---------------------------------------------------------------------------- ligand binding (a citation may include results from several previous) some overlap with mechanism file, as these are hard to separate; includes entire studies, so some positions are found NOT to directly bind ---------------------------------------------------------------------------- Krautwurst et al. Cell 95:917 (1998) olfactory receptor 17 mouse,rat specific for heptanal,octanal, respectively residue 206 V:5 I V Han et al. PNAS USA 94:13442 (1997) rhodopsin Schiff base attachment K296 VII:11 " " counterion E113 III:3 9-Me of retinal contacts G121 III:11 (steric push -> activation) ring of retinal contacts W265 VI:16 (Xlink to analog) retinal contacts Y268 VI:19 Dukkipati et al. Biochem 40(50):15098 (2001) short-wavelength-sensing cone opsin of Xenopus S85 II:21 is important for spectral properties, may H-bond to Schiff base counterion D108 III:3 Rao et al. Nature 367:639 (1994) rhodopsin Schiff base counterion can be subst by G90D II:21 Zhukovsky et al. Biochem 31(42):10400 (1992) rhodopsin Schiff base counterion can be subst by A117D III:7 or partially substituted by G120D III:10 Han et al. JBC 271(50):32337 (1996) Han et al. JBC 272(37):23081 (1997) rhodopsin retinal and/or G121 III:11 contact F261 VI:12 (complementation) Lin et al. JBC 273(38):24583 (1998) visual pigment opsin shifts (A) green->red mostly due to IV:14 A164 S (bovine rhodopsin numbering) VI:12 F261 Y VI:20 A269 T (B) green->blue mostly due to II:17 M 86 L II:21 G 90 S III:7 A117 G III:12 E122 L III:14 A124 T VI:16 W265 Y VII:7 A292 S VII:10 A295 S VII:14 A299 C and not to II:18 V 87 C (III:-3) P107 R III:-2 T108 H "...dipolar groups can redshift or blueshift...depending on whether they are situated near the ring or the Schiff base, respectively..." Nakayama et al. JBC 266(7):4269 (1991) rhodopsin bovine retinal pocket III:12,16 E122,W126 VI:16,19 W265,Y268 Fraser et al. Mol Pharm 36:840 (1989) m1R rat mutating conserved D -> N decreased ag/antag binding III:1 III:7 Lu and Hulme JBC 274(11):7309 (1999) m1R rat Ala scan of III:2-23 100-121 L W L A L D Y V A S N A S V M N L L L I S F x x x x x x x >~10x decr Ach affinity III:3,III:4,III:7,III:8,III:11,III:12,III:21 Ward et al. Mol Pharm 56:1031 (1999) m1R rat Ala scan of VI:19-25 381-387 YNIMVLV, positions inferred important: antag agonist NMS aff Ach aff Ach potency est Ach efficacy decr >10x? VI:19 Y381A decr decr 40x decr 2750x decr 50x F ~wt decr 30x decr 70x n VI:20 N382A decr decr 6x decr 10-40x n VI:24 L386A ~wt decr 4x decr 30x decr 20x Allman et al. Mol Pharm 58:175 (2000) m1R rat scan of residues 188-186 V:2-10; muts that decr ACh aff > 5x I188C V:2 T189S V:3 T192A,C V:6 also labeled by BrACh, thus <-> methyl of acetyl group A196G,C V:10 Lu et al. JBC 276(36):34098 (2001) m1R rat Ala scan of IV:3-25 142-164 and VII:5-24 402-421 antag agonist only muts with >10x changes listed here NMS aff Ach aff Ach potency est Ach efficacy IV:14 S153A decr 13x decr ~10x decr ~10x IV:17 V155A decr >10x IV:18 W157A decr 123x decr ~100x decr >1000x decr 160x IV:20 P159A decr 13x decr ~100x decr ~1000x IV:22 I161A decr 3x decr ~10x decr ~10x VII:7 Y404A decr 25x decr >10x decr >100x VII:10 C407A decr 19x decr >10x decr >10x VII:11 Y408A decr 48x decr >10x decr ~1000x VII:14 S411A decr 12x VII:17 N414A incr >10x zero efficacy VII:18 P415A incr >10x decr 12x VII:21 Y418A incr >10x decr 160x Wess Critical Reviews in Neurobiology 10:69 (1996) muscarinic receptors S II:21 -> antagonist W III:3 -> agonist/antagonist D III:7 -> agonist/antagonist N+ Y III:8 -> agonist T V:3 -> agonist T V:6 -> agonist W VI:16 -> agonist/antagonist Y VI:19 -> agonist N VI:20 -> antagonist/some agonists Y VII:7 -> agonist/antagonist Y VII:11 -> agonist Wess TiPS 14:308 (1993) muscarinic R Y's III:8,VI:19,VII:7,11 and T's V:3,6 H-bond to AcCh ester activation mechanism may involve D II:14, T V:6, Y VI:19, P VII:18 also Wess et al. EMBO J 12:331 (1993) m3R IV:20 P->A maintains expression, efficacy, but lowers ag/antag affinity P->A at V:14, VI:18, or VII:18 decreases expression; P's IV:20, V:14, VI:18 not needed for activation, but V:14 P->A decreases ag affinity and VII:18 P->A severely impairs coupling m3R W->F mutations at IV:11, VI:16 but not VII:8 decrease ag/antag affinity Kurtenbach et al. JBC 265(22):13702 (1990) m1R C98 III:0 involved in SS bond in native receptor D105 III:7 X-links to antagonist PrBCM Huang et al. Mol Pharm 56:775 (1999) m1R human VI:26 S388Y incr agonist aff/pot, GTP-insens binding, slight constit act VI:27 T389P agonist aff ~wt, antag MNS aff decr 16x Vogel et al. Arch Biochem Biophys 361:283 (1999) m2R porcine D69N III:1 decr agonist affinity 2-25x D103N III:7 effects consistent with function as counterion to ligands plus a mechanistic role in signal transduction Krejci and Tucek Mol Pharm 60:761 (2001) human muscarinic receptors interaction with allosteric modulators, where alpha represents cooperativity with NMS; >1 negative, <1 positive coop NMS aff gallamine aff, alpha alcuronium aff, alpha m2R 0.75 nM pKa 6.4 11 pKa 6.1 0.3 m3R 0.29 pKa 4.7 4.3 pKa 3.9 2.6 m3R/m2-EC2 0.27 incr 13x (vs m3R) 4.8 incr 8x 3.1 m3R/m2-EC3 0.74 incr 50x 25 incr 3x 0.3 Wong et al. JBC 263(17):7925 (1988) betaAR turkey X-linking to pindolol derivatives W330 VII:8 Dixon et al. Cold Spring Harbor Symp Quant Biol 53:487 (1988) betaAR mutations that decr isoproterenol binding (some not in Strader list below...disproven or proven indirect?) D113 III:7 critical for both agonist and antagonist binding S204 V:7 antag ICYP aff ~wt, decr agonist isoproterenol aff S207 V:10 antag ICYP aff ~wt, decr agonist isoproterenol aff S319 VII:14 antag ICYP aff ~wt, decr agonist isoproterenol aff F289A VI:19 antag ICYP aff ~wt, agonist isoproterenol aff decr 1000x F290M VI:20 antag ICYP aff ~wt, agonist isoproterenol aff decr 10x Y326L VII:21 antag ICYP aff ~wt, agonist isoproterenol aff decr 10x Strader et al. Ann Rev Biochem 63:101 (1994) betaAR agonist binding D113 III:7 -> amine (also antagonist) S204 V:7 -> meta-OH S207 V:10 -> para-OH F290 VI:20 -> aromatic ring (but see above/below) transduction D79 II:14 antagonist binding N312 VII:7 -> aryloxy O HH2R histamine binding D186,T190 V:6,V:10 muscarinicR ag binding T231,T234 V:3,V:6 NK1R agonist binding N85,89,92 II:21,25,28 (* see below) Y287 VII:3 transduction E78 II:14 Y205 V:11 nonpept antag bindg Q165 IV:21 H197 V:3 H265 VI:20 Y287 VII:3 specificity (human vs. rat) modulation V/I 116 III:11 I/S 290 VII:6 Wu et al. Biochem J 354:485 (2001) beta2AR Chinese hamster photoaffinity labeled by antagonist (reactive at aryl portion) at V:2 Y199 Wieland et al. PNAS USA 93:9276 (1996) beta2 AR Asn VI:23 <-> isoproterenol beta-OH may also be involved in activation mechanism; affinity decrease for N293L correlated with intrinsic activity of agonist Zuurmond et al. Mol Pharm 56:90. (1999) beta2AR N293 VI:23 <-> catecholamine beta-OH N293L <-> methyl replacing OH in isoproterenol (but not clenbuterol); this OH->methyl decreases agonist efficacy considerably Green et al. JBC 2 71:24029 (1996) "exosite" for long-acting B2AR agonist salmeterol resides within IV:2-11 (IV:2,5-9 differ from B1AR, which does not exhibit exosite binding) Isogaya et al. Mol Pharm 54:616 (1998) Isogaya et al. Mol Pharm 56:875 (1999) beta2AR vs. beta1AR selectivity (formoterol,procaterol,salmeterol) involves several residues in II and beta2AR Y308 VII:3 beta1AR vs. beta2AR selectivity (denopamine,T-0509) involves beta1AR L110, T117, V120 II:20,(27,30) EC1 Granneman et al. Mol Pharm 53:856 (1998) beta3 vs. beta1 selectivity response: catecholamines beta3-selective agonists beta1 VII:5,6 FF->AL like wt beta1 much increased beta3 VII:5,6 AL->FF like wt beta3 much decreased Gros et al. Eur J Biochem 251:590 (1998) beta3 AR III:7 D117L strongly decr ligand binding VI:23 N312A decr max effect of some agonists; transduction mech? Hwa and Perez JBC 271(11):6322 (1996) TM5 serines beta2 AR V:7,10 (not 6) bind catechol OH's; both 7->meta,10->para important alpha2a V:10->para more important in function than V:6->meta alpha1a V:6->meta more important in affinity than V:10->para when both present; when 6 is mutated, 10 can sub to maintain affinity but not function; V:6->para resulting in partial agonism by ligands w/o meta-OH Hwa et al. JBC 170(39):23189 (1995) alpha AR subtype ligand-binding selectivity mainly due to two residues that alpha1a alpha1b may interact with each other as well as ligs V:3 V185 A204 VI:23 M293 L314 Hamaguchi et al. Biochemistry 35:14312 (1996) dihydropyridine antagonist selectivity for alpha1A over alpha1D AR's due to II:28 F86 M Cockcroft et al. J Neurochem 74:1705 (2000) alpha AR human subtype 1a-selectivity of agonist UK14,304 1a 1b V:7 C201 S C201A compensated nearly fully by S204C V:10, partly by S200C V:6 or V197C V:3; mutation effects seen only for low-affinity state of R Waugh et al. JBC 175(16):11698 (2000) alpha1aAR rat muts to beta2 residues decr agonist aff but not antag aff IV:23 F163Q decr epinephrine aff 12.5x V:5 F187A decr epinephrine aff 8x postulate direct hydrophobic interactions Waugh