---------------------------------------------------------------------------- data on possible TM-TM contacts and/or relative orientations (note that in some cases there are counter-data, not included here) ---------------------------------------------------------------------------- I-VII/VIII Liu et al. JBC 270:19532 (1995) m2/m5 chimeras - restoration of agonist binding and signaling suggests interaction between I:7 and VII:4 (compatible: A/T as in m2R, T/H as in m5R, T/E, T/N; expressed at surface but not viable: T/T, T/A) Yang et al. Biochem 35:14040 (1996) bovine rhodopsin (I:28) <-> Cterm (VII:31) nitroxide spinlabels 65<->316 dark state 10 +/- 3 A, activated 15 +/- 4 A apart unlabeled Cys at these positions capable of disulfide bond formation Struthers et al. Biochem 39:7938 (2000) rhodopsin Cys mutant still functions with SS bond 65-316 I:28<->VIII Klein-Seetharaman et al. Biochemistry 40:12472 (2001) rhodopsin Cys mutant pairs 316 + 55-75 examined for dark-state SS formation VIII 316 I:24 61 ++ VIII 316 I:24 62 + VIII 316 I:24 64 + VIII 316 I:28 65 ++++ VIII 316 IC1 66 ++ VIII 316 IC1 67 ++ VIII 316 IC1 68 +++ VIII 316 IC1 69 ++ Cai et al. Biochemistry 40:12479 (2001) rhodopsin Cys mutant pairs examined for dark-state SS formation: 246 + 311-314; 250 + 311-314; 65 + 306-321 I:28 65 VII:25 310 + I:28 65 VII:25 311 + I:28 65 VII:25 312 ++ I:28 65 VII:25 313 + I:28 65 VII:25 314 + I:28 65 VII:25 315 +++ I:28 65 VII:25 316 ++++ Hamdan et al. Biochemistry 41:7647 (2002) m3R rat disulfide formation between double Cys constructs (low Cys backgrd) in situ (native membrane environment) 532 VII:10 + 71-80 I:4-13; only crosslinked pair (not affected by ag,antag) I:10 77 VII:10 532 these two also crosslinked by bifunctional linker p-PDM ~10 A but not o-PDM ~6 A or BMOE ~8 A Li, Wess, et al. JBC epub Jul 10, 2007 m3R rat crosslinks inhib by agonist (carbachol), stim by antag (atropine) I:24 91 VII:27 549 (probably in helix VIII) I:25 92 VII:28 550 (probably in helix VIII) Yeagle et al. Biochem 36:9649 (1997) NMR structure of complex of rhodopsin intracellular portions - like R*? salt bridge (I:29) <-> Cterm K66-D330 II-III Elling et al. EMBO J 15:6213 (1996) antagonistic His2-Zn++ bridge Elling et al. Fold Des 2:S76 (1997) NK1R (II:28) <-> III:3 ()=not in Baldwin 3D model Lagerstrom et al. JBC published online 09/30/03 MC4R human activating Zn++ bridges I104H <-> D122 II:28 <-> III:0 E100 <-> I125H II:24 <-> III:3 II-VII Rao et al. Nature 367:639 (1994) II:21 rhodopsin bovine G90D can act as VII:11 Schiff base counterion Zhou et al. Mol Pharm 45:165 (1994) Flanagan et al. JBC 274(41):28880 (1999) complementation/rescue of expression GnRHR II:14 N87 (D) interacts with VII:17 D318 (N) Sealfon et al. JBC 270:16683 (1995) 5HT2AR II:14 interacts with VII:17 IPmax II/VII wt(D/N) 100% > D/D,N/D ~50% > N/N,D/A,A/N 0% Donnelly et al. Biochem J 339:55 (1999) NK2R activation complementation of II:14 D79N by VII:17 N303D Miettinen et al. JBC 274(39):27934 (1999) FPR human complementation of II:14 D71N by VII:17 N297D