FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2013289991> ?p ?o ?g. }

• W2013289991 endingPage "24327" @default.
• W2013289991 startingPage "24322" @default.
• W2013289991 abstract "We have completed a systematic search of the intracellular loops of a muscarinic acetylcholine receptor for domains that govern G-protein coupling. A unique feature of the second intracellular (i2) loop was an ordered cluster of residues where diverse substitutions cause constitutive activation. A second group of residues in i2 was identified where mutations compromised receptor/G-protein coupling. The residues of each group alternate and are spaced three to four positions apart, suggesting an α-helical structure where these groups form opposing faces of the helix. We propose that the constitutively activating face normally constrains the receptor in the “off-state,” while the other face couples G-proteins in the “on-state.” Therefore, the i2 loop functions as the switch enabling G-protein activation. We have completed a systematic search of the intracellular loops of a muscarinic acetylcholine receptor for domains that govern G-protein coupling. A unique feature of the second intracellular (i2) loop was an ordered cluster of residues where diverse substitutions cause constitutive activation. A second group of residues in i2 was identified where mutations compromised receptor/G-protein coupling. The residues of each group alternate and are spaced three to four positions apart, suggesting an α-helical structure where these groups form opposing faces of the helix. We propose that the constitutively activating face normally constrains the receptor in the “off-state,” while the other face couples G-proteins in the “on-state.” Therefore, the i2 loop functions as the switch enabling G-protein activation. transmembrane guanosine 5′-(β,γ-imido)triphosphate. Muscarinic acetylcholine receptors are members of a large family of receptors that mediate signal transduction by coupling with G-proteins (1Hulme E.C. Birdsall H.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1087) Google Scholar, 2Kubo T. Fukuda K. Mikami A. Maeda A. Takahashi H. Mishina M. Haga T. Haga K. Ichiyama A. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1986; 323: 411-416Crossref PubMed Scopus (663) Google Scholar, 3Peralta E.G. Winslow J.W. Peterson G.L. Smith D.H. Ashkenazi A. Ramachandran J. Schimerlink M.I. Capon D.J. Science. 1987; 236: 600-605Crossref PubMed Scopus (333) Google Scholar, 4Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1215) Google Scholar, 5Bonner T.I. Young A.C. Brann M.R. Buckley N.J. Neuron. 1988; 1: 403-410Abstract Full Text PDF PubMed Scopus (656) Google Scholar). Most members of this family display significant sequence homology, particularly within regions predicted to form 7-transmembrane domains (TM1–7)1 (6Donnelly D. Findlay J.B.C. Blundell T.L. Receptors and Channels. 1994; 2: 61-78PubMed Google Scholar). Current models of receptor structure arrange the transmembrane domains in a counterclockwise circle centered around TM3 forming a pocket where ligands could bind (7Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar, 8Schwartz T.W. Curr. Opin. Biotechnol. 1994; 5: 434-444Crossref PubMed Scopus (280) Google Scholar, 9Unger V.M. Hargrave P.A. Baldwin J.M. Schertler G.F.X. Nature. 1997; 389: 203-206Crossref PubMed Scopus (479) Google Scholar). Consistent with this proposed structural configuration, mutational data indicate that residues located on TM2, TM3, TM5, TM6, and TM7 are needed for ligand interactions with muscarinic receptors (10Wess J. Gdula D. Brann M.R. EMBO J. 1991; 10: 3729-3734Crossref PubMed Scopus (125) Google Scholar, 11Wess J. Nanavati S. Vogel Z. Maggio R. EMBO J. 1993; 10: 331-338Crossref Scopus (151) Google Scholar, 12Blüml K. Mutschler E. Wess J. J. Biol. Chem. 1995; 269: 18870-18876Abstract Full Text PDF Google Scholar, 13Matsui H. Lazareno S. Birdsall N.J.M. Mol. Pharmacol. 1995; 47: 88-98PubMed Google Scholar, 14Fraser C.M. Wang C. Robinson D.A. Gocayne J.D. Venter J.C. Mol. Pharmacol. 