et al. JBC 276(27):25366 (2001) alpha1aAR rat mutations that decr alpha1 antag and imidazoline ag but not phenethylamine ag affinities VII:3 F308A decr aff for 3/7 alpha1 antagonists (4-30x) 0/3 imidazoline agonists VII:3 F308L decr aff for 3/7 alpha1 antagonists (3-25x), 2/3 imidazoline agonists (4-13x) VII:7 F312N decr aff niguldipine only (1200x) 1/3 imidazoline agonists (25x) VII:7 F312A decr aff for 4/7 alpha1 antags tested (5-1000x) 3/3 imidazoline agonists (8-150x) Cavalli et al. FEBS Lett 399:9 (1996) alpha1bAR hamster alanine mutants suggest the following bind ligands: III:7 D125 <-> agonist/antagonist amine V:2 Y203 also efficacy V:6 S207 but S at :7 OR 10 needed for efficacy VII:11 Y338 Chen et al. JBC 274(23):16320 (1999) alpha1bAR hamster VI:19 F310 muts decr ag/antag binding; size-correlated; W ~wt, L decr potency, A decr potency and efficacy (Emax) VI:20 F311 muts decr expression/folding, ag/antag binding; not size-correl; A decr potency, L decr potency and efficacy - VI:19 but not 20 SCAM-accessible and in ligand-binding pocket - constit act mut A293E VI:2 "dominant," VI:2,19 double mutants also constit act, although with decreased agonist pot/eff as in single mutants (which are not constit act) - postulate VI:19 more likely catecholamine aromatic-aromatic partner than 20 Scheer et al. Mol Pharm 57:219 (2000) alpha1bAR hamster mutations in "DRY" and IC3 III:24 D142A constit act, increased agonist affinity III:25 R143K constit act, increased agonist affinity III:25 R143H,D slight constit act, impaired ag stim, increased ag aff III:25 R143A,N impaired ag stim, increased agonist affinity III:25 R143I,E no coupling, increased agonist affinity D142A/R143A impaired ag stim, increased agonist affinity VI:2 A293E constit act, increased agonist affinity R143A/A293E slight constit act, impaired ag stim, increased ag aff VI:-3/VI:0 R288A/K291A, R288E/K291E impaired ag stim, increased ag aff Wang et al. Mol Pharm 40:168 (1991) alpha2A AR human II:14 D79N high affinities maintained but coupling abolished III:7 D113N decr ag/antag affinity (maybe decr expression) III:24 D130N uncoupled to low-affinity state (Emax same/decr) V:6 S200A uncoupled to low-affinity state (Emax sl decr) V:10 S204A <->p-OH; uncoupled to low-aff state (Emax mostly same) Frang et al. JBC 276(33):31279 (2001) alpha2aAR human III:11 C117V decr phentolamine (imidazoline antag) aff 45x Rudling et al. British J Pharmacol 127:877 (1999) alpha2A AR both serines V:6,10 important in binding/activation V:6 S200A more sensitive to p-octopamine (thus V:6 -> catecholamine m-OH) V:10 S204A more sensitive to m-octopamine (thus V:10 -> catecholamine p-OH) Link et al. Mol Pharm 42:16 (1992) alpha2AR subtypes with V:7 C201 rather than S201 have greater affinity for the bulky alpha2-selective antagonists yohimbine and rauwolscine Suryanarayana et al. JBC 266(23):15488 (1991) alpha2AR VII:7 F412N decr (alpha2-sel antag) yohimbine affinity 350x, incr (beta antag) alprenolol affinity 3000x by binding oxygen on arom ring Guan et al. Mol Pharm 41:695 (1992) 5HT1AR VII:7 N385 responsible for binding aryloxyalkylamine beta-adrenergic antagonists (see also Suryanarayana et al. above) Ho et al. FEBS Lett 312:259 (1992) 5HT1AR mutations decr 5HT affinity 60-100x, antagonist pindolol aff ~wt II:14 D82N III:7 D116N V:6 S198A (affinity too weak to measure for V:7 T199A) Parker et al. J Neurochem 60:380 (1993) Oksenberg et al. Nature 260:161 (1992) 5HT1BR human rat VII:7 T355 N responsible for pharmacological differences Granas et al. J Receptor & Signal Trans Res 18:225 (1998) 5HT1R's human 1A form 1B form V:6 S->A decr 5HT aff 5HT aff ~wt but decr aff of similar agonist 5CT Granas et al. Eur J Pharmacol 421:69 (2001) 5HT1BR human mutations below do not change the affinity of 5HT for the high-aff state or antagonist affinity for the low-aff state % high-aff sites 5HT aff for low-aff state 5HT % inhib AC wt 48 100 nM 87 IV:22 F185A 13 decr 6x 80 IV:22 F185M 10 decr 3x 74 V:6 S212A 20 ~wt 68 VI:20 F331A 53 incr 11x 53 VI:23 S334A 28 decr 4x 72 VII:6 F354A 25 incr 2x 59 VII:6 F354Y 43 incr 2x 58 Choudhary et al. Mol Pharm 48:568 (1995) 5HT2AR binding ergolines (ergonovine, etc.), ergopeptines (ergotamine, etc.) VI:20 F340L,A not Y signif decr ergoline but not ergopeptine affinity VI:19 F339L,A and VI:20 F340Y decr aff ketanserin but not others Roth et al. Mol Pharmacol 52:259 (1997) 5HT2AR mutations in aromatic residue; significant decr in agonist affinity, efficacy IV:11 W200A VI:16 W336A VI:20 F340L VII:8 W367A VII:11 Y370A smaller effects on agonist affinity VI:19 F339L VII:6 F365L (also decr efficacy) no significant effects II:19 F125L VII:24 F383A Almaula et al. JBC 271(25):14672 (1996) 5HT2AR III:11 S <-> serotonin -NH3+ (as well as III:7 D) agonist partial agonist partial agonist affinity: 5HT N,N-dimethyl-5HT (bufotenin) LSD S159A decr 17.6x decr 4.0x ~wt S159C decr 4.9x decr 1.4x ~wt Almaula et al. Mol Pharm 50:34 (1996) 5HT2R's human 2A form 2C form V:10 S A responsible for subtypes' ergoline prefs Johnson et al. Mol Pharm 45:277 (1994) 5HT2AR human rat H V:10 S242 A suspect Ser-O...HN- of N-unsubst ergolines, tryptamines Shapiro et al. Mol Pharm 58:877 (2000) 5HT2AR rat affinity Kact PI hydrol 5HT derivs DOI antags 5HT derivs DOI V:7 S239A decr 6-13x incr 5x incr 3x incr 5-32x incr 4x V:8 F240A ~wt ~wt incr 2x ~wt ~wt V:11 F243A decr 2-50x inc 40x dec 5-200x inc 200-300x inc 200x Vmax 31-44% 49% V:12 F244A decr 3-10x decr 2x dec 3x-inc 3x incr 1-7x incr 3x Vmax 45-65% ~wt Kristiansen et al. JPET 293:735 (2000) 5HT2AR rat III:7 D155A,N,Q surf-expr but lig binding undetectable D155E decr surf expr, decr aff most ligands but incr aff gramine (one C shorter) thus <-> N+ of ligands VII:4 N363A active but like above mutants, not constit act argues against III:7<->VII:4 salt bridge maintaining OFF state Mialet et al. British J Pharmacol 130:527 (2000) 5HT4(a)R human radiolab antag aff 5HT other (all muts expr) II:14 D66N ~wt ~wt decr 6-26x III:7 D100N ~wt lg decr decr 3-63x IV:14 P149A - ? but signals IV:15 T150V ~wt ~wt ~wt IV:18 S153A ~wt ~wt ~wt V:6 C196A sm incr ~wt sm incr/decr V:7 S197A - ? low signal VI:19 F275A - ? but signals VI:20 F276V ~wt ~wt ~wt VI:23 N279L sm decr lg decr ~wt/ sm decr VII:11 Y302A - ? low signal Kokkola et al. BBRC 249:531 (1998) mel1aR human yeast-based assay for functional response to agonists III:7 M107T ~wt III:7 M107D decr agonist potency III:24 N124A decr V:7,10 V192T/H195A no response (possibly not expressed) V:10 H195A incr VI:18 P253A decr VII:6 S280A ~wt VII:6,10 S280F/A284G decr (probably due to S180F) Kokkola et al. Biochem Pharmacol 65:1463 (2003) mel1aR human expressed in COS-7 cells (poor binding below could be poor expr) III:7 M107T ~wt III:7 M107D very poor binding III:24 N124A very poor binding V:7,10 V192T/H195A no binding V:10 H195A ag aff decr ~3x; incr expr V:10/VII:6 H195A/S280A ag aff decr ~10x; incr expr VI:18 P253A very poor binding VII:6 S280A ~wt VII:6,10 S280F/A284G very poor binding Conway et al. BBRC 239:418 (1997) melatonin mel1aR ovine mel aff V:7 V208A decr 2x V:7 V208L decr 6x V:10 H211F decr 6x; no activation V:10 H211L decr 8x; no activation mutations at V:10 did not decr aff for agonists w/o 5-OCH3 Conway et al. BBRC 282:1229 (2001) mel1R human aff of 2 agonists, 4 partial agonists, antag luzindole tested III:3 S103A affinities ~wt III:7 M107T affinities ~wt III:10 S110A ag aff decr 3-15x, luzindole aff ~wt III:14 S114A ag aff decr 3-15x, luzindole aff ~wt Conway et al. JBC 275(27):20602 (2000) mel1R human VI:23 G258T decr melatonin aff >100,000x Mazna et al. J Neurochem 91:836 (2004) mel2R human iodomelatonin affinity V:6 V204A loss of binding V:7 V205A ~wt V:11 F209A ~wt VI:23 G271T signif decr VI:24 L272A loss of binding VII:11 Y298A loss of binding Pollock et al. JBC 267 (25):17780 (1992) D1R human affinity Emax expression V:10 S202A ~wt but decr dopamine! " " good Mansour et al. Eur J Pharmacol 227:205 (1992) D2R human aff vs. wt N-0437 other ags raclopride other antags III:7 D114N,G no binding no binding III:9 M116L ~wt ~wt III:10 M117C ~wt ~wt V:7 S194A ~wt decr .5-3x ~wt small shifts V:10 S197A ~wt decr 2-48x decr 4x small shifts V:7/V:10 S194A/S197A no binding since SAR suggests m-OH more imp than p-OH, propose m-OH interacts w/ V:10 Cho et al. J Neurochem 65:2105 (1995) D2R human, long isoform mutation ->A (found mechanistic: L VI:17, F VII:6) VI:19 F ag/antag binding VI:20 F ag/antag binding Wiens et al. Mol Pharm 54:435 (1998) D2sR role of serines in TM5; tested S->A mutants V:6 decr affinity/efficacy of catecholamines but not other agonists V:7 decr affinity/efficacy of catecholamines but not other agonists V:10 no effect Lundstrom et al. J Receptor and Sig Trans Res 18:133 (1998) D3R VI:23 H349L decreased dopamine affinity >10x VII:7 T369V increased affinity of some agonists Schetz et al. Mol Pharm 57:144 (2000) D4R rat by subst of D2R residues (relatively minor change in dopamine aff) II:24 L87W incr affinity of extended ligands II:25 F88V signif decr affinity of D4-selective antag L750,667 III:2-6 region modulatory, perhaps indirect Wieland et al. JBC 