----------------------------------------------------------------------------- G protein residues implicated in binding to receptor A = helix, B = strand ----------------------------------------------------------------------------- ALPHA Kostenis et al. JBC 272(31):19107 (1997) removal of the 6-residue Nterm extension of Gq/11 enables more promiscuous coupling (to some but not all receptors normally coupled to q/11, i/o, or s) Mody et al. Mol Pharm 57:13 (2000) alpha16 is relatively promiscuous (couples to many q,i,s-coupled receptors); swapping tail of alphaz for that of alpha16 (16z44>16z25) increases coupling to some i-coupled receptors, decreases coupling to some s-coupled receptors Krieger-Brauer et al. JBC 274(40):28308 (1999) alphas 100-119 (alpha-helical domain: end of alphaA,loop) may contact and mediate activation by agonist-bound B2AR; - polyclonal Ab to (100-119) activates alpha- and betagamma-med responses - peptide (100-119) inhibits isoproterenol activation of alpha- and betagamma-mediated responses - tail peptide (385-394) only inhibits isoproterenol activation of alpha-mediated responses Bae et al. JBC 274(21):14963 (1999) 5HT1BR couples to Gi/o not Gt although Cterms are very similar; chimeras used to identify determinants i1 t in A4 Q304 K300 E308 L304 Bae et al. JBC 272(51):32071 (1997) based on alphai1/t chimeras, the alphai1 A4-B6 region (299-318) is important for coupling to the 5HT1BR as assessed by agonist affinity shift and Galpha GTPgS binding; the Nterm 219 residues of alphai1 also contribute to the interaction Rasenick et al. JBC 269(34):21519 (1994) alphas peptides block BAR coupling and stabilize the high-agonist-affinity in a partially additive manner: 354-372 (end of A4,loop,B6) 384-394 (Cterm) but not alphas 15-29 or three analogous alphai peptides Grishina and Berlot, Mol Pharm 57:1081 (2000) alphas A3/B5 loop (280,284,285) but not A4/B6 loop involved in B2AR/isoproterenol-mediated activation; probable receptor contact Garcia et al. Embo J 14:4460 (1995) Ala scanning Gtalpha last ten residues, susceptibility to act by rhodopsin moderate impairment K341A severe impairment L344A, G348A, L349A very mild impairment N343A, F350A no effect E342A, K345A, D346A, C347A Onrust et al. Science 275:381 (1997) Ala scanning Gtalpha, residues implicated in binding rhodopsin: A2-B4 loop: V214 A4-B6 loop: R309,D311,V312,K313 A5: F330,F332,D337,I338,I340,K341 Cterm: N343,L344,C347,G348,L349,F350 Marin et al. JBC 276(29):27400 (2001) Ala scanning A5 helix of Gtalpha (325-339) decreased rate of R*-catalyzed activation (possible R*-binding residues) F330A, V331A, D337A, I338A rate of R*-catalyzed activation ~wt Q326A, K329A, D333A, V335A, T336A increased rate of R*-catalyzed activation (also basal rate) T325A, Q327A, V328A, F332A, I339A (note results for 331,332 differ from those of Onrust above) Natochin et al. JBC 274(12):7865 (1999) Ala scanning Gt/i alpha chimera (16 residues in A3-B5 region from alphai) in the A4/A4-B6 loop region (293,294,297-306,308-314); residues implicated in binding R* (rhodopsin MII): importance D311>R310 Natochin et al. JBC 275(4):2669 (2000) alphas QRMHLRQYELL alphat IKENLKDCGLF by substitution of transducin residues into the Cterm of alphas, alphat residues important in activation by R* (rhodopsin MII): alphat (340-350) sufficient; D346 modestly involved C347, G348 essential Martin et al. JBC 271(1):361 (1996) Aris et al. JBC 276(4):2333 (2001) transducin Cterm peptide (340-350) SAR for stabilizing rhodopsin MII: 340 I hydrophobic; N-acetylated slightly better K L>R>K; Q,C also in potent peptides E E,Q,K N N,D,V L hydrophobic K R sometimes better D D,E,S C C,Abu,M,V>A>S,D G G,D-Ala>A>L (type II' beta-turn?) L hydrophobic 350 F hydrophobic; amidated slightly worse Hamm et al. Science 241:832 (1988) transducin peptides 311-328 block transducin stabilization of MII: - 8-23 does not stab MII; could bind rhodopsin and/or disrupt heterotrimer - 311-328 but not {311-19 + 320-328} or 311-328[C321S]; acetyl-311-329 has 20x higher affinity and can stab MII - 340-350 can stab MII Ernst et al. JBC 175(3):1937 (2000) transducin alpha peptide (340-350) (IC50 50 microM) stabilizes MII but (340-350)K341R/L349A does not; stabilization lost for rhodopsin mutant 310-312 NKQ -> SPD (as in B2AR) Cai et al. PNAS USA 98:4877 (2001) rhodopsin photoactivatable crosslinker attached at S240C (IC3, VI:(-9)); MII bound