1989; 36: 840-847PubMed Google Scholar, 15Kurtenbach E. Curtis C.A.M. Pedder E.K. Aitken A. Harris A.C.M. Hulme E.C. J. Biol. Chem. 1990; 265: 13702-13708Abstract Full Text PDF PubMed Google Scholar, 16Spalding T.A. Birdsall N.J.M. Curtis C.A.M. Hulme E.C. J. Biol. Chem. 1994; 269: 4092-4097Abstract Full Text PDF PubMed Google Scholar). In particular, a central role of Asp105 in TM3 of the m1 muscarinic receptor for interactions with both agonists and antagonists has been demonstrated (14Fraser C.M. Wang C. Robinson D.A. Gocayne J.D. Venter J.C. Mol. Pharmacol. 1989; 36: 840-847PubMed Google Scholar, 15Kurtenbach E. Curtis C.A.M. Pedder E.K. Aitken A. Harris A.C.M. Hulme E.C. J. Biol. Chem. 1990; 265: 13702-13708Abstract Full Text PDF PubMed Google Scholar, 16Spalding T.A. Birdsall N.J.M. Curtis C.A.M. Hulme E.C. J. Biol. Chem. 1994; 269: 4092-4097Abstract Full Text PDF PubMed Google Scholar). Numerous experiments have shown that receptor/G-protein coupling occurs through the intracellular loops. By preparing chimeras between subtypes from the two functional classes (e.g. chimeric m1/m2 and m3/m2 receptors), the third cytoplasmic loop (i3) has been shown to be the region that defines subtype specificity for distinct G-proteins (17Wess J. Brann M.R. Bonner T.I. FEBS Lett. 1989; 258: 133-136Crossref PubMed Scopus (78) Google Scholar, 18Wess J. Bonner T.I. Brann M.R. Mol. Pharmacol. 1990; 38: 517-523PubMed Google Scholar, 19Kubo T. Bujo H. Akiba I. Nakai J. Mishina M. Numa S. FEBS Lett. 1988; 241: 119-125Crossref PubMed Scopus (124) Google Scholar, 20Lechleiter J. Hellmiss R. Duerson K. Ennulat D. David N. Clapham D. Peralta E. EMBO J. 1990; 9: 4381-4390Crossref PubMed Scopus (149) Google Scholar). The central portion of the i3 loop can be deleted without impairing coupling to G-proteins, indicating that only the N- and C-terminal regions of the i3 loop (Ni3 and Ci3, adjacent to TM5 and TM6, respectively) are required for function (21Shapiro R.A. Nathanson N.M. Biochemistry. 1989; 28: 8946-8950Crossref PubMed Scopus (58) Google Scholar, 22Lameh J. Philip M. Sharma Y.K. Moro O. Ramachandran J. Sadee W. J. Biol. Chem. 1992; 267: 13406-13412Abstract Full Text PDF PubMed Google Scholar, 23Arden J.R. Nagata O. Shockley M.S. Philip M. Lameh J. Sadee W. Biochem. Biophys. Res. Commun. 1992; 188: 1111-1115Crossref PubMed Scopus (28) Google Scholar, 24Kunkel M.T. Peralta E.G. EMBO J. 1993; 12: 3809-3815Crossref PubMed Scopus (56) Google Scholar). In contrast to results with i3, work on i2 indicates less of a role in governing G-protein coupling specificity and more of a role in governing overall strength of coupling (25Wong S.K.F. Parker E.M. Ross E.M. J. Biol. Chem. 1990; 265: 6219-6224Abstract Full Text PDF PubMed Google Scholar, 26Wong S.K.F. Ross E.M. J. Biol. Chem. 1994; 269: 18968-18976Abstract Full Text PDF PubMed Google Scholar). In fact, the DRY motif at the junction of TM3 and i2 is one of the most highly conserved regions of G-protein-coupled receptors (6Donnelly D. Findlay J.B.C. Blundell T.L. Receptors and Channels. 1994; 2: 61-78PubMed Google Scholar), thus it is likely that the i2 loop plays a fundamental role in G-protein coupling for all receptors. Contemporary pharmacological theory states that G-protein coupled receptors spontaneously interconvert between active (R*) and inactive (R) states (known as the two-state model or extended ternary complex model, see Refs. 27Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 28Leff P. Trends Pharmacol. Sci. 1995; 16: 89-97Abstract Full Text PDF PubMed Scopus (463) Google Scholar, 29Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 30Spalding T.A. Burstein E.S. Wells J.W. Brann M.R. Biochemistry. 1997; 36: 10109-10116Crossref PubMed Scopus (55) Google Scholar). Consistent with this theory, we have observed that overexpression of compatible G-proteins constitutively activates muscarinic receptors by promoting formation of R* (31Burstein E.S. Spalding T.A. Bräuner-Osborne H. Brann M.R. FEBS Lett. 1995; 363: 261-263Crossref PubMed Scopus (74) Google Scholar, 32Burstein E.S. Spalding T.S. Brann M.R. Mol. Pharmacol. 1997; 51: 312-319Crossref PubMed Scopus (111) Google Scholar, 33Burstein E.S. Bräuner-Osborne H. Spalding T.A. Conklin B.R. Brann M.R. J. Neurochem. 1997; 68: 525-533Crossref PubMed Scopus (24) Google Scholar). This concept also explains numerous observations that receptors are capable of activating G-proteins and eliciting responses in the absence of agonists (29Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 34Hilf G. Jakobs K.H. Eur. J. Pharmacol. 