274(42):29994 (1999) H1R guinea pig histamine receptor III:7 D116 <-> protonated amine of ags/antags IV:17 W167 <-> aromatic part of antags V:3 K200 <-> carboxylate in newer antags, imidazole of histamine V:10 N207 <-> imidazole of histamine VI:20 F433 <-> aromatic part of antags VI:23 F436 <-> aromatic part of antags, imidazole of histamine Ligneau et al. Brit J Pharmacol 131:1247 (2000) H3R rat 10x higher affinity for antagonist ciproxifan than FUB349; opposite at H3R human; due to III:12 A119 (human T) III:15 V122 (human A) Kim et al. JBC 270(23):13987 (1995) Jiang et al. Mol Pharm 50:512 (1996) Jiang et al. J Med Chem 40:2588 (1997) adenosine 2a R human hypothesis adenine <-> V:7, VI:20,27 ribose <-> VII:10,14 decr ag/antag affinity ~wt other III:7 V84 A,D | L | III:11 T88 A>R>S especially ags | | III:12 Q89 N>S>R>H>L decr affinity | | A incr affinity, esp ags III:13 S90 | A | III:14 S91 | A | V:5 F180 | A | V:6 N181 | | S decr affinity some ags V:7 F182 A | | Y,W " VI:20 H250 A | N | F,Y " VI:23 N253 A,S,Q | | VI:24 C254 | A | (VI:27) F257 A | R | VII:4 Y271 A | F,H,R | VII:7 I274 A | | VII:10 S277 | N,T,C | A decr ag not antag affinity VII:11 H278 A,Q,F,Y,K,N | | VII:14 S281 A | N | T incr affinity, espec ags Kim et al. Mol Pharm 49:683 (1996) EC2 E151 A,Q,D | | EC2 E161 | A | EC2 E169 A | | Q incr N6-subst, decr others EC2 D170 | K | EC2 P173 | R | EC3 C262 | G | Rivkees et al. JBC 274(6):3617 (1999) adenosine A1R human I:5 G14T agonist binding (affinity incr 10-100x); DPCPX aff decr 2x I:7 E16 agonist binding I:16 P25L agonist binding (affinity decr 3-60x); DPCPX aff decr 1.5x III:6 P86F ag aff decr 10-100x, N6-subst ags ~100x, but maybe indirect III:8 L88A <-> N6 region (ag aff decr 30-500x, N6-subst ags 75-500x) III:11 T91A <-> adenine (ag aff decr 20-200x) III:12 Q92A <-> adenine (ag aff decr 20-200x, non-N6-subst ags 200x) III:14 S94 ligand binding VI:20 H251 antagonist binding VII:3 270 species-specific aff differences for A1-selective ligands VII:10 277 <-> 5' position of ribose VII:11 H278 ligand binding Olah and Stiles review Pharmacology & Therapeutics 85:55 (2000) adenosine A1R (also A2R data but already shown above) I:7 E15A human ag aff decr 4-40x, antag aff ~wt II:14 D55A human ag aff incr, antag aff ~wt, loss of Na+ regulation III:14 S94A human ag/antag binding undetectable (restored with T) VI:20 H251L bovine ag aff ~wt, antag aff decr 4x VII:3 contrib to canine (M270) vs. bovine (I270) binding specificity VII:10 T277A human decr NECA (5'-subst ag) aff >400x, small effects on R/S-PIA (other ags) (restored with S); contrib to canine/bov specfy VII:11 H278L bovine ag/antag binding nearly abolished Chen et al. BBRC 284:596 (2001) adenosine A3R human all well surface-expressed; EC50, aff changes >5x and percentages <75 or >125 noted PLC act: AC inhib: basal % EC50 Emax % basal agonist affinities I:18 N30A ~wt ~wt ~wt ~wt decr 8-36x III:5 C88F ~wt incr 41x 54 ~wt decr 7-14x III:24 D107N ~wt ~wt ~wt ~wt ~wt III:24 D107K ~wt decr 5x ~wt ~wt ~wt III:24 D107R ~wt ~wt ~wt ~wt ~wt III:25 R108K 130 ~wt ~wt incr ~wt III:25 R108A 150 decr 25x ~wt incr ~wt III:25 R108N ~wt ~wt ~wt ~wt ~wt III:25 R108E ~wt ~wt ~wt ~wt ~wt III:25 R108H ~wt ~wt ~wt ~wt ~wt III:26 Y109F ~wt incr 12x 68 ~wt ~wt D107K/R108K ~wt unmeas 23 ~wt ~wt D107K/R108E ~wt unmeas 21 ~wt ~wt VI:2 A229E 160 decr 37x ~wt incr ~wt VII:21 Y282F ~wt incr 29x 57 ~wt decr 8-9x Hoffmann et al. JBC 274(21):14639 (1999) each R,K,D,E,C in EC regions -> A P2Y1R human (binds AMP,ADP,ATP) EC mutations that incr PLC act EC50 >10x: either or both SS bond partners III:0-EC2 (C124A,C202A) expr very low either or both SS bond partners NTD-EC3 (C42A,C296A) EC2 D204A,N,E EC2 E209A (D,Q,R <10x) may interact with 3'OH EC3 R287A,K,Q,E may interact with alpha-phosphate Jiang et al. Mol Pharm 52:499 (1997) P2Y1R human TM mutations, effect on PLC act EC50's III:4 R128A no activity III:7 F131A incr 7-15x III:8 H132A incr 7-12x III:12 Y136A incr 5-18x V:6 T221A incr 5-90x V:7 T222A incr 3-33x V:11 F226A incr 5-16x VI:20 H277A incr 13-79x VI:23 K280A incr 572-1409x VII:4 Q307A incr 121-358x VII:7 R310A no activity VII:7 R310K incr 38-678x VII:7/11 R310S/S314R no activity VII:11 S314A no activity VII:11 S314T incr 6-9x VII:14 S317A incr up to 2x Moro et al. J Med Chem 41:1456 (1998) P2Y1R human mutations effect on antagonism by nucleotide analog MRS 2179 antagonism lost: V:11 F226A VI:23 K280A VII:4 Q307A moderate decrease due to mutations at: III:8 H132 III:12 Y136 V:7 T222 antagonism similar to that at wild type: III:7 F131A V:6 T221A VI:20 H277A VII:7 R310K VII:14 S317A Erb et al. JBC 270:4185 (1995) P2Y2 (=P2UR) mouse (binds ATP,UTP) mutations' effects on [Ca++]i increase III:1 K107I not important III:4 R110L not important VI:20 H262L important incr EC50 105x VI:23 R265L important incr EC50 457x VII:7 R292L important incr EC50 851x Chin et al. J Neurochem 70:366 (1998) Tao et al. Mol Pharm 55:605 (1999) CB1R human agonist CP-55,940 agonist WIN55,212-2 III:3 K192R maintained affinity maintained affinity ->Q,L,E signif decr affinity maintained affinity but >10x incr EC50 CB2R human III:3 K109R,A maintained affinity/efficacy for agonists tested III:3,6 K109A/S112G decr aff ags except WIN55,212-2, decr efficacy, exprsn Shire et al. Life Sci 65:627 (1999) CB1R human (the II:14 and III:3 mutants had little effect within the CB2R) II:14 D163 N,E signif decr aff WIN but not other agonists III:3 K192 A,L,Q,E not R signif decr aff most agonists, some decr aff WIN V:3 Y275 aromatic residue important for binding several ligands Song et al. Mol Pharm 56:834 (1999) WIN55212-2 >10x higher affinity for CB2 than CB1, due (possibly solely) to V:10 V282 in CB1, F197 in CB2 (other agonists' affinities unchanged) Rhee et al. FEBS Lett 466:300 (2000) CB2R human HU-243 HU-243, WIN55212-2 expr, ag bind (homogenate) ag-dep AC inhib (whole cell?) III:24 D130A y 20% ND (not detectable) III:25 R131A y 80% decr Emax to 55-83% III:26 Y132A y 70% decr Emax to 47% triple mutant y 15% ND (not detectable) Rhee et al. J Neurochem 75:2485 (2000) CB2R human cannabinoid binding signaling to Gi IV:11 W158F retained retained IV:11 W158Y,A lost - IV:25 W172F,Y retained retained IV:25 W172L,A lost - Sagan et al. JBC 274(34):23770 (1999) NK1R human agonist SP (RPKPQQFFGLM-amide); sidechains of FF (7,8), M (11) and M-carboxamide important for binding Turcatti et al. JBC 272(34):21167 (1997) NK1R human agonist SP tolerates fluorescein at Nterm and dansyl at 3 or 11 but not 8; the fluorophores at N-term, 3, or 11 are fairly solvent-exposed when SP is bound to the NK1R; dansylated antag CP96,345 is more buried and is in the vicinity of F264,F267,F268 (VI:19,22,23) Li et al. JBC 270:1213 (1995) NK1R NTD (residues 1-21) crosslinks to 3-Bpa SP derivative (potent agonist) Bremer et al. JBC 276:22857 (2001) NK1R NTD (11-21) and EC2 (173-177) crosslink to 3-Bpa SP derivative Li et al. JBC 276:10589 (2001) NK1R EC2 M174 crosslinks to SP derivative Bpa4 Boyd et al. PNAS USA 93:433 (1996) Kage et al. JBC 271 (42):25797 (1996) NK1R rat M181 (EC2 next to SS-bonded C180) crosslinks to 8-Bpa SP derivative (potent agonist) Macdonald et al. Biochem 40:2530 (2001) NK1R EC2 crosslinks to SP derivatives Bpa4 crosslinks to M174 (but M181 in M174A mutant) Bpa8 crosslinks to M181 (~67%)and M174 (~33%) Pellegrini et al. JBC 276(25):22862 (2001) NK1R human/SP model based on the crosslinks above; suggests NTD folds over the binding pocket Holst et al. Mol Pharm 53:166 (1998) NK1R VI:23 F268 mutations decr SP affinity: A 11x H 4.7x incr: W 5x Rosenkilde et al. JBC 269(45):28160 (1994) NK1R; contend that II:14,21,25, (28,32), rather than interacting directly with SP, modulate isomerization to the agonist-binding form of the receptor Huang et al. Biochem 33:3007 (1994) and Huang et al. Biochem 34:10048 (1995) agonist peptide binding to NKR's involves in NK1R not NK2R NK2R not NK1R both III:3 H108/Q109* IV:24 Y168/169 E1 (Nterm) 23/24,25/26 IV:21 Q165/166 V:3 H197/198 E2 VII:3 Y287/289 VI:20 H265/267 II:21,25,28,32 (* see above) (VII:0) Q284/286 peptide Cterm "message" (activity) may bind II:21 Huang et al. Biochem 34:16467 (1995) NK1R activation and ligand specificity V:22 Y216->A increased affinity for some agonists, prevented activation ->W,L,E impaired activation Bhogal et al. JBC 269(44):27269 (1994) NK2R binding NKA III:3 Q109* V:3 H198 also binding antagonist SR48968 V:7 I202 not NK1R VI:26 G273 increased affinity for NKB, SP (decreased specificity) VII:6 L292S but not L292I; mutation of corresponding I290 in NK1R no effect on selectivity Robin-Jagerschmidt et al. Mol Cell Endocrinol 139:187 (1998) NPY1R rat surface expression but abolished NPY binding II:14 D85N,A,E EC1 D103A abolished NPY binding (surface expression not verified) EC3 D286A Sautel et al. Mol Pharm 50:285 (1996) NPY1R human (antagonist BIBP3226 based on Cterm of NPY) mut -> alanine significantly decreases affinity for: EC1 Y100 NPY EC1 D104 NPY EC1 H105 - IV:11 W163 NPY and BIBP3226 IV:18 S170 - IV:21 F173 NPY and BIBP3226 IV:22 L174 - EC2 V178,D200,D205,S206,H207,S210 (singly) - EC2 Y211 BIBP3226 (may bind diphenylmethyl of this