Wilson et al. Mol Pharm 59:929 (2001) alpha2A AR coupl/stab partial complementation of II:14 D79N and VII:17 N422D Xu et al. FEBS Lett 447:318 (1999) mu opioidR rat bind/act complementation II:14 D114N and VII:17 N332D Gardella et al. JBC 271:12820 (1996) PTHR, by Lichtarge alignment, II:17 R233H (also Q,N for human PTH(1-34) deriv) bovine PTH(1-34) binding defect but not signaling complemented by VII:13 Q451K (TM-TM contact or ligand contact??) Thomas et al. Mol Endocrinol 2008 Feb 7 Epub PTHR, by Lichtarge alignment, VII:9 F447C crosslink formation to II:22 F238C disrupted by PTH binding II:24 K240C enhanced by PTH binding II:25 D241C nonexistent (no crosslink) II:26 A242C enhanced by PTH binding Mills et al. JBC 273(17):10428 (1998) FPR & related (II:28) K and VII:4 D correlation suggests possible ion pair Marie et al. JBC 269(33):20815 (1994) AT1R rat II:14 D74 and VII:11 Y292 may interact in the ON state or during its formation; D74E,N and Y292F maintain ag/antag affinities but impair coupling severely (evidence weak) Donohue et al. Biochem 38:9366 (1999) gastrin-releasing peptide GRPR mouse (rhodopsin family) salt bridge D98-R309 II:25-VII:7 in ON state (coupling to Gq) III-V Elling et al. EMBO J 15:6213 (1996) antagonistic His2-Zn++ bridge Elling et al. Fold Des 2:S76 (1997) NK1R III:4 <-> (V:-1) ()=not in Baldwin 3D model Lu and Hulme, JBC 275(8):5682 (2000) m1R rat Zn++ bridge between His substs (based on inhibition of NMS binding) did NOT form between III:18 and V:22 or between III:22 and V:22 Yu et al. Biochem 38:12028 (1999) Yu and Oprian Biochem 38:12033 (1999) rhodopsin bovine, SS bond formation within split receptors; spectra suggest SS-bonded receptors are folded normally; with oxidant III:30 C140 <-> V:21 C222 in dark, incr rate of formation in light BUT prevents activation of transducin ---------------------------------------------------- tried (w/o oxidant) found ---------------------------------------------------- 140 vs. 218-225 140-225 III:30-V:24 130-142 vs. 222 136-222 III:26-V:21 inhib Gt activation 136-142 vs. 225 140>139>136-225 III:30>III:29>III:26-V:24 136 vs. 218-225 136-225>222>>223 III:26-V:24>V:21>>V:22 Struthers et al. Biochem 39:7938 (2000) rhodopsin Cys mutant still functions with SS bond 140-225 III:30<->V:24 also note native SS bond 110-187 III:0<->EC2 Sommers and Dumont JMB 266:559 (1997) STE2 yeast M218T (V) suppresses loss-of-function E143K or T144P (III) III-VI Han et al. JBC 271(50):32337 (1996) Han et al. JBC 272(37):23081 (1997) rhodopsin G121->bigger III:11 complemented by F261->smaller VI:12 (direct interaction, or through chromophore or other residues??) Sheikh et al. Nature 383:347 (1996) rhodopsin mutant His-Zn++-His bridge 138-251 III:28 <-> VI:2 inhibitory Sheikh et al. JBC 274(24):17033 (1999) CA-CA dist in Bldwn B2AR mut inhibitory His-Zn++-His bridges III:28 <-> VI:-2 8.7 III:28 <-> VI:-1 10.3 III:28 <-> VI:2 8.8 ...but not III:28 <-> VI:0 12.6 III:28 <-> VI:1 10.2 III:28 <-> VI:3 12.5 PTHR mut inhibitory His-Zn++-His bridges III:26 <-> VI:2 7.3 (Lichtarge alignment) III:26 <-> VI:3 10.3 ...but not III:26 <-> VI:-2 10.6 III:26 <-> VI:-1 10.6 III:26 <-> VI:0 12.5 III:26 <-> VI:1 10.4 Lu and Hulme, JBC 275(8):5682 (2000) m1R rat Zn++ bridge between His substs (based on inhibition of NMS binding) III:18 <-> III:22 (intrahelical) III:18 <-> VI:12 ...but not III:22 <-> VI:12 Farrens et al. Science 274:768 (1996) distances (A) rhodopsin mutants/spinlabels dark state activated SSbond CA-CA in Bldwn 139 III:29 + 247 VI:-2 ? Y 7.0 139 III:29 + 248 VI:-1 12-14 23-25 Y 7.5 139 III:29 + 249 VI:0 15-20 15-20 Y 10.4 139 III:29 + 250 VI:1 15-20 12-14 Y 9.1 139 III:29 + 251 VI:2 12-14 23-25 Y 6.9 139 III:29 + 252 VI:3 15-20 23-25 N 10.2 all SSbonds inhibitory to transducin activation Zeng et al. JBC 274(23):16629 (1999) m3R rat background construct m3R' with 10/13 C->A or S, glycosylation sites N->Q, and altered IC3 was found to be fully functional; additional Cys muts ability to SSbond and activate G prot oxid vs. red (except as noted Emax 40-90% of m3R' when not xlnkd) CA-CA in Bldwn 139 III:29 + 484 VI:-3 + decr signif 10.8 139 III:29 + 485 VI:-2 Emax v low + decr signif 7.0 139 III:29 + 486 VI:-1 ++ decr signif 7.5 139 III:29 + 487 VI:0 ++ abolished 10.4 139 III:29 + 488 VI:1 ++ abolished 9.1 139 III:29 + 489 VI:2 ++ abolished 6.9 139 III:29 + 490 VI:3 ++ decr signif 10.2 139 III:29 + 491 VI:4 + abolished 12.2 139 III:29 + 492 VI:5 - unchanged 10.8 139 III:29 + 493 VI:6 - unchanged 11.5 Sommers and Dumont JMB 266:559 (1997) STE2 yeast Y266C (VI) suppresses loss-of-function E143K (III) III-VII Elling et al. PNAS USA 96(22):12322 (1999) beta2AR activating metal-ion site D113H III:7 and N312C VII:7 Holst et al. Mol Pharm 58:263 (2000) NK1R activating metal-ion site P112D or H III:7 and M291C VII:7 Lu and Hulme, JBC 275(8):5682 (2000) m1R rat Zn++ bridge between His substs (based on inhibition of NMS binding) III:18 <-> VII:17 ...but not III:18 <-> VII:21 III:22 <-> VII:17 III:22 <-> VII:21 Robinson et al. Neuron 9:719 (1992) rhodopsin counterion Glu113 III:3 to Schiff base Lys296 VII:11 salt bridge helps maintain OFF state Zhukovsky et al. Biochem 31:10400 (1992) rhodopsin Schiff base VII:11 counterion can be supplied by D117 III:7 and to a lesser extent D120 III:10 Porter et al. JBC 271:28318 (1996) alpha 1B AR salt bridge between Asp125 III:7 and Lys331 VII:4 helps maintain OFF state; bulky residue at III:10 thought to disrupt this (somewhat weak; no swap/complementation) Balmforth et al. JBC 272(7):4245 (1997) AT1R since mutating either N->S apparently constitutively activates, postulate OFF state interaction between N111 and N295, III:10 and VII:14 (evidence weak) IV-VI Yeagle et al. Biochem 36:9649 (1997) NMR structure of complex of rhodopsin intracellular portions - like R*? salt bridge (IV:0) <-> VI:-4 E150-K245 V-VI Elling et al. Fold Des 2:S76 (1997) antagonistic His3-Zn++ bridge NK1R (V:-1) and/or V:3 <-> (VI:27) ()=not in Baldwin 3D model Thirstrup et al. JBC 271(14):7875 (1996) antagonistic His3-Zn++ bridge kappa-opioid R (V:-1),V:3 <-> (VI:27) ()=not