1992; 225: 245-252Crossref PubMed Scopus (73) Google Scholar, 35Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (475) Google Scholar, 36Tian W. Duzic E. Lanier S.M. Deth R.C. Mol. Pharmacol. 1994; 45: 524-531PubMed Google Scholar, 37Barker E.L. Westphal R.S. Schmidt D. Sanders-Bush E. J. Biol. Chem. 1994; 269: 11687-11690Abstract Full Text PDF PubMed Google Scholar, 38Leeb-Lundberg L.M. Mathis S.A. Herzig M.C.S. J. Biol. Chem. 1994; 269: 25970-25973Abstract Full Text PDF PubMed Google Scholar, 39Jakubik J. Bacakova L. El-Fakahany E.E. Tucek S. FEBS Lett. 1995; 377: 275-279Crossref PubMed Scopus (74) Google Scholar). The observable levels of constitutive activity of wild-type receptors are relatively low indicating that wild-type receptors exist predominantly as R. However many mutationally activated G-protein coupled receptors have been described which have levels of constitutive activity that are significantly higher than the original wild-type receptors (40Spalding T.A. Burstein E.S. Bräuner-Osborne H. Hill-Eubanks D. Brann M.R. J. Pharmacol. Exp. Ther. 1995; 275: 1274-1279PubMed Google Scholar, 41Cohen G.B. Yang T. Robinson P.R. Oprian D.D. Biochemistry. 1993; 32: 6111-6115Crossref PubMed Scopus (214) Google Scholar, 42Kjelsberg M.A. Cotecchia S. Ostrowski J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1430-1433Abstract Full Text PDF PubMed Google Scholar, 43Hogger P. Shockley M.S. Lameh J. Sadee W. J. Biol. Chem. 1995; 270: 7405-7410Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 44Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (195) Google Scholar). In general, activating mutations are thought to alter the equilibrium between R and R* to increase the proportion of receptor that exists as R*. By inference, residues where mutations are activating therefore may play a restraining role, maintaining receptors in an inactive conformation (27Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 42Kjelsberg M.A. Cotecchia S. Ostrowski J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1430-1433Abstract Full Text PDF PubMed Google Scholar). Conceptually, receptor domains that undergo large conformational changes upon transitioning from R to R* are “switch regions,” and would be expected to contain many such residues which restrain the receptor in R, as well as residues that are required in R*, for example, to couple G-proteins. In a systematic search for residues that affect G-protein coupling, both the i2 and i3 loops of the human m5 muscarinic receptor have been randomly mutated, and tested for their functional properties. Previously we reported that the functionally important residues of Ni3 and Ci3 are distributed with a periodicity of 3 to 4 positions, suggesting these regions form α-helical extensions of their adjacent transmembrane domains (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 47Hill-Eubanks D. Burstein E.S. Spalding T.A. Bräuner-Osborne H. Brann M.R. J. Biol. Chem. 1996; 271: 3058-3065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Similarly, in i2 we find the distribution of functionally important residues suggests an α-helical structure extending from TM3. Uniquely, a cluster of constitutively activating residues was identified in i2 which are also distributed in a pattern consistent with an α-helix, and which alternate with residues required for G-protein activation. We propose that the i2 loop has two protein faces, one comprised of residues important for attaining the active state of the receptor, and the other comprised of residues important for maintaining the inactive state of the receptor, and that the i2 loop operates as a conformational switch enabling G-protein coupling. Mutations were randomly introduced into the i2 loop from residues 127 to 151 using oligonucleotides doped at a 15% rate with an equimolar mixture of the four bases as described previously (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) except that cassettes containing point mutations were ligated into compatible cohesive ends using an existing uniqueEcoRI site, and an SpeI site created by introducing silent mutations into Leu121 (CTT → CTA) and Leu122 (CTG → CTA) of the coding sequence for the human m5 receptor (pCDE2-m5) rather than blunt-end ligated. A second library containing mutations from 129 to 143 was constructed based upon results with the first library including the