antagonist) V:3 T212 - V:10 Q219 NPY and BIBP3226 VI:20 T280 - VI:23 N283 NPY and BIBP3226 VI:24 T284 - EC3 F286 NPY and BIBP3226 EC3 D287 NPY and BIBP3226 (may bind guanidino sidechain in each) EC3 W288 NPY EC3 N289,H290,Q291 (singly) - EC3 H298 NPY Du et al. Prot Engr 10:109 (1997) NPY1R human residues when mutated <15% agonist PYY binding relative to wt II:14 D86 maybe allosteric, conf change mechanism II:28 Y100 II:32 D104 EC1 W106 III:-3 E110 III:0 C113 probably in disulfide with EC2 III:7 Q120 EC2 D190 EC2 D194 EC2 D200 V:10 Q219 V:12 F221 VI:22 F282 VI:23 N283 VI:27 D287 VII:7 H306 Kanno et al. Peptides 200:405 (2001) NPY1R human mutations resulting in "lost affinity" (??) for... 1229U91 (peptide antagonist) VII:4 L303A PYY (peptide agonist) I:7 Y47A also 1229U91 II:26 F98A II:32 D104A III:12 T125A EC2 D200A EC2 D205A V:6 L215A V:10 Q219A VI:19 L279A VI:22 F282A VI:23 N283A VI:26 F286A VI:28 W288A VII:-1 H298A VII:0 N299A also 1229U91 PYY, 1229U91, and J-104870 (nonpeptide antagonist), but surf-expressed IV:11 W163A VI:27 D287A none of the above (minor effects on binding) I:1 F41A EC2 Y176A II:17 V89A EC2 Q177A III:-3 E110A EC2 D181A III:6 V119A EC2 E182A III:10 S123A EC2 D190A IV:17 L171A EC2 D194A IV:19 F173A EC3 N297A IV:21 S169A V:2 Y211A V:3 T212A V:7 L216A VI:20 T280A VI:24 T284A VII:3 F302A VII:7 H306A VII:9 T308A Akeson et al. JBC 272(28):17405 (1997) Sainz et al. JBC 273:(26):15927 (1998) four residues contribute to high affinity of NMBR rat for neuromedin B and high affinity of gastrin-releasing peptideR mouse for bombesin whereas the related bombesinR subtype 3 human has low affinity for both in NMBR rat and GRPR mouse in BRS3 human III:7 Q R121 EC2 P S199 VI:(26) R H288 VII:6 A S308 Fathi et al. JBC 268(20):14622 (1993) NMBR NMB selectivity over GRPR involves V:6 L216 (S in GRPR) Tokita et al. JBC 276(1):295 (2001) NMBR rat antagonist peptoid PD168368 selectivity over GRPR mouse involves V:10 Y220 (F219 in GRPR) with lesser contributions from VI and EC3 Lin et al. JPET 294:1053 (2000) GRPR mouse binds bombesin analogs BN (agonist) and ME (antagonist) IV:25 S180A ag,antag aff ~wt (IV:26) D181A " V:3 S212A " V:6 S215A " V:7 F216A " VI:16 W278A " VI:23 Y285A decr ag but not antag aff VII:11 F313A decr antag but not ag aff Pellegrini and Mierke Biochem 38:14775 (1999) CCKAR human peptide 1-47 41 residues and I:1-6 NMR of complex with nonsulfated CCK8 shows NOE's W39 <-> Y27,M28 of CCK Giragossian and Mierke Biochem 40:3804 (2001) CCKAR human peptide 329-357 (EC3 region) NMR of complex with CCK8 shows NOE's N333,A334,Y338 <-> W30,M31,D32 of CCK Kennedy et al. JBC 272(5):2920 (1997) 25 33 CCK9 = CCK(25-33), CCK8 = CCK(26-33) CCKAR NTD W39,Q40 <-> first two residues of CCK9: R-D-Y(SO3H)-M-G-W-M-D-F-NH2 * * Silvente-Poirot and Wank, JBC 271(25):14698 (1996) CCKAR high aff for CCK; CCKB high aff for gastrin, CCK, nonsulfated CCK CCK ....RDY-MGW-MDF-NH2 Y sulfated gastrin ....EA-YGW-MDF-NH2 Y sulfated or not CCKBR rat gastrin selectivity due largely to EC2: CCKBR 204-209 QcMHRW -> McRFLL decr gast17 aff much more than CCK8 aff CCKAR 195-200 McRFLL -> QcMHRW no bindg CCK8 or gast17; antag aff ~wt Silvente-Poirot et al. Mol Pharm 54:364 (1998) CCK/CCKR affinity Gigoux et al. JBC 273(23):14380 (1998) Gigoux et al. Prot Sci 8:2347 (1999) (agonist affinity, rat receptor and CCK-8 unless otherwise noted) CCKAR CCKBR NTD W39F decr 13x (CCK-9) L52A no change NTD Q40N decr 21x (CCK-9) E53A no change I:1 A55L no change I:2 I56A no change I:3 R57A decr 21x hum R57Q decr 6x (CCK) EC1 N102A no change N115A decr 16x EC1 L103A severe decr L116A decr 6x EC1 hum K105L decr 4x (CCK) EC1 F107A severe decr F120A severe decr EC1 F109A no change F122A decr 8x III:0 C127A severe decr (same for C205A, likely SS bond partner; not surface-expressed) EC2 hum M195L decr 33x, shift to low-aff state;->CCK9 sulfoTyr EC2 hum R197M decr >100x, shift to low-aff state;->CCK sulfate EC2 F198A no change H207A,F severe decr (-> D32) V:3 hum S219H decr 5x (CCK) VII:7 H381L no change hum H376L decr 29x (CCK) Gigoux et al. JBC 274(29):20457 (1999) CCKAR human interacting with CCK9 analog VI:23 N333 <-> terminal -CONH2; -COCH3 relatively insens to N333A VI:26 R336 <-> D32; ->R complements R336D in binding affinity (but not activity); ->A relatively insensitive to R336M Dong et al. JBC 274(8):4778 (1999) CCKAR rat EC1 labeled by photolabile residue corresponding to CCK-24 within agonist, partial agonist, and antagonist peptides Hadac et al. JBC 273(21):12988 (1998) CCKAR rat EC3 H347, L348 labeled by photolabile residue corresponding to CCK-29 within agonist CCK8 analog (H362, L363 in cckr_rat.sw) Ji et al. JBC 272(39):24393 (1997) CCKAR labeled at W39 in NTD by photolabile residue corresponding to CCK-33 in agonist analog of CCK8 - disagrees w/ above (or both ends bind same region) ( I:-2 W54 in cckr_rat.sw) Ding et al. JBC 276(6):4236 (2001) CCKAR rat labeled by agonist CCK Bpa-24 analog within 10-37 NTD and E345 EC3 (25-52 and E360 in cckr_rat.sw) while the analog retains potent activity at receptor with 1-30 deletion, no crosslinks form their model: Ding et al. Mol Pharmacol 61:1041 (2002) Silvente-Poirot et al. JBC 274(33):23191 (1999) CCKBR rat mutations at EC2 H207 combined with Ala scanning of CCK9 analog demonstrates interaction H207 <-> D32; H207A insens to D32A,E,F,L Anders et al. Biochemistry 38:6043 (1999) CCKBR human L52-Y61 I:-2-7 crosslinks with pBz at Nterm of sulfated CCK-8 analog (potent agonist) Kopin et al. JBC 270(10):5019 (1995) CCKBR human, 58 TM residues -> CCKAR residues nonsel B-selective agonist those with >5x increase in IC50 for CCK8 or gastrin I:3 R57Q 6.1 8.0 fold increase III:4 S131T 1.4 8.5 V:3 S219H 5.7 4.1 VII:7 H376L 29 17 Blaker et al. Mol Pharm 58:399 (2000) CCKBR human mutants well-expressed, Emax ~wt for CCK4,CCK8 affinity: x=IC50 incr >5x | Emax if sig diff from wt receptor CCK4,CCK8 PD135158,PD136450| PD135158 PD136450 L740-093S (peptides) (peptoids) | (20% at wt) (42%) (30%; nonpept) I:7 Y61A x | 16 12 II:24 F110 | II:25 T111A x | 16 6 III:4 S131 | III:8 G135 | IV:18 M186A x x | 41 (incr) 67 (incr) 9 IV:25 T193A x | V:3 S219A | V:10 L226A | V:11 F227A | 0 8 VI:16 W346A | 43 (incr) 12 VI:19 V349A x | 3 3 72 (incr) VI:20 Y350A | 0 0 0 VI:23 N353L x | 15 12 VI:24 T354A x | 5 VII:10 S379A | Mouledous et al. Mol Pharm 57:495 (2000) nociceptinR (ORL1R) human noc affinity noc potency other effects III:7 D130A,N,E lg decr (ND) decr >5000x III:8 Y131A decr 25x decr 49x lofentanil->antag III:8 Y131F decr 2.2x ~wt V:7 F220A decr 1.7x decr 9x V:11 F224A lg decr (ND) decr 200x VI:16 W276A decr 3.6x decr 14x lofent,HP5->partial ags VI:26 Q286A decr 1.5x no coupling VI:26 Q286E decr 2x decr 2.5x Meng et al. JBC 271(50):32016 (1996) altering rat orphanin FQ (nociceptin) R (ORL1R) to also recognize dynorphins V:3 A213K VI:19-21 276-8 VQV->IHI VII:7 T302I Mollereau et al. Mol Pharm55:324 (1999) altering KOR1 to recognize nociceptin as well as wt ligand dynorphin A; chimera with Nterm->EC1 and EC2 of ORL1R Metzger et al. Neurochem Res 21:1287 (1996) naltrexone binding to opioidR III:8 V:11 VI:16,20 Befort et al. Mol Pharm 49:216 (1996) opioid receptor III:7* mutations mu D->A decreased expression, decreased agonist affinity, antagonist affinity ~same delta D->A decreased expression, affinity ~same for most ligands (ag/antag, sel/nonsel, pept/nonpept), except in Na+-induced low-affinity state in which agonist affinity decreases more for mutant than wt delta D->N expression ~same, decreased affinity for many ligands postulate that at least in the deltaOR, III:7 is not the counterion for opioid N+ but is involved in forming the binding pocket Befort et al. JBC 271(17):10161 (1996) delta OR mouse mutations of aromatic TM residues; affinity decr >20x for III:8 Y129A 5-7-res agonist peptides, nonselective alkaloids (ag & antag) F deltorphin II (7-res delta-specific agonist peptide) IV:11 W173A some 5-31-res agonist peptides V:7 F218A (none) 11 F222A (none) VI:16 W274A nonselective alkaloids (ag & antag) VII:11 Y308F (none) Cavalli et al. Neuroscience 93:1025 (1999) delta OR mouse expression constit act DPDPE (ag) aff naloxone aff III:7 D128A ~wt yes ~wt ~wt D128H low yes ND ND D128K low yes decr ~1000x decr ~100x but becomes agonist! Valiquette et al. JBC 271(31):18789 (1996) delta OR human residues important in binding delta-selective ligands SNC-80 (nonpept ag), DPDPE and deltorphin II (pept ags), naltrindole (nonpept antag) Ala-scanned 14/16 EC3 residues; all mutants retained ~wt affinity for nonsel ligand bremazocine; most important positions: VI:26 W284 EC3 near Cterm V296 EC3 near Cterm V297 Hosohata et al. Life Sci 68:2233 (2001) delta OR human mutant VI:26 W284L tested vs delta-selective agonists affinity decr SNC80 15-30x, DPDPE 1-2x, (-)TAN67 5-9x, SB21825 7x Emax (ag-dep GTPgS bdg) incr for SNC80, ~wt for DPDPE, decr for other two Na+, GPTgS effects on affinities incr Surratt et al. JBC 269(32): 20548 (1994) mu opioid receptor affinity coupling DAMGO (mu-sel ag) morphine naloxone II:14 D->A "reduced binding" N dec 5277x dec 74x ~same dec E dec 1333x dec 33x ~same ~same III:7 D->A,N "reduced binding" dec E dec 5x dec 6x ~same VI:20 H->A "reduced binding" inc Bonner et al. Eur J Pharmacology 403:37 (2000) mu OR rat effects on binding 3 nonsel, 6 mu-sel, 3 delta-sel ligands VI:26 K303E (as in kappa) aff within 5x wt but DAMGO EC50 incr 382x VI:26 K303Q aff within 5x wt but DAMGO EC50 incr 37x VI:26 K303W (as in delta) aff within 5x wt but DAMGO EC50 incr 77x and morphine EC50 became unmeasurable (->v weak agonist) VII:3 W318K decr aff of nonsel and mu-sel up to 18x, incr DAMGO EC50 much more than its aff, incr delta-sel aff up to 29x VII:3 W318L (as in delta) decr aff of mu-sel up to 7x, incr CAMGO EC50 much more than its aff, incr delta-sel aff up to 63x (thus VII:3 is important in mu vs. delta selectivity) Mansour et al. J Neurochem 68:344 (1997) mu OR rat mutations affecting ligand binding III:10 N150A agonist affinity incr 3-20x, antag affinity unchanged VI:20 H297A general marked affinity decr VII:11 Y326F broad range decr affinity (a little to a lot) Seki et al. Eur J Pharmacol 350:301 (1998) opioid R DAMGO (mu-selective agonist) discriminates at positions delta mu kappa II:27 K108 N127 EC1 VI:26 K303 E297 EC3 VII:1 V316 S310 EC3 VII:3 W318 Y312 EC3 VII:4 H319 Y313 EC3 Xu et al. Synapse 32:23 (1999) mu OR rat VII:4 H319A decr affinity most ligands (up to 48-fold) Schioth et al. FEBS Lett 410:223 (1997) human MCRs tolerate Nterm deletions without changes in lig binding or expr MC1R 27-residue deletion MC3R 25 MC4R 28 MC5R 20 Yang et al. JBC 272(37):23000 (1997) MC1R human interaction with agonists including alpha- and gamma-MSH II:24 E94 (see also cams file) III:0 D117 (see also cams file) III:4 D121 (see also cams file) Frandberg et al. BBRC 202:1266 (1994) MC1R human mutations that signif decr alpha-MSH affinity III:0 D117A VI:22 H260A little effect IV:21 F179A V:24 H209A Chhajlani et al. BBRC 219:521 (1996) MC1R human mutations that signif decr alpha-MSH affinity NTD S6A EC2 D184A EC3 E269A EC3 T272A little effect EC1 E102A EC1 R109A Frandberg et al BBRC 281:851 (2001) alpha-MSH Ac-SYS-M-EH-F-RWGKPV-NH2 (first 13 residues of ACTH) NDP-MSH Nle f (differences) MC1R human cysteine residues studied; DTT decr alpha-MSH aff 36x (3.4->120 nM), NDP-MSH aff ~wt (~1.5 nM) althought there was a biphasic "loss of NDP-MSH binding" (loss of sites?) C->G mutation effects on alpha-MSH and NDP-MSH binding and signaling loss of NDP-MSH binding I:-3 C35G S some binding VI:29 C267G S some binding EC3 C273G S some binding EC3 C275G S some binding normal affinities, no cAMP signal (like previously described IC2 mut R151C) V:6 C191G IC3 C215G VIII? C315G (probable palmitoylation site) normal affinities, normal alpha-MSH signaling, NDP-MSH -> antagonist II:8 C78G no significant effect on alpha-MSH or NDP-MSH binding or signaling II:5 C75G II:9 C79G III:8 C125G III:16 C133G VI:15 C253G VII:12 C289G Yang et al. JBC 274(20):14100 (1999) agouti-related protein (AGRP) select. for MC4R not MC1R assessed by chimeras; contribution EC3 > EC2 (and approx. additive; other regions contribute) Gu et al. Diabetes 28:635 (1999) MC4R human mutation decr agonist aff, Emax, incr EC50; found in obese person III:15 I137T Yang et al. Biochemistry 39:14900 (2000) MC4R human mutations that incr Ki >5x NDP-MSH alpha-MSH AGRP (antag) notes II:14 D90A 4x 11x ~wt also uncoupl. III:10 D122A 6x 21x 2x =>R8 of alpha-MSH D122N 4x 4x ~wt III:14 D126A unmeas unmeas 9x D126N unmeas unmeas 5x V:10 M200A 2x 17x ~wt V:14 M204A 2x unmeas ~wt VI:16 W258A 3x 23x ~wt VI:19 F261A 5x 5x ~wt VI:22 H264A unmeas unmeas ~wt lesser effects by mutations I:5 E49A I:17 E61A II:24 E100A III:8 C130A IV:1 K164A IV:2 R165A V:6 C196A V:9 T199A V:13 T203A V:24 H214A VI:25 F267A VI:26 Y268A VII:2 H283A VII:4 N285A VII:17 D298A (VII:24) R305A Oosterom et al. JBC 276(2):931 (2001) affinity changes agouti NDP-a-MSH Nle4-g2-MSH D-Tyr4-MTII (MC4-sel) MC4R human VI:26 Y268I decr >10x ~wt incr decr MC3R rat VI:26 I265Y decr (not sig) ~wt decr 40x incr 4x Nickolls et al. Mol Pharm 304:1217 (2003) MC4R human postulate III:0 D122A decr aff of peptides with L-Phe not D-Phe <-> Arg8 III:3 I125F some decr aff of MC4R-sel ligands III:15 I137T decr aff of cyclic peptide agonists indirect IV:21 F184M little effect VI:19 F261A decr aff of peptides with L-Phe not D-Phe <-> Phe7 VII:3 F284A decr aff of peptides with L-Phe not D-Phe <-> Phe7 MC3R human III:3 F157I some incr aff of MC4R-sel ligands MacDonald et al. Mol Pharm 58:217 (2000) melanin-concentrating hormone DFDMLRCMLGRVYRPCWQV with C's disulfide-bonded MCHR human, -fold EC50 incr of mutations III:7 D123A,E,R,K,S ND (binding undetectable although surf-expressed) propose interaction with MCH R11 since R6, R14 shown unimportant III:10 S126A 6 IV:14 S171A 2 IV:17 S174A 2 V:3 T209A 4 V:6 Q212A 5 VI:23 Q276A 4 VI:26 Q279A 2 VII:-1 F289A 4 VII:3 Y293A 16 VII:4 N294A 3 Ji et al. JBC 268(31):22971 (1993) LHR rat EC1 (II:28) D397 <-> hCG alpha K91 important for act not binding LHR 397 EC50's (nm) for incr cAMP hCG 91 wt(D) A K R wt(K) 0.02 2.7 - - M 1.8 - - - (Kd's all ~wt, 0.2-0.6 nM) D >6.5 - 2.1 2.5 E >7.3 - 2.4 3.1 Ryu et al. Mol Cell Endo 125:93 (1996) LHR rat VII:3 K583 important for activation but not binding; Ala scan 573-583 results suggest EC3 may be a beta-strand hCG, FSH, TSH share common alpha subunit; Cterm residues are His90 Lys91 Ser92 imp in all, to imp in hCG and in imp in TSH, not others some extent; FSH signaling, H90S incr FSH pot not others - alpha(83-92) peptide can activate LHR but not FSHR - ABG-alpha(83-92) can activate and crosslink with LHR - ABG-alpha(full-length) together with beta can activate LHR; receptor binding decreases extent of alpha-beta crosslinking Puett et al. Mol Cell Endo 125:55 (1996) Bhowmick et al. Endocrinology 140:4558 (1998) LHR NTD mutations that strongly decrease hCG binding: E132K, D135K K158E not R,Q,G K183E not R,Q,G E184K not N D206K not E,Q the following do NOT affect hCG binding or signal transduction much: NTD E57K, E189K, K190E III:7 T424A VI:16 M560L VI:20 S564A VII:10 Y590A not surface-expressed: NTD E80K, E181K important for signaling rather than binding: NTD E332, D333 Hong et al. JBC 273(22):13835 (1998) LHR rat NTD by Cterm truncation of this domain and Ala scanning, L20,C22,G24 each important for binding hCG Phang et al. JBC 273(22):13841 (1998) LHR rat NTD photoactivatable peptide analog of 18-36 labeled both subunits of hCG, alpha more than beta Roche et al. Endocrinology 131:268 (1992) LHR rat hCG-binding peptides; of 32 NTD tested plus EC1, EC2, EC3, 12 inhibited hCG/LHR binding, defining four regions: 21-38 nonhomol, may bind beta 102-115 } 253-272 } homol, may bind alpha EC3 573-583 } Remy et al. Mol Cell Endo 125:79 (1996) Couture et al. Eur J Biochem 241:627 (1996) hCG epitopes 15-17,73-75 "alpha tip:" exposed in free and porcine LHR-ectodomain-bound but occluded in pLHR(full length)-bound, by EC2 or EC2 peptide 481-497 (EC2 peptide inhibits hCG-induced incr cAMP but not hCG high-aff binding) "beta tip:" exposed in free and pLHR-bound alpha/beta interface: exposed in free, occluded in pLHR-ectodomain- bound and pLHR-bound Couture et al. J Mol Endo 16:15 (1996) LHR porcine NTD peptides effects on pLH/PLHR binding and transduction IC50's (microM) binding incr cAMP 7-24 130+/-70 125+/-25 21-36 88+/-11 300+/-50 25-40 225+/-75 6.5+/-3.5 102-111 240+/-70 500+/-50 107-121 >1000 70+/-30 102-121 207+/-103 650+/-50 their interpretation: more imp for binding transduction 7-24,102-107 25-40,111-121 Dattatreyamurty and Reichert Mol Cell Endo 87:9 (1992) FSHR rat peptide 9-30-Tyr-NH2 binds oFSH, bFSH, hFSH but not LH, TSH Dattatreyamurty and Reichert Mol Cell Endo 93:39 (1993) FSHR rat peptide 9-30-Tyr-NH2 binds beta subunit of hFSH Abdennebi et al. J Mol Endo 22:151 (1999) FSHR porcine peptides from NTD elicit antibodies in ewes that are... vs. 18-27, 25-34 --> competitive FSH antagonists vs. 29-38 --> noncompetitive FSH agonist(s) Morris et al. JBC 268(15):10900 (1993) TSHR human TSH-binding peptides; of 26 NTD tested plus EC1, EC2, EC3, the following inhibited TSH/TSHR binding: 256-275 > 16-35, 106-125 > 226-245 > 286-305 (all NTD; others inactive) |________________________| also inhibit hCG-LHR binding, thus may bind common hormone subunit alpha Sealfon et al. Endocrine Reviews 18:180 (1997) GnRHR II:25 Asp <-> GnRH His2 II:29 Asn <-> GnRH Gly10-NH2 (probably H-bond to carbonyl) III:7 Lys K->R maintains lig affinity, increases coupling while K->Q decreases agonist but not antag affinity VII:0 Asp <-> GnRH Arg8 TRHR interactions with TRH III:8,12 (Y,N) important in binding, III:7 (Q) not so important Flanagan et al. Biochem 39:8133 (2000) GnRHR human II:25 D98 <-> GnRH His2 more experimental evidence Davidson et al. Biochem 36:2881 (1997) GnRHR NTD C14 xlinks to photoactivatable group at position 6 of agonist; causes irreversible activation, disconnects C14-C200 disulfide bridge Hoffman et al. Mol Endo 14:1099 (2000) GnRHR human Ala mutation of known binding residues and EC2/V region no binding/signaling (possibly not surf-expr) II:14 N87A II:17 E90A E90Q ~wt II:25 D98A EC2 R179A EC2 W206A V:2 Y211A V:5 F214A V:6 T215A mutation ag EC50 