mainly transducin alpha 310-313 (A4-B6 loop) 342-345 (Cterm continuation of A5) Itoh et al. PNAS USA 98:4883 (2001) rhodopsin crosslinker attached at S240C (IC3, VI:(-9)) reacts with neutral Lys (thus triggered by raising pH); MII bound mainly transducin alpha 19-28 (Nterm helix) Lee et al. Mol Pharm 47:218 (1995) C5aR couples to G16 not G11; 11/16 chimeras tested for coupling 1-237/241-374 no activity 1-216/220-374 65% activity 207-359/1-209 40% activity 151-359/1-153 no activity thus specificity determinants in mult regions Conklin et al. Nature 363:274 (1993) A1R and D2R do not couple to alphaq, but couple to qi4,qi5,qz5, and qo5 q E Y N L V i2 D C G L F o G C G L Y z Y I G L C Conklin et al. Mol Pharm 50:885 (1996) in q/i chimeras, -3 and -4 are critical in coupling specificity; chimeras with the last 5 residues swapped do not always couple to the expected receptors (q5s couples to V2R but not B2AR; z5q couples to bombesin and V1aR but not oxytocinR) Kostenis et al. Biochem 26:1487 (1997) mutated residues in Cterm of q -> i, looked for coupling to wt m2R Gq E Y N L V | | | each of these 3 single mutants could couple to m2R Gi1,2 D C G L F to some extent Gi3 E C G L Y Kostenis et al. JBC 272(38):23675 (1997) Gs Q Y E L L Gq/11 E Y N L V mutated residues in Cterm of Gsalpha, looked for coupling to Gq/11-coupled receptors: m3R V1a vasopressin gastrin-releasing peptide Gs wt n n n Gs -5 Q->E y y n Gs -3 E->N y y y Wurch et al. Mol Pharm 60:666 (2001) Go C351I G I G L Y Gs Q Y E L L rat alphao-C351I and o5s single amino acid exchanges investigated for coupling to alpha2aAR human (GTPgS loading assay) G->Q incr clonidine efficacy relative to epinephrine, incr/decr absolute efficacy depending on context, same/incr agonist affinity G->E markedly decr clonidine efficacy absolute and relative to epinephrine, decr agonist but not antagonist affinity Y->L little effect on clonidine efficacy, affinity Bahia et al. Biochem 37:11555 (1998) Gi1 C351 (-4) mutations affect coupling to alpha2A-AR I>L>F>W>A>Y,V,C>S,Q,N>H>T>E,G,D>P>M,R,K ~150% ~100% ~50% ~0% Dupuis et al. Neuropharmacology 40:36 (2001) Gi3 rat C351 mutations affect coupling to 5HT1AR, increasing constitutive and agonist-stimulated activity: M,L,I,Y > F,V,W,S,A > C,H,Q ~200% ~150% ~100% Kellett et al. Mol Pharm 56:684 (1999) fusion protein 5HT1AR->Gi1 alpha(C351,wt) is constitutively active, as is (C351I) but not (C351G); 5HT equally potent with C351 and C351G Taylor et al. JBC 269(44):27618 (1994) Taylor et al. JBC 271(7):3336 (1996) Wade et al. Mol Pharm 50:351 (1996) alpha2A AR IC3 Nterm peptide "P" CRIYQIAKRRTRV, Cys + V:24-32 + 3 residues -> N- and C-term (stim partly blocked by PTX) of Galphao/i proposed role: coupling specificity (along with IC2) and IC3 Cterm peptide "Q" RWRGRQNREKRFTC, 6 residues + VI:-4-2 + Cys -> N-term of Galphao/i (xlinker at end of Q labels 1-17), Cterm of Gbeta (xlinker at end of Q labels 281-341) proposed role: coupling effector (membrane-distal), modulator (mem-prox) Blahos et al. JBC 273(40):25765 (1998) family 3 receptors, chimeras of q + last 5 amino acids of Gi2, et al. mGlu2R -> qi5,qo5,qz5[I-4C] not qo5[C-4I] (but does couple to alpha15, which has -4I but not the preceding G) mGlu4R -> qi5,qo5,qz5 positions -1 and -5 also influence coupling efficiency ----------------------------------------------------------------------------- BETA Hou et al. JBC 276(23):19982 (2001) m2R-mediated activation (+ carbachol) of alpha GTPase rate in reconst system, reflecting the rate of nucleotide exchange: alphao-beta4-gamma2 > alphao-beta1-gamma2 suggests that the few positions with nonconservative differences between beta4 and beta1 may contact the receptor directly (31,35,37,39,302,303,305) ----------------------------------------------------------------------------- GAMMA Ernst et al. JBC 175(3):1937 (2000) transducin gamma peptides (50-71)-far (IC50 285 microM) and (60-71)-far stabilize MII but (60-71) and (60-71)F64A/L67A-far do not; stabilization lost for rhodopsin mutant 310-312 NKQ -> SPD (as in B2AR) but enhanced for rhodopsin mutant C322S/C323S (not palmitoylated) Kisselev et al. JBC 270(43):25356 (1995) Kisselev et al. JBC 269(34):21399 (1994) rhodopsin MII stab by Gt; max stab obtained with >=12-14 Cterm residues of gamma1 and farnesyl (C15) > geranylgeranyl (C20) > geranyl (C10) -----------------------------------------------------------------------------