observations that positions 144–149 could be freely mutated without affecting the functional properties of the receptor, whereas mutations at residues 127 and 128 severely affect receptor function. The lower mutation rate at residues 150 and 151 was due to inefficient priming of the 3′ end of the oligonucleotide in the polymerase chain reaction (47Hill-Eubanks D. Burstein E.S. Spalding T.A. Bräuner-Osborne H. Brann M.R. J. Biol. Chem. 1996; 271: 3058-3065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Therefore, the oligonucleotide used for the second library contained wild-type sequence at the 3′ end (codons 144 and 145) to eliminate polymerase chain reaction artifacts. Point mutations at each of four residues, Phe130, Arg134, Tyr138, and Arg142 were constructed as described (46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) using oligonucleotides doped with an equimolar mixture of the four bases at each individual codon rather than at a 15% rate over multiple codons as described above for random saturation mutagenesis of the i2 loop.Escherichia coli (DH5α) were transformed, and individual transformants were picked, amplified, and plasmid DNA isolated for all subsequent studies. Clones were sequenced after positive identification and characterization in the functional assays so that in essence, functional studies were done blindly. NIH 3T3 cells (ATCC number CRL 1658) and tsA cells (human embryonic kidney cell line tsA 201, Ref. 48Chahine M. Bennet P.B. George A.L. Horn R. Pfluegers Arch. 1994; 427: 136-142Crossref PubMed Scopus (108) Google Scholar) were incubated at 37 °C in a humidified atmosphere (5% CO2) in Dulbecco's modified Eagle's medium supplemented with 4500 mg/liter glucose, 4 mml-glutamine, 50 units/ml penicillin G, 50 units/ml streptomycin (A.B.I.) and 10% calf serum for 3T3 cells or 10% fetal bovine serum (Life Technologies, Inc.) for tsA cells. Receptor selection andamplification technology (R-SATTM) assays were performed as described (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) using 0.2 μg of receptor and 0.5 μg of p-SI-β-galactosidase (Promega, Madison WI) transfected into 2 × 105 cells per well of a 6-well rack for dose-response analysis, except that cells were combined with ligands in Dulbecco's modified Eagle's medium supplemented with 2% cyto-SF3 synthetic supplement (Kemp Laboratories) instead of calf serum. For screening, R-SAT assays were performed entirely in 96-well plates, using proportionately less DNA. Clones were screened in the presence of 100 μm carbachol, 2 μmatropine, or no drug. Plates were read at 420 nm on a plate-reader (Bio-Tek EL 310 or Molecular Devices). Data from R-SAT assays were fit to the equation: r = A + B(x/(x + c)), where A = minimum response, B = maximum response minus minimum response, c = EC50, r = response, and × = concentration of ligand. Curves were generated by least-squares fits using the program KaleidaGraph™ (Abelbeck Software). Maximum response values were normalized according to an internal standard based on the fact that NIH 3T3 cells endogenously express prostanoid FP receptors which produce a robust R-SAT response when exposed to the prostanoid agonist cloprostanol (Kayman Chemicals). We assayed each batch of transfected cells with 10 nm cloprostanol (EC50= 1 nm) 2T. Messier and M. Brann, personal communication. + 1 μm atropine (to suppress any constitutive muscarinic activity), and used the resulting response to normalize the dose-response data obtained using carbachol and atropine. The studies were performed as described (46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Cells were harvested 72 h after transfection and stored at −80 °C. Membranes were prepared in binding buffer containing 25 mm sodium phosphate (pH 7.4), 5 mm magnesium, and 50 μm Gpp(NH)p immediately before use. The second intracellular loop (i2) is highly conserved, with 13 of 25 amino acids being identical among the muscarinic subfamily (1Hulme E.C. Birdsall H.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1087) Google Scholar). To investigate structure-function relationships of m5 receptor/G protein coupling in the i2 loop we employed random-saturation mutagenesis in combination with a high throughput functional assay (R-SATTM see Refs. 45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 49Bräuner-Osborne H. Brann M.R. Eur. J. Pharmacol. 