incr antag IC50 incr rel Bmax (# = >10x) I:1 K36A 1-2.9x 1.1-1.6x 0.34 II:28 W101A # 730-6000x # 37-39x 0.17 II:29 N102A # 16-240x 4.9-5.9x 1.2 N102Q ~wt EC2 Q204A 2.1-3.6x 3.6-3.8x 0.81 EC2 W205A 0.9-2.8x 0.8-1.0x 0.36 V:-2 H207A 0.7-1.4x 0.7-1.5x 0.22 V:-1 Q208A 1.1-1.5x 1.6-2.3x 1.1 V:1 F210A 0.2-0.5x 0.5-0.6x 1.5 V:3 N212A # 13-30x # 20-48x 0.16 V:3 N212Q 3.7-16x 1.2-1.5x 0.29 V:4 F213A 1.6-4.3x 0.7-1.3x 0.65 V:7 F216A 1.5-2x 0.8-0.9x 0.4 V:7 F216Y # 3.2-19x 9.1-9.3x 0.72 V:8 S217A 0.2-0.5x 0.6-0.7x 0.64 S217R no bind/sig Hovelmann et al. Biochem 41:1129 (2002) GnRHR human Ala mutations in VI no binding/signaling (possibly not surf-expr) VI:19 Y283A VI:20 Y284A Y284C ~wt binding, decr expr and signaling VI:27 W291A mutation ag EC50 incr antag IC50 incr rel Bmax (# = >10x) VI:25 W289A 4.9-16x 1.8-3.7x 0.25 VI:26 Y290A # 200-1000x 2.6-3.6x 0.23 VI:28 F292A 1.7-3.6x 3.1-5.1x 0.27 Blomenrohr et al. FEBS Lett 501:131 (2001) GnRHR catfish cGnRH-II: IC50 EC50 (IP's) Emax membrane expr II:14 D90N unmeas unmeas unmeas 49% wt II:14/II:17 D90N/M93E incr 16x incr 16x ~wt 159 III:7 K124M incr 148x incr 708x 41% wt 31 D90N/M93E/K124M incr 776x incr 708x 23 ~wt Han et al. Biochem 34:13412 (1995) TRHR rat IV:26 N289 <-> pyroGlu* (TRH = pyroGlu-His-ProNH2) Han and Tashjian Mol Endo 9:1708 (1995) TRHR mutations effect on MeTRH affinity (all mutants expressed) III:-5 Y93A decr >20x, antag aff decr similar EC2 Y181F binding undetectable V:-1 Y188A binding undetectable V:-1 Y188F decr 4-5x, antag aff ~wt V:7 F196A decr 4-5x, antag aff ~wt V:10 F199A binding undetectable VI:26 N289A binding undetectable VI:27 S290A binding undetectable mutations without significant effect on MeTRH affinity NTD del(2-22) III:-3 Y95A EC2 N186A,E V:-2 Y187F,A EC3 S293A EC3 Q297A VII:-1 E298A VII:0 N299A Perlman et al. Biochem 35:7643 (1996) TRHR mouse interactions with TRH III:8,12 Y106, N110 <-> pyroGlu* IV:19 Y282 <-> His VII:7 R306 <-> terminal carboxamide (ProNH2) Perlman et al. Biochem 36(50):15670 (1997) TRHR EC2 Y181 <-> pyroGlu* suggest "entry channel" Perlman et al. JBC 270(42):24682 (1995) TRHR mouse disulfide bond C98-C179 III:0-EC2 needed for high-affinity agonist binding but not receptor activation Feighner et al. Mol Endo 12:137 (1998) GHSR (growth hormone secretagogue receptor, different than GHRHR) III:0 C116 needed for binding, probably structural (SS bond) III:8 E124 <-> agonist primary amine V:3 M213 <-> some agonists Noda et al. JBC 270(48):28511 (1995) Noda et al. Biochem 335:16435 (1996) Miura et al. JBC 274(11):7102 (1999) AT1R rat angiotensin II H2N-DRVYIHPF-COOH III:10 N111 <-> Tyr4 receptor muts G>A not I,F constit (but see file dists; Balmforth postulates interaction with VII:14 N295) size and aromaticity important at 4 position (Cha decr affinity >200x and Emax ~50%, diiodoTyr decr affinity >300x) EC2 H183 <-> Asp1 weak (agonist ang III lacks Asp1) V:6 K199 <-> Phe8 COO- affinity, orientation of Phe8 sidechain (below) also arom interaction with tetrazoles Noda JBC 270(5):2284 (1995) VI:19 H256 <-> Phe8 agonism/efficacy aromaticity important at 8 position (Cha ~same affinity but decr Emax to ~40%); aromaticity at 256 needed for full act (H,Y but not Q,A) (but Han et al. Mol Endo 12:810 (1998) say mechanistic, not lig bind) EC3 D278 <-> Arg2 relatively weak Feng et al. JBC 270(21):12846 (1995) VII:0 D281 <-> Arg2 agonism/efficacy Hunyady et al. JBC 270(28):16602 (1995) AT1aR rat mutations -> Ala in conserved NPxxY; all internalize ~wt; indirect binding effect by F301A? also decr aff losartan, nonpept antag affinity: pept antag angII Gq cplng expr (wt=1) VII:17 N298A ~wt ~wt lg decr 1 VII:18 P299A ~wt ~wt decr 1/3 VII:19 L300A ~wt ~wt incr 1/3 VII:20 F301A ~wt decr >10x ~wt 1/3 VII:21 Y302A ~wt ~wt decr 1 Yamano et al. JGC 270:14024 (1995) AT1AR rat mutations significantly decreasing ATII affinity EC2 R167A V:6 K199A VI:16 W253A VI:22 F259A VI:26 D263A little affect on affinity EC2 H166A EC2 E173A EC2 E185A Hjorth et al. JBC 269(49):30953 (1994) AT1R residues important for binding ATII include NTD I14 I:-4 H24 I:-1 I27 II:32 Y92 EC3 D278 VII:0 D281 Ji et al. PNAS USA 92:9240 (1995) AT1bR rat binds losartan with high affinity, unlike amphibian xATaR; thirteen important residues (identified by chimera with xATaR) are II:13 D73 III:7 V108 III:8 S109 III:13 A114 III:14 S115 IV:1 A163 V:-1 P192 V:5 T198 VI:11 F248 VI:15 S252 VII:14 N295 VII:19 L300 VII:20 F301 Boucard et al. Biochemistry 39:9662 (2000) AT1R human Val3->Bpa ATII analog (agonist) crosslinks to EC2 (IV:30) M172 in I172M mutant Laporte et al. Mol Endo 13:578 (1999) AT1R human Asp1->Bpa ATII analog (agonist) crosslinks to VII:4-14 285-295 Phe8->Bpa ATII analog (antagonist) crosslinks to EC2 (IV:24-V:6) 166-199 Perodin et al. Biochemistry 41:14348 (2002) AT1R human Phe8->Bpa ATII analog crosslinks to VII:12,13 F293,N294 Clement et al. JBC epub May 12, 2005 AT1R human Phe8->Bpa ATII analog crosslinks to III:11,12 L112M,Y113M VI:12,16,19,23 F249M,253M,H256M,T260M VII:12-16 F293M,N294M,N295M,C296M,L297M Servant et al. JBC 272:8653 (1997) AT2R rat crosslinks with AngII Bpa1 analog<->NTD, Bpa8 analog<->III:12-21 Turner et al. Biochem Biophys Res Comm 257:704 (1999) AT2R rat V:6 K215 binding some peptide ligands VI:20 H273 binding some peptide ligands Fathy et al. JBC 273(20):12210 (1998) bradykinin (BK): RPPGFSPFR des-Arg BK: RPPGFSPF kallidin (KD): KRPPGFSPFR des-Arg KD: KRPPGFSPF BRB1R human high affinity for KD, des-Arg KD BRB2R human high affinity for BK, KD BRB1R not BRB2R high aff for kinins w/o Cterm R; by B1/B2 chimeras, III:8 B1 K118, B2 S138 <-> kinin Cterm (propose K+<->des-Arg COO-) Fathy et al. Mol Pharm 57:171 (2000) BRB2R not BRB1R high aff for kinins w/o Nterm K; by B1/B2 chimeras, EC3 (both halves) <-> kinin Nterm Leeb et al. JBC 272(1):311 (1997) B2 high aff for BK, low aff for des-R KD VI:19 (B1 Y266) B2 F286 VI:23 (B1 A270) B2 T290 Nardone and Hogan PNAS USA 91(10):4417 (1994) BRB2R rat mutations decreasing BK affinity VI:19 F261V 1600-fold VI:20 Q262A modest decr VI:23 T265A 700-fold VI:26 D268A modest decr VI:27 T269A modest decr Vincent et al. TIPS 20:303 (1999) neurotensin receptors NTS1R, NTS2R NTS1R NTD near I binding NT D139 EC1 binding NT Y347 EC3 binding NT R327 VI:22 binding NT, SR48692 (NTS1R-specific antagonist) Y351 VII:3 binding SR48692 Barroso et al. JBC 275(1):328 (2000) neurotensin NTS1R rat ND = not measurable or detectable agonist NT(8-13) = RRPYIL (SAR: last 3 residues more imp than first 3) effect on affinity interaction suggested by mutant mutation SR4862 (antag) NT(1-13) (ag) cycle anal w/ NT(8-13) analogs ----------------------------------------------------------------------------- D139A III:-3 ND ND ND (maybe conf, maybe ->R8,R9) M208A IV:25 ND decr 10x -> I12 > Y11, L13 R327M VI:22 ND ND -> COO- (Cterm; also in antag) F331A VI:26 ND decr 10x -> R9, L13 W339A EC3 ~wt decr 10x -> Y11, P10 F344A EC3 ~wt decr 5x -> Y11 Y347A EC3 ~wt decr 6000x -> Y11 (its major contact) Y347M EC3 ~wt decr 1000x Y347F EC3 ~wt decr 20x ----------------------------------------------------------------------------- apparently not NT-binding: III:1 R143 EC3 F346 EC3 Y349 other SR28692 contacts: VI:19 Y324 VII:3 Y351 VII:6 T354 VII:10 F358 VII:11 Y359 Postina et al. JBC 271(49):31593 (1996) oxytocinR binds oxytocin (OT) and arginine vasopressin (AVP) with high affinity; V2R binds AVP but not OT with high affinity S-S / \ AVP CYFQNCPRG-NH2 OT I L V2R selectivity against I3 involves EC2,TMD's; against L8 involves NTD,EC1 Hawtin et al. Biochem 39:13524 (2000) V1aR rat 37-47 in NTD very important for binding agonists and signaling but not for binding antagonists (cylic or linear peptides or nonpeptides); V1aR 1-54 can be subst with OTR 1-42, but separately expressed V1aR peptide 1-86 or 25-47 cannot make up for deletion of 37-47. Chini et al. EMBO J 14:2176 (1995) V1aR rat II:32 Y115 <-> R8 of AVP because Y115D (as in V2R) and Y115F (as in OTR) switch selectivity accordingly Ufer et al. FEBS Lett 362:19 (1995) V2R human, bovine, rat selectivity of agonist dDAVP (deamino D-Arg) due to II:32 D103 <-> D-Arg (dDAVP poor agonist at V1R's, porcine V2R) Hibert et al. J Receptor Sig Trans 19:589 (1999) V1aR selectivity for R8, against L8 of OT due largely to Y115 (EC1; F in OTR) V1aR rat (human #'s) role AVP binding Ki and efficacy Kact II:21 Q104A bind/mech? incr 6x incr 50x II:25 Q108A bind incr 290x incr 256x III:4 K128A bind incr 63x incr 53x III:7 Q131A bind incr 40x incr 43x IV:21 Q185A bind incr 1220x incr 1100x V:7 T221A ? decr 2x decr 2x VI:23 Q311A bind incr 8x incr 12x OTR human residues -> V1aR equivalents effect on AVP binding and efficacy V:11 Y509->F225 mech/efficacy affinity ~same partial ag -> full ag VI:12 F609->Y300 mech/efficacy affinity ~same partial ag -> full ag (AVP is partial ag at OTR in some systems; postulate positions 2 and 3 of the hormones interact with these aromatic residues in the receptor) Kojro et al. Biochem 32:13537 (1993) V2R bovine and agonist analog with photoactivatable group at R8 position crosslink via EC1 residues R106 > T102 EC1: 98-AWDATDRFRGPD-109 x X Chen et al. Biochem Biophys Res Comm 