in Baldwin 3D model Fowler et al. Biochem 43:8700 (2004) engineered Zn++ site mu-opioid R rat I234C V:4, F237C V:7, H297 VI:20, V300C VI:23 Elling et al. Biochem 39:667 (2000) NK1R SS bridge V:3 <-> (VI:27) 25% spontaneous, more with oxidant Struthers et al. Biochem 38:6597 (1999) Yu et al. Biochem 34:14963 (1995) split rhodopsin (in IC3) SS bridges 198 (V:-3), 200 (V:-1), or 204 V:3 with 276 (VI:27); function fairly normally Struthers et al. Biochem 39:7938 (2000) rhodopsin Cys mutant still functions with SS bond 204-276 V:3 <-> (VI:27) Hwa et al. JBC 271:7956 (1996) alpha-AR V:3 V/A interact with VI:23 M/L, respectively, to maintain OFF state (alpha 1A V185,M292; alpha 1B A204,L314) Ward et al. JBC 277(3):2247 (2002) m3R rat disulfide formation between double Cys mutants (in low Cys backgrd) in situ (native membrane environment) 254 V:26 + 484-493 VI:-3-6 basal and agonist-dependent V:26 254 VI:2 489 + incr V:26 VI:3 490 - incr V:26 VI:4 491 - incr V:26 VI:5 492 - incr V:26 VI:-3-1,6 484-488,493 (no disulfide formation) Dube et al. JBC 275(34):26492 (2000) STE2 yeast not constit act alone, only when combined (V-VI pairs) V223C L247C also spontaneously formed SS bond V223C L248C V223C S251C L226C M250C L226C S251C Lee, Naider, and Becker. JBC Epub 2005 Nov 28 STE2 yeast N205 (V) and Y266 (VI) interact in active state only, based on effects of double mutants including SS bond formation in Cys mutants VI-VII/VIII Cai et al. Biochemistry 40:12479 (2001) rhodopsin Cys mutant pairs examined for dark-state SS formation: 246 + 311-314; 250 + 311-314; 65 + 306-321 VI:-3 246 VIII 311 +++ VI:-3 246 VIII 312 +++ VI:-3 246 VIII 313 + VI:1 250 VIII 312 + Lu and Hulme, JBC 275(8):5682 (2000) m1R rat Zn++ bridge between His substs (based on inhibition of NMS binding) VI:12 <-> VII:17 VI:12 <-> VII:21 Kosugi et al. Mol Pharm 53:894 (1998) LHR human D VI:12 <-> N VII:17 in OFF state, complmtation of activating muts Govaerts et al. JBC 276(25):22991 (2001) TSHR human VI:12 <-> VII:17 in OFF state, N674D constit act reverted by D633N Yeagle et al. Biochem 36:9649 (1997) NMR structure of complex of rhodopsin intracellular portions - like R*? salt bridge VI:0 <-> VII:26 E249-K311 VII-other Mizobe et al. JBC 271:2387 (1996) beta2/alpha2 chimeras suggest VII:5 faces outward, VII:6 faces III and VI, and VII:7 faces I and II ---------------------------------------------------------------------------- III-tail Yu et al. Biochem 38:12028 (1999) rhodopsin bovine, SS bond formation within split receptors III:30 C140 <-> Ctail C316 in light state only IC3-tail Cai et al. PNAS USA 92:14267 (1997) rhodopsin mutant SS bonding between C338 (tail) and C242 (IC3) slower in illuminated state Granier et al. JBC Epub 2007 Mar 8 B2AR FRET between fluorophore attached at C265 and FlAsH at two sites in the C-term tail indicate distances 57A, 62A (unbound); all ligands shorten distance to the proximal FlAsH site, some ligands increase distance to the distal site ----------------------------------------------------------------------------