1996; 295: 93-102Crossref PubMed Scopus (103) Google Scholar, and 50Messier T.L. Dorman C.M. Bräuner-Osborne H. Eubanks D. Brann M.R. Pharmacol. & Toxicol. 1995; 76: 308-311Crossref PubMed Scopus (26) Google Scholar). Randomly doped oligonucleotides were used in a polymerase chain reaction based construction method (see “Materials and Methods”) to generate libraries of mutant m5 muscarinic receptors. The libraries were screened for functional receptors using R-SATTM, and these receptors were sequenced and subjected to concentration-response analysis. Overall, 63 functional receptors were isolated from libraries containing over 900 unique receptors. Three basic phenotypes of receptors were observed (see Fig. 1) including activated receptors (constitutive activity ≥ 15%), wild-type like receptors (EC50 ≤ 10-fold, EC50WT and max response ≥ 70% WT), and compromised receptors (EC50 > 10-fold EC50 WT or max response < 70% WT or both). To facilitate comparison of the molecular differences between activated, wild-type like, and compromised receptors, the associated mutations were tabulated by position and compiled into lists (see Table I).Table ISequences and functional phenotypes of m5 receptors randomly mutated across i2 Open table in a new tab A striking finding was the large number of constitutively activated receptors were identified in the i2 library (Table I and Fig. 1 A). These receptors mediated ligand independent responses which could be reversed by the negative antagonist atropine. In general, the EC50 values of carbachol at the activated receptors were lower than the value for wild-type m5. Although mutations were introduced randomly into i2, mutations associated with constitutive activity were not randomly distributed, but were instead clustered at Phe130, Arg134, Thr137, Tyr138, and Arg142. In contrast, analysis of the i3 loop by random mutagenesis identified very few constitutively activated receptors (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 47Hill-Eubanks D. Burstein E.S. Spalding T.A. Bräuner-Osborne H. Brann M.R. J. Biol. Chem. 1996; 271: 3058-3065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), 3E. S. Burstein, T. A. Spalding, and M. R. Brann, unpublished data. suggesting that mutations in the i2 loop have a greater propensity to cause constitutive activity. Receptors which were phenotypically similar to wild-type m5 also contained a non-random pattern of mutations (see Table I and Fig. 1 B). While the majority of residues in i2 tolerated multiple radical substitutions, eight residues were either never mutated or were substituted infrequently and only with conservative amino acids. No mutations were observed at Asp127, Arg128, or Phe135 in wild-type like or activated receptors. Since the random mutagenesis procedure certainly produced receptors containing mutations to these residues (46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), mutations at these residues probably disrupt receptor function. Several other residues were also identified where non-conservative mutations may disrupt receptor function. Tyr129 was only mutated to phenylalanine and cysteine which are found at the analogous position in other G-protein coupled receptors and are well tolerated substitutions in the m1 muscarinic receptor (51Lu Z. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (84) Google Scholar). Ile132and Thr143 were only conservatively mutated to valine and alanine, respectively. Only bulky hydrophobic substitutions were observed at Leu136, a structural requirement previously predicted for this residue (52Moro O. Lameh J. Hogger P. Sadee W. J. Biol. Chem. 1993; 268: 22273-22276Abstract Full Text PDF PubMed Google Scholar). Arg139 was only mutated to tryptophan which is often seen to occupy analogous positions to arginine in three-dimensional structures of homologous proteins as illustrated by the PAM250 matrix (53Pearson W.A. Methods Enzymol. 