258:689 (1999) SSTR1 mouse 1 9 14 subtype-specif agonist somatostatin (SS): AGCKNFFWKTFTSC CH275: CKFFw-Iamp-TFTSC |__________| |______________| II:17 L107 (subtype-specific) <-> isopropyl group on CH275 Iamp III:7 D137 <-> (+) charge of SS K9, CH275 Iamp Kaupmann et al. EMBO J 14:727 (1995) SSTR2-selectivity of octreotide fCFwKTCT (lowercase means D-amino acid; -CH2OH instead of Cterm COOH) |____| due mainly to SSTR2 SSTR1 (octreotide binds well to SSTR1 hum Q291N/S305F) VI:23 N276 Q291 VII:3 F294 S305 Rose et al. FEBS Lett 362:243 (1995) Webb et al. Biochem 35:2548 (1996) ETAR (endothelin receptor) human; ET1 and ET3 are peptide agonists II:14 D126A no coupling to PLC, ET1 aff ~wt (but note ETAR does not have a GTP-analog-induced ET1 affinity shift), incr ET3 aff 160x II:17 Y129 mutations decr ET1 aff up to 10x (Y129A decr Emax 50-75%), incr ET3 aff up to 1367x, decr antag aff up to 1250x II:21 D133A decr aff of antag BMS-182874 80x Rose et al. FEBS Lett 362:243 (1995) ETBR human II:14 D147A no coupling to PLC; ligand affinity ~wt II:17 H150A,Y minor effects on ligand affinity II:21 D154 mutation ligand affinity ~wt Leong et al. JBC 269(30):19343 (1994) IL8AR (CXCR1) human muts -> Ala in EC regions (mainly single-IL8-conc evals) signif decr IL8 binding, but signaling >40% wt NTD T18, P21, Y27 (TM1 starts at 40) EC1 W103, G106 EC2 Y178, L191, G192, M200 EC3 M268, R269, I273, E275, T276, R280 signif decr IL8 binding and signaling NTD C30 III:0 C110 (disulfide C110-C187) EC2 C187, R199 V:3 R203 EC3 D265 (VI:(26)), C277 Skelton et al. Structure 7:157 (1999) IL8AR (CXCR1)-derived peptide complexed to IL8 NMR structure consistent with affinity changes for mutating the corresponding receptor residues NTD P21A decr IL8 affinity 31x Y27A 20x P29A 12x Rosenkilde et al. Curr Top Med Chem 6(13):1319 (2006) CCR1, CCR2, CCR5 propose Glu VII:7 anchors N+ of nonpeptide ligands Mirzadegan et al. JBC 275(33):25562 (2000) incr Kd (sat) IC50 CCR2 human hMCP1 hMCP1 spiropiperidine antags 2CP antags (VII:0) D284A 4x 5x 3-4x 0.64x (VII:0) D284N 26x 6x 1x 0.35x (VII:0) D284K >50x - - - VII:7 E291A 10x 9x >180x 2x VII:7 E291Q 12x 6x >180x 0.5x Howard et al. JBC 274(23):16228 (1999) CCR5 human RANTES bind MIP-1B bind chemotaxis NTD I12T n n n NTD C20S n n n NTD A29S n n n I:12 I42F aff incr 4x ~wt y, less desens at high conc I:25 L55Q aff incr 4x ~wt y, less desens at high conc II:11 A73V aff incr 7.8x ~wt y, less desens at high conc Govaerts et al. JBC 276(16):13217 (2001) CCR5 IC50s incr >10x EC50s incr >10x II:20 T82V (none) MCP2, MIP1b II:20 T82C (none) MCP2 II:20 T82A,S (none) (none) II:22 P84A MCP2, MIP1a, MIP1b MCP2, MIP1a, MIP1b for all these mutants, RANTES IC50, EC50 ~wt Blanpain et al. JBC 274(27):18902 (1999) CCR5 human extracellular Cys form disulfides III:0 - EC2 C101-C178 conserved in most family 1 receptors NTD - EC3 C20-C269 conserved in chemokineR's singly or collectively -> A: even after correction for moderately decr expression, abolished ligand binding/signaling, decr HIV1 coreceptor act Mills et al. JBC 273(17):10428 (1998) peptide crosslinking to FPR human fMLF analog fMXF, where X is a photoactivatable crosslinker, is an agonist and labels II:26-28 (83-85 VRK) Miettinen et al. J Immunol 159:4045 (1997) FPR mutations that retain expression and - decrease [uncoupled-state] affinity for f-Nle-LF-Nle-YK-fluorescein II:21 L -> A III:8 D -> N decr coupling D->A decr surface expression III:11 L -> A IV:18 T -> A V:2 R -> A decr coupling V:5 I -> Y V:6 R -> A decr coupling VI:16 W -> A VI:19 Y -> A Y->F little effect VII:11 F -> A - have little effect on affinity for f-Nle-LF-Nle-YK-fluorescein I:7 F -> L I:8 A -> G II:14 D -> A decr coupling II:17 F -> A III:3 L -> I III:4 F -> H III:5 T -> I III:7 V -> A IV:11 W -> A partly decr surface expression V:7 F -> A VI:20 Q -> A VII:4 D -> N VII:5 V -> P VII:7 S -> A Sun et al. Prot Sci 8:2304 (1999) C3aR human mutants' C3a binding, Ca++ signaling II:14 D68A ~wt ~wt EC1 (II:27) H81A decr decr III:1 K96A decr decr IV:25 R161A none very weak (expressed) EC2 (IV:26) E162A ~wt ~wt V:6 R240A none very weak (expressed) VI:20 H394A ~wt ~wt EC3 E406A ~wt ~wt VII:3 D417A none very weak (expressed) VII:4 H418A ~wt ~wt DeMartino et al. JBC 270(27):15966 (1995) C5aR V:6 R206 may interact with C5a R74 Cterm COO- Raffetseder et al. Eur J Biochem 235:82 (1996) C5aR mutations that retain expression but have decreased C5a binding; postulate indirect effect due to modulation of high/low affinity states affinity decrease affinity ~wt II:14 D82 A N V:6 R206 A,Q K VII:3 D282 A N Chen et al. JBC 273(17):10411 (1998) C5aR binding site in N-terminal domain of receptor "address" residues 21-30 Monk et al. JBC 270(28):16625 (1995) Crass et al. Biochem 38:9712 (1999) C5aR human EC2 E199 may interact with C5a K68 Vogen et al. J Peptide Res 53:8 (1999) C5aR human EC2 E199 may interact with K68 equivalent in agonist decapeptides based on C5a(65-74); apparently depends on tissue; receptor could have subtypes or conformational differences due to other proteins Pease et al. Mol Immunol 31:733 (1994) C5aR human - not part of binding surface: D82 (II:14), E179, E180 (EC2) suggested by little change in affinity with D->N, E->Q mutations Cain et al. Biochemical Pharmacology 61:1571 (2001) C5aR human muts->murine, effects >10x vs C5a, C5a-desR, peptide ags & antags; none of these changed IC50 >10x EC1 P103Y (none) III:-4 G105D MeFKP-dCha-W-dR EC50 incr 19x EC1/III:-4 P103Y/G105D C5a-desR EC50 incr 48x Nanevicz et al. JBC 170(37):21619 (1995) thrombinR Xenopus vs. human ligand specificity (caps more important) 87 (NTD) 260 (EC2) agonist peptide Xenopus pirrNitk dLkd TFRIFD human qlpaFise nEtl 260<->5a SFLLRN Blackhart et al. Mol Pharm 58:1178 (2000) thrombinR PAR-1 several point mutations decr efficacy of peptide agonists but not affinity of peptide agonists or efficacy of thrombin (which generates tethered peptide agonist) even if same sequence tethered/free EC50 thrombin EC50 peptide Ki SFLLRNP peptide NTD I88A ~wt incr >833x incr 2.5x NTD S89A ~wt incr 250x incr 2.5x NTD L96A ~wt incr 333x ~wt EC2 D256A incr 10x incr >500x ND EC2 D256N incr 8x incr 58x incr 2x EC2 E260A incr 5x incr 4x ~wt EC3 E347A incr 10x incr >500x incr 2x EC3 E347Q incr 9x incr >833x incr 3x mutations disrupting the conserved III:0-EC2 disulfide, however, significantly incr the EC50s of both thrombin and peptide Ishii et al. JBC 272(12):7846 (1997) PAFR guinea pig decr PAF binding: V:4 H188A VI:19 H248A postulate the three histidines bind the phosphate VI:20 H249A VI:23 Q252A VII:4 Q276A VII:6 Q278A incr PAF binding: II:9 N58A II:14 D63A III:10 N100A constit act III:11 T101A III:14 S104A VII:17 D289A Parent et al. JBC 271(38):23298 (1996) Parent et al. BBRC 219:968 (1996) PAFR human concluded none of the following directly contact ligand II:14 D63 important for coupling III:7/III:8 FF->GG only decr PAF aff 3x VII:13 N285I abolishes lig bind although expressed at surface; A ~wt VII:17 D289A uncouples yet incr PAF aff; N ~wt Huang and Tai Biochem J 307:493 (1995) PGE2 receptor mouse EP3 subtype VII:8 R309K but not E,V tight binding of PGE2; suspect salt bridge COO- Kobayashi et al. JBC 275(32):24294 (2000) mouse IPR (PGI R) vs. DPR (PGD R) I:7 S50 G IPR broad specif for PGI,PGE II:18 T K75 DPR high affinity for PGD2 II:26 F L83 DPR specificity for PGD Kedzie et al. Mol Pharm 54:584 (1998) human EP2R VII:10 L304Y PGE potency ~wt, iloprost (PGI2 analog) potency incr 100x VII:8 R302Q,E decr potency all prostaglandins tried (<->carboxylate) D'Angelo et al. JBC 271:6233 (1996) TXA2R (TPR) human platelet mutation of Cys residues NTD C11S ~wt II:8 C68S ~wt EC1 C102S relatively small changes in lig affinity (decr ~2-5x) III:0 C105S or EC2 C183S no lig binding (necessary SS bond; not expr?) VI:15 C257S ~wt Dorn et al. JBC 272(19):12399 (1997) agonist I-BOP selectivity for rat TPR (Kd 0.6nM) over human TPR (4.0 nM) mostly due to I:12 A36 V36 I:13 L37 V37 I:16 G40 A40 ---------------------------------------------------------------------------- secretin family (family B) (Baldwin numbering from Lichtarge alignment) (PTHR = PTH1R) ---------------------------------------------------------------------------- Gardella et al. Endocrinology 135:1186 (1994) PTH(1-34) V2R mutant has specificity for the rat vs. the opossum receptors: (but PTH(1-34) wt does not) in rat in oppossum - greater affinity for the rat receptor mainly 427 VI:22 L T and 371 V:15 V I - greater agonism at the opossum receptor partly 370 V:14 S A Turner et al. JBC 273(7):3830 (1998) PTH1R binds PTHrP and PTH, PTH2R is PTH-specific. Regions discriminating against PTHrP in PTH2R and corresp. PTH1R residues: III:8 I L VII:5 C Y VII:8 F L Bergwitz et al. JBC 272:28861 (1997) PTH1R and PTH2R both bind/respond to PTH and PTHrP, but PTH2R only binds/responds to PTH PTH and PTHrP position 5 (I or H) modulates specificity of activation whereas position 23 (W or F) modulates specificity of binding PTH1R/PTH2R chimeras reveal III:8 and V:7 (L, I in PTH1R; I, Y in PTH2R) may interact with ligand residue 5 to produce activation. However, the reciprocal mutations in PTH1R