-------------------------------------------------------------------------- MTSEA (hydrophilic Cys-specific) labeling of adenosine A1R human Dawson and Wells Mol Pharm 59:1187 (2001) water-accessible from EC side (reacts with MTSEA): VII:3,5,6,7,9,11,21 (-2-29 tested) -------------------------------------------------------------------------- cannabinoid CB1R human Fay et al. Biochemistry 44(24):8757 (2005) water-accessible from EC side (reacts with MTSEA, MMTS): VII:10 C386 -------------------------------------------------------------------------- SCAM studies of the AT1R human Martin et al. JBC 279:51415 (2004) TM3 Boucard et al. JBC 278:36628 (2003) TM7 Domazet et al. JBC Epub Mar 9 2009 TM2 Domazet et al. JBC Epub Sep 22 2009 TM5 water-accessible from EC side (reacts with MTSEA): II:14,21,25,28,29 III:3,11 (high) 10 (low) (2-26 tested) the pattern is changed in the presence of constit act mut N111G III:10, III:3,11,15 (high) 8 (low) (and strangely, MTSEA incr lig binding to the III:12 mutant) V:4,7,8,10,11 (most positions in range -3-24 tested) the pattern is changed in the presence of constit act mut N111G III:10, V:8 became more accessible, V:10 less accessible VII:-4,1 (high) -1,5,10,20 (med) 2 (low) (-5-21 tested; -3,0,3,4,14 not used since Cys mutation disrupted binding of radiolabeled ligand) the pattern is changed in constit act mut N111G III:10, especially incr reactivity for VII:10 and decr for others and incr for II:26 and decr for II:14,21 -------------------------------------------------------------------------- phenoxybenzamine (PB; hydrophilic Cys-specific) labeling of adrenergicRs Frang et al. JBC 276(33):31279 (2001) III:10 NOT labeled in beta2AR human, alpha2aAR human F116C/C117V III:11 labeled in alpha2a,b,c AR human, beta2AR human V117C -------------------------------------------------------------------------- chloroethylclonidine (CEC, agonist) and MTSEA labeling of alpha2aAR human Marjamaki et al. Mol Pharm 53:370 (1998) Marjamaki et al. JBC 274(31):21867 (1999) CEC MTSEA V:3 197 + + V:4 198 - not determined V:5 199 - not determined V:6 200 + > + V:7 201 + + V:8 202 - - V:9 203 - - V:10 204 + + "-" nonzero but signif less inhib than "+", likely due to other positions; differences between labels could be due to size, shape, R->R* due to agonism -------------------------------------------------------------------------- IANBD-labeled beta2AR human mutants (fluorescent) Kobilka et al. Gether et al. EMBO J 15:6737 (1997) decreased fluorescence (decreased hydrophobicity of environment) upon act: III:19 125 VI:15 285 little change II:12 77 III:10 116 (VI:-5) 265 VII:22 327 tail 378, 406 Jensen et al JBC 276(12):9279 (2001) fluorescence change upon agonist act I- accessibility, change upon act III:19/VI:15 decrease (decr hydrophob) low not much VI:-1 269 apparently small VI:0 270 apparently small VI:1 271 increase (incr hydrophob) med sl decrease VI:2 272 increase (incr hydrophob) med sl decrease -------------------------------------------------------------------------- fluorescein labeling of beta2AR human Kobilka et al. Ghanouni et al. PNAS USA 98(11):5997 (2001) fluorescein attached at VI:-5 C265; upon activation in micellar system decr fluorescence intensity (incr intrareceptor quenching) decr quenching by KI (aqueous) incr quenching by NHS attached at K224 V:(27) incr quenching by lipid headgroup (but not by lipid middle region) -------------------------------------------------------------------------- MTSEA (hydrophilic Cys-specific) labeling of beta2AR human Javitch et al. Javitch et al. JBC 272(30):18546 (1997) CAM: VI:-4,-3,-1,2 mutated Rasmussen et al. Mol Pharm 56:175 (1999) III:24 D130N mutant accessible in constitutively active but not unstimulated wt receptor: VI:15 C285 -------------------------------------------------------------------------- SCAM studies of the D2R (various Cys-specific MTS reagents) Javitch et al. Shi et al. Biochem 40:12339 (2001) TM1 Javitch et al. Biochem 38:7961 (1999) TM2 Javitch et al. Neuron 14:825 (1995) TM3 Javitch et al. Biochem 39:12190 (2000) TM4 Javitch et al. Biochem 34(50):16433 (1995) TM5 Javitch et al. Biochem 37(4):998 (1998) TM6 Fu et al. Biochem 