1990; 183: 63-98Crossref PubMed Scopus (1638) Google Scholar). Overall these data define a consensus of residues which are likely to be crucial for receptor/G-protein coupling and are therefore important for the active state of the receptor. Receptors with compromised phenotypes (EC50 > 10-fold EC50 WT or max response < 70% WT or both) are shown in Table I. These receptors had a more random distribution of mutations than the other groups of receptors, and most of the compromised receptors (14 out of 21) contain mutations at residues which were never mutated in the fully functional receptors (see Table I), providing more evidence for the functional importance of these residues. Functional receptors containing mutations at Arg128 were never observed, consistent with its pivotal role in G-protein coupling (see Ref. 54Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (66) Google Scholar). Receptors appear to function by switching between active and inactive conformations (27Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 28Leff P. Trends Pharmacol. Sci. 1995; 16: 89-97Abstract Full Text PDF PubMed Scopus (463) Google Scholar, 29Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 30Spalding T.A. Burstein E.S. Wells J.W. Brann M.R. Biochemistry. 1997; 36: 10109-10116Crossref PubMed Scopus (55) Google Scholar). The effect of constitutively activating mutations is to increase the preference of the receptor for the active state and this can be achieved either by disrupting interactions important for maintaining the inactive state, or by strengthening interactions needed for active state. To distinguish between these possibilities, libraries of receptors were constructed which contained point mutations at each of four residues associated with the activated phenotype. The libraries were screened for constitutive active receptors which were then sequenced and subjected to concentration-response analysis with carbachol and atropine. As shown in Table II and Fig. 2, at each position diverse substitutions representing most of the major classes of amino acids were constitutively activating. Given this diversity, it is likely that the primary effect of these mutations is to disrupt interactions needed to maintain the inactive state rather than to strengthen interactions needed in the active state.Table IIConcentration-response parameters from receptors containing point mutations to residues Phe, Arg, Tyr, and ArgSubstitutionConstitutive activity (%)Maximum response (%)Carbachol, EC50 × 10−7mAtropine, EC50 × 10−9m%Wild-type151001.2aF130Q1101550.0317F130C751400.24F130M701000.051F130S601400.13F130E601300.15F130H601350.34F130K601200.26F130R551700.42F130G501700.76F130T301550.610R134D601100.25R134N501200.14R134E501100.19R134T501000.15R134Q451300.15R134A401000.26R134C30850.87R134V251300.5aR134G201101.1aY138A401300.218Y138C451600.83Y138T401500.58Y138S401250.39Y138M351150.39Y138V351200.77Y138G351551.11R142L801150.036R142N751200.14R142H701000.082R142T651300.11R142G551500.32R142Q501500.33R142F451900.62R142S451100.067R142D401900.84R142A351100.15R142C301000.15R142W201600.9aEC50 denotes the concentration of ligand which produces a half-maximal response from the receptor (either to increase or reverse activity) in the absence of other ligands. Maximum response and constitutive activity are defined as in Table I. Maximum response and constitutive activity values are rounded to the nearest 5%. Substitutions are denoted using the single letter amino acid code.a indicates the EC50 values for atropine could not be determined. Open table in a new tab EC50 denotes the concentration of ligand which produces a half-maximal response from the receptor (either to increase or reverse activity) in the absence of other ligands. Maximum response and constitutive activity are defined as in Table I. Maximum response and constitutive activity values are rounded to the nearest 5%. Substitutions are denoted using the single letter amino acid code.a indicates the EC50 values for atropine could not be determined. Ligand binding studies revealed that receptors having constitutively activated, wild-type, and compromised phenotypes were expressed at similar levels, thus these diverse phenotypes are unlikely to be due to changes in expression levels (Table III). Two compromised receptors (clones 19 and 151, see Table I for functional phenotypes) bound carbachol with similar affinity as wild-type m5, thus the reduced potency of carbachol seen in functional assays is almost certainly because of impaired G-protein coupling. As in the functional assay, carbachol affinity was greater for activated receptors than for wild-type m5 and tended to be greatest at the most constitutively activated receptors, a predicted consequence of constitutive activity (27Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 30Spalding T.A. Burstein E.S. Wells J.W. Brann M.R. Biochemistry. 