did NOT confer specificity. Hoare et al. JBC 275(35):27274 (2000) peptide TIP39 100x selectivity for PTH2R (where it is an agonist) over PTH1R (antagonist) is not due to the receptor Nterm/I, but the rest of the receptor interacting with the TIP Nterm; TIP(8-39) is an antagonist at both receptors and selective for PTH1R over PTH2R: affinity of TIP(8-39) vs TIP39 G-coupled uncoupled for PTH1R incr 6x incr 12x for PTH2R decr 70x decr 32x Bisello et al. JBC 273(35):22498 (1998) PTH1R human - photoreactive PTH agonist (crosslinker subst for Ala 1) labels VI:20 M425 crosslinker Bpa tried at 1-6 of PTH(1-34): affinity activation Bpa1 ~wt ~wt 2 decr decr 3-5 strong decr strong decr 6 decr ~wt Behar et al. JBC 275(1):9 (2000) PTH1R human - photoreactive PTHrP agonists, Bpa tried at 1-6 of PTHrP(1-36) analog: Bpa pos affinity activation Xlink 1 ~wt ~wt VI:20 M425 2 ~wt none (antag) VI:20 M425 and elsewhere in VI 3 decr 4 no bindg 5 no bindg 6 ~wt none (antag) but iodination decr binding thus Xlinking not examined Adams et al. Mol Endo 12:1673 (1998) PTH1R human - photoreactive PTH(1-34) agonist (crosslinker at Lys 13) labels within 182-189 (I:-2-0),I:1-5 probably at R186 I:2 Greenberg et al. Biochem 39:8142 (2000) PTH1R human - photoreactive PTH(1-34) agonist (crosslinker at Lys27) labels L261 EC1 Mannstadt et al. JBC 273(27):16890 (1998) PTH1R rat - photoreactive PTHrP agonist (crosslinker at 23), [Ile5,Bpa23,Tyr36] PTH-related protein(1-36)-amide, labels receptor within residues 23-40 (23 is presumed Nterm after signal cleavage) mutation decreased affinity of: NlePTH PTHrP T33A 5.0x 14x Q37A 2.3x 48x Shimizu et al. Endocrinology 142:3068 (2001) PTH peptide potency increased by substitutions 2A,12A,10A,11homoR,14W; analog of PTH(1-11) full agonist, pot/eff strongly decr by PTH1R human F184A I:0 L187A I:3 Carter et al. JBC 274(45):31955 (1999) PTH1R rat muts PTH(1-34) EC50 Emax (% wt) PTH(1-14) Emax (% wt) E182A I:-2 ~wt 115 113 V183A I:-1 ~wt 97 65 F184A I:0 incr 8x 94 14 hydrophobic best F184D I:0 incr 80x 78 4 F184E I:0 incr 29x 66 6 F184G I:0 incr 45x 70 9 F184H I:0 incr 8x 87 14 F184I I:0 ~wt 112 55 F184K I:0 incr 80x 77 5 F184L I:0 ~wt 105 46 F184M I:0 ~wt 90 47 F184R I:0 incr 24x 95 8 F184V I:0 incr 6x 84 17 D185A I:1 ~wt 93 133 D185I I:1 ~wt 96 161 D185K I:1 ~wt 108 181 R186A I:2 ~wt 100 9 R186I I:2 ~wt 94 25 R186K I:2 ~wt 104 123 L187A I:3 ~wt 93 5 hydrophobic best L187E I:3 incr 350x 19 9 L187H I:3 incr 22x 74 11 L187I I:3 ~wt 91 41 L187R I:3 incr 290x 27 10 L187V I:3 ~wt 92 26 G188A I:4 ~wt 102 146 M189A I:5 ~wt 106 93 I190A I:6 ~wt 86 9 Turner et al. JBC 271:9205 (1996) PTH and secretin receptors - reciprocal mutations in TM2 allow binding and response to the "other" agonist without attenuating response to the cognate agonist: opossum PTHR I234N and rat SR N192I, both II:21; PTHR R230 II:17 already shown important in PTH binding and signaling Vilardaga et al. Mol Endo 15:1186 (2001) PTH1R/secretinR chimeras lead to functional assignments 1-62 Nterm high PTH affinity 105-186 Nterm some PTH binding sites within; important for activation 146-186 Nterm especially important for activation by PTH V PTH binding III,VI activation by PTH Lee et al. Mol Endo 9:1269 (1995) PTHR homolog scanning with SecR sequences decreased agonist PTH(1-34) but not antagonist (weak partial agonist) PTH(3-34) binding: Nterm/I I:(-2-0),1-6 III:1-5,8-13 V:(-2-2),3,4 VI:20-25,(26-35) especially W437 (32) and Q440 (35) Bergwitz et al. JBC 271(43):26469 (1996) using calcitonin/PTH hybrids and chimeric receptors, determined that - Cterm of full-agonist peptides binds receptor Nterm, accounting for most of the binding affinity - hormone Nterm binds receptor TMD's, is required for activation, and contributes some affinity Conner et al. Mol Pharmacol 67:20 (2005) calcitonin-receptor-like receptor, with RAMP1 is CGRP receptor; all prolines mutated to alanine; two of these mutations impaired CGPR binding and receptor activation: P321A VI:10 phenotype rescued by additional mutation I325P P331A VI:20 van Eyll et al. Peptides 17:565 (1996) GLP1R effects of NTD mutations on GLP1 Kd (wt 0.3 nM) Q37K/K38A decr 5x (incr affinity) K38A incr 6x W39A,F no binding although surface-expressed R40A incr 2x E41D incr 18x Dong et al. JBC 274(2):903 (1999) SecR rat labeled within first 30 residues of Nterm domain by rat secretin analog [Tyr10,Bpa22]Sec-27 (potent full agonist) Dong et al. JBC 274(27):19161 (1999) SecR rat labeled at Val4 by rat secretin analog [Bpa6,Tyr10]Sec-27 (potent full agonist) Dong et al. JBC 275(25):26032 (2000) SecR labeled at L17 (NTD) by Sec Bpa22 analog (potent agonist, see 1999 ref) SecR labeled at L36 (NTD) by Sec Bpa26 analog (potent agonist) Dong et al. Mol Pharmacol 2008 May 8 Epub SecR rat labeled at F349 (EC3) by Sec Bpa5 analog Di Paolo et al. Receptors Channels 6:309 (1999) Di Paolo et al. FEBS Lett 424:207 (1998) SecR rat residues (numbering w/o signal pept) NTD -> middle, Cterm portions of secretin I:7 Y124 indirect? I:11 Y128 -> Asp3 II:17 R166 -> Asp3 II:24 K173 -> Asp3 II:25 D174 indirect? Gaylinn GH & IGF Res 9:37 (1999) GHRHR photoXlinker at GHRH positions 1 (analog has high aff but decr efficacy) <-> Cterm half of receptor Lys12 <-> NTD 21 <-> Cterm half Couvineau et al. JBC 271(22):12795 (1996) VIP1R human (rat) has low (high) affinity for PHI; chimeras identify human rat nonadditive discriminants EC1 Q207 H208 EC1 G211 A212 III:4 M219 V220 Lins et al. JBC 276(13):10153 (2001) VIP1R (VPAC1R) human residues very important for VIP binding NTD E36, M66, W67, D68, W73, P74, G109, W110 I:4 K143 II:25 D196 somewhat important NTD P87, F90 mutations with little effect NTD D38A, K65A, R78A, K91A, F93A, R99A, E108A Knudsen et al. FEBS Lett 503:126 (2001) VPAC1R human rel to wt, VIP IC50 EC50 GTP sensitivity (aff shift) IV:14 P266A incr 3x incr 24x ~wt V:10 P300A incr 2x decr 3x incr VI:8 L346A incr 2x incr 20x ~wt VI:10 P348A incr 3x decr 10x incr VI:11 L349A incr 3x incr 104x decr Solano et al. JBC 276(2):1084 (2001) VIP1R human binds agonist VIP, whose Nterm region mediates agonism certain mutations decr VIP affinity but incr VIP[D3N], VIP[D3Q] affinity; wt D3 interaction with basic residues apparently important for efficacy II:17 R188Q,L II:24 K195Q,I Gaudin et al. Biochem Biophys Res Comm 122:901 (1995) VIP1R human C->G mutants, all expressed at surface; disrupt VIP binding? 7 in Nterm domain 6/7 EC1 C208 no III:0 C215 no EC2 C288 yes Vertongen et al. Brit J Pharmacol 133:1249 (2001) VPAC2R human postulate R172 <-> VIP Asp3 VIP VIP-D3N VIP-D3Q EC50 EC50 Emax(%VIP) EC50 Emax(%VIP) wt 7 75 52 II:17 R172L 150 12 400 8 360 II:17 R172Q 190 2 150 2 150 II:24 K179I (inactive) decr decr II:24 K179Q 4000 decr decr ---------------------------------------------------------------------------- family 3 receptors ---------------------------------------------------------------------------- Litschig et al. Mol Pharm 55:453 (1999) mGlu1R-selective noncompetitive antag CPCCOEt binds human mGlu1R at VII:0 T815 VII:3 A818 corresp to human mGlu5R M802,S805; swapping these swaps selectivity Pagano et al. JBC 275(43):33750 (2000) mGlu5R-selective noncompetitive antag MPEP binds III:? P655 ? S658 VII:8 A810 corresp to human mGlu1R S668,C671,V823; swapping these swaps selectivity; note CPCCOEt and MPEP are competitive wrt each other Brauner-Osborne et al. JBC 274(26):18382 (1999) NTD residues important for binding agonist: mGlu1R S165 T188 and the aligned residues CaR (S147) S170 ()=less important, perhaps indirect/modulatory Hampson et al. JBC 274(47):33488 (1999) NTD residues known to be important for binding agonist: mGlu4R R78 S159 (S181) T182 ()=less important corresp to mGlu1R S165 T188 GBR1a S246 (S269) (GABA(B)R subunit) Galvez et al. JBC 274(19):13362 (1999) GABABR S246 (S269) (S270) Y470 and these incr ag aff, decr antag aff indirectly by favoring closed state S247A Q312A Galvez et al. JBC 275(52):41166 (2000) GABAB1R rat coexpressed with GABAB2R; mutants membr-expressed but incr EC50 in IP assay at least 5x for agonist GABA or agonist baclofen lobe I: S246A,P,T,N unmeasurable unmeasurable S269A 5x <1x S270A 8x 10x E465A 39x 16x D471A,E unmeasurable unmeasurable but not mutations at 247,249,250,265,266,268,187,192,458-60,463-5,470 lobe II: Y366A 33x becomes antagonist! E367A 2x 6x but not mutations at 312,313,315,316,365 postulate D471 interacts with GABA N+ and S246,Y366 interact with COO- ---------------------------------------------------------------------------- STE2 S. cerevisiae mutations that reduce alpha-factor affinity I F55V decr 10x V S219P decr 8x VI S259P decr >10x Abel et al. Biochim Biophys Acta 1448:12 (1998) STE2 S. cerevisiae alpha-factor (WHWLQLKPGQPMY) receptor alanine scanning 262-270 VI Y266A no transduction but alpha-factor affinity only decr 6x; dominant negative (sequesters G protein); vs wt, incr affinity for alpha-factor analogs with ala subst 1-4 decr affinity for alpha-factor analogs with ala subst 5-13 Y266S,L,K also signaling-defective Y266F,W retained function Lee et al. Biochemistry 41:13681 (2002) ----------------------------------------------------------------------------