat N294A decr coupling without major affinity changes Parent et al. JBC 271(38):23298 (1996) PAFR human N285A wt but N285I no ligand binding although surf-expressed; inconclusive as to role VII:14 S/C Laue et al. Mol Endo 10:987 (1996) LHR human S616Y decr expr,expr-norm couplg VII:15 Miettinen et al. JBC 274(39):27934 (1999) FPR human C295S signif decr couplg VII:17 N/D Barak et al. Biochem 34:15407 (1995) B2AR human N->A but not D uncoupled Hunyady et al. JBC 270(28):16602 (1995) AT1aR rat N298A lg decr Gq couplng Donnelly et al. Biochem J 339:55 (1999) NK2R human N303A,S but not D impaired Satoh et al. BBRC 255:164 (1999) EP3R mouse D318L uncoupled from Gi Flanagan et al. JBC 274(41):28880 (1999) GnRHR D->N,A but not E uncoupled Parent et al. JBC 271(18):23298 (1996) PAFR D->A uncoup but incr PAF aff Lu and Hulme, JBC 275(8):5682 (2000) m1R rat N414H PI no coupl to PI hydrol Gales et al. JBC 275(23):17321 (2000) CCKBR rat N391A no coupl but binds Gq Govaerts et al. JBC 276(25):22991 (2001) TSHR human N674A Wilson et al. Mol Pharm 59:929 (2001) alpha2A AR N422D decr coupl and instab Yang et al. Biochemistry 39:14900 (2000) MC4R human D298A He et al. J Immunol 166:4099 (2001) FPR human N297A no coupling, decr ag aff *counterexample Miettinen et al. JBC 274(39):27934 (1999) FPR N297A ~wt Mitchell et al Nature 391:411 (1998) N not D needed for coupl to SMALL Gprot VII:18 P Wess et al. EMBO J 12:331 (1993) m3R P540A decr coupling Barak et al. Biochem 34:15407 (1995) B2AR human P->A sl decr coupling VII:21 Y Wang et al. Mol Pharm 52:306 (1997) alpha1BAR hamster Y->A Barak et al. Biochem 34:15407 (1995) B2AR human Y->F,A Lu et al. JBC 276(36):34098 (2001) m1R rat Y->A Puett et al. Mol Cell Endo 125:55 (1996) LHR rat Y601A Miettinen et al. JBC 274(39):27934 (1999) FPR human Y301A signif decr couplg He et al. J Immunol 166:4099 (2001) FPR human Y301A sl decr PLCbeta act but no MAPK act, internalization, signif decr chemotaxis; Y301F sl decr PLCbeta act but signif decr MAPK act and internalization, maintained chemotaxis (but shifted to higher agonist conc) VII:23 Min and Ascoli Mol Endo 14:1797 (2000) LHR human I625K impaired hCG response Cterm tail palmitoylation site O'Dowd et al. JBC 264(13):7564 (1989) B2AR human C341G Eason et al. PNAS USA 91:11178 (1994) alpha2A AR human C442F *normal coupling* but abolished downregulation ----------------------------------------------------------------------------- SECRETIN RECEPTOR FAMILY II:7 Turner et al. Mol Endo 10:132 (1996) SecR H178D decr AC stim Turner et al. JBC 273(7):3830 (1998) note H220R PTH1R, H180R PTH2R CA II:13 Turner et al. Mol Endo 10:132 (1996) PTHR S226A, SecR S184A decr AC stim II:17 Turner et al. Mol Endo 10:132 (1996) PTHR R230A,E, SecR R188E decr AC stim Turner et al. JBC 273(7):3830 (1998) PTH1R R230E, PTH2R R190E decr AC stim -----------------------------------------------------------------------------