1997; 36: 10109-10116Crossref PubMed Scopus (55) Google Scholar). In contrast, antagonist affinities (and potencies, see Table II) were largely unaffected. Since the i2 loop resides in the cytoplasm, it is unlikely that the observed increases in carbachol affinity are due to direct changes in the ligand-binding site but probably result from allosteric effects.Table IIILigand binding parameters of mutant receptorsK 1(NMS)B maxIC50 (Cch)Fold change×10−10mpmol/mg× 10−6mm52.07.41771R142H1.77.5921R142N2.49.31018F130Q1.86.61512Y138T1.83.3316F130K1.43.7375F130M2.77.5633F130C2.06.1593R134E1.54.3722Clone 190.84.7394Clone 1511.05.51461IC50 values for carbachol were determined in the presence of 500 pm N-methylscopolamine. The Hill numbers for determination of carbachol IC50 values were m5, 0.86; R142H, 0.62; R142N, 0.65; F130Q, 0.65; Y138T, 0.81; F130K, 0.81; F130M, 0.70; F130C, 0.83; R134E, 0.71; clone 19, 0.74; and clone 151, 0.60. Clones 19 and 151 are functionally compromised receptors (see Table I). Fold-change was calculated as the ratio of IC50,Cch values for each mutant to the IC50,Cch value for wild-type m5. Open table in a new tab IC50 values for carbachol were determined in the presence of 500 pm N-methylscopolamine. The Hill numbers for determination of carbachol IC50 values were m5, 0.86; R142H, 0.62; R142N, 0.65; F130Q, 0.65; Y138T, 0.81; F130K, 0.81; F130M, 0.70; F130C, 0.83; R134E, 0.71; clone 19, 0.74; and clone 151, 0.60. Clones 19 and 151 are functionally compromised receptors (see Table I). Fold-change was calculated as the ratio of IC50,Cch values for each mutant to the IC50,Cch value for wild-type m5. We have shown that the i2 domain contains a group of residues necessary for holding the receptor in an inactive conformation (the off-state) and another group necessary for G-protein coupling (the on-state). These residues occur with a periodicity of 3 to 4 positions and when mapped on a perfect α-helix, form opposing faces, offset approximately 100° (see Fig. 3). Given the clear division between constitutively activating residues and functionally conserved residues, we speculate that i2 switches conformation to alternatively prevent or promote G-protein coupling through these two groups. The two putative protein faces formed by these groups could engage/disengage G-proteins, other accessory proteins, or other domains of the receptor. G-protein coupling to muscarinic receptors occurs mainly through the i2 and i3 loops (55Burstein E.S. Spalding T.A. Brann M.R. Biochem. Pharmacol. 1996; 51: 539-544Crossref PubMed Scopus (13) Google Scholar), therefore with this and previous studies (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 47Hill-Eubanks D. Burstein E.S. Spalding T.A. Bräuner-Osborne H. Brann M.R. J. Biol. Chem. 1996; 271: 3058-3065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 56Burstein E.S. Spalding T.S. Brann M.R. Biochemistry. 1998; 37: 4052-4058Crossref PubMed Google Scholar), we have completed analysis of the major G-protein coupling domains of the m5 muscarinic receptor by random mutagenesis (see Fig. 3). Comparison of these results reveals several patterns. G-protein coupling occurs primarily at the transmembrane/cytoplasmic borders between TM3/i2, TM5/Ni3, and TM6/Ci3, respectively. All of the functionally important residues in i2 and i3 identified by random mutagenesis are restricted to segments adjacent to these transmembrane domains whereas the remaining residues can be freely mutated without affecting function. The functionally critical residues in each domain are predominantly distributed with a periodicity of 3 to 4 positions, consistent with the hypothesis that these regions form α-helical extensions of the adjacent transmembrane domains (also see Refs. 57Liu J. Blin N. Conklin B. Wess J. J. Biol. Chem. 1996; 271: 6172-6178Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 58Blüml K. Mutschler E. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7980-7984Crossref PubMed Scopus (43) Google Scholar, 59Duerson K. Carroll R. Clapham D. FEBS Lett. 1993; 324: 103-108Crossref PubMed Scopus (20) Google Scholar). There is generally conservation of hydrophobic residues within i2 and