35(35):11278 (1996) TM7 water-accessible from EC side (reacts with MTSEA): I:4,17,18,24,25 (4-25 tested) 21 maybe false neg, found with MTSHTEA II:14,15,17,21,23-26,28-30 (11-32 tested) III:1-4,8,11,14,15,18,22 (1-23 tested) IV:11,14,15,17,18,20-24,26 (5-26 tested) V:2-11,13,14,22 (0-23 tested) VI:8,12,16,18-20,22-24,27 (7-28 tested) VII:2-8,11,13,17,18,21 (1-26 tested) Shi and Javitch PNAS USA 101(2):440 (2004) EC2 consistent with structure like that in rhodopsin -------------------------------------------------------------------------- bromoACh (weak partial agonist) and MTSET labeling of m1R rat Cys mutants Allman et al. Mol Pharm 58:175 (2000) BrACh MTSET V:2 I188 - - V:3 T189 - - V:4 F190 - + V:5 G191 - - V:6 T192 + + V:7 A193 - + V:8 M194 - - V:9 A195 - - V:10 A196 - - "-" nonzero but signif less inhib than "+"; "+" signif greater than wt -------------------------------------------------------------------------- MTSEA (hydrophilic Cys-specific) labeling of opioidRs Javitch et al. Xu et al. Biochem 39:13904 (2000) overall sensitivity reflected in second-order rates: 34.3 human kappa-OR 3.7 rat mu-OR 0.42 human delta-OR sensitivity of each mostly due to cysteine at VII:6 Xu et al. Biochem 40:8018 (2001) SCAM opioidRs x = accessible in all three, VI:18,20 muts no radiolig bdg rat mu human delta human kappa compare D2R (ref above) VI:6 VI:7 VI:8 x - - x VI:9 VI:10 x - - VI:11 VI:12 x x x x VI:13 VI:14 VI:15 VI:16 x - x x VI:17 VI:18 x VI:19 x x x x VI:20 x VI:21 VI:22 x x x x VI:23 x x x x VI:24 x x x x VI:25 x x x VI:26 x x x VI:27 x x x x VI:28 - x x -------------------------------------------------------------------------- spinlabel-Cys mutants of bovine rhodopsin Khorana, Hubbell Altenbach et al. Biochem 38(25):7945 (1999) 59-75 IC1 Farahbakhsh et al. Biochem 34(27):8812 (1995) 136-155 IC2 Altenbach et al. Biochem 35(38):12470 (1996) 225-256 IC3 Altenbach et al. Biochem 38(25):7931 (1999) 306-322 "IC4" apparent aqeuous/hydrophobic boundaries: I:26-27 63-64 apparently helical -> 64 I:27 II:2-3 71-72 apparently helical 71 <- II:2 III:28 138 apparently helical -> 140 III:30 IV:2 152 not determined V:26-30 227-231 apparently helical -> 237 (V:36) VI:1-5 250-254 apparently helical 240 <- (VI:-9) VII:23-24 308-309 apparently helical -> 314 (VII:29) increased motional freedom upon activation: III:26,28-30 136,138,139,140 IC3 244 VI:1,2,4 250,251,254 Cterm 313,316(?) decreased motional freedom upon activation: IC2 147,149 IV:3,4 153,154 IC3 227,231 -------------------------------------------------------------------------- reagent accessibility of Cys mutants of bovine rhodopsin Dunham and Farrens JBC 274:1683 (1999) PyMPO dark MII mBBr, dark->MII VI:-1 248 + + ~same VI:0 249 + + ~same VI:1 250 - + + incr. hydrophob. yet incr. I- quenching VI:2 251 + + ~same VI:3 252 + + ~same VI:4 253 - + - VI:5 254 - - VI:6 255 + - VI:7 256 - - Klein-Seetharaman Biochem 38(25):7938 (1999) 4-PDS added to dark state mutants 56-75 I:18-27 and 11 more residues four reactivity groups: high 63,65-70,73 I:26, IC1, II:4 intermediate 60,62,64,72,74 I:23,25,27, II:3,5 low 61,71 I:24, II:2 none 56-59, 75 I:19-22, II:6 reactivity of labeled Cys to DTT removal of label incr upon activation: 68 IC1 70-72 II:1-3 in Baldwin model face "DRY" III:24-26 decr upon activation: 69,73 II:0,4 Cai et al. Biochem 38(25):7925 (1999) 4-PDS added to dark state mutants 306-321 VII:21-26 and 10 more residues high 310,312,313,315 intermediate 308,309,311,314,316,319 low 306,307 none 317,318,320,321 (322,323 are palmitoylation sites) -------------------------------------------------------------------------- MTSEA-biotin SCAM variant on Ste2p S. cerevisiae (alpha-factor receptor) Lin et al. Biochem 42(2):293 (2003) estimated EC end of V, 199-208: accessibility drops gradually over 206-212 estimated EC end of VI, 266-275: accessibility drops more abruptly over 268,269 all Cys mutants functional except 204,266 sterile/dom neg and 205,273 only partially responsive to alpha-factor by halo assay Hauser et al. JBC (2007) Feb 9 Epub EC1 changes upon binding agonist and antagonist summarized in Table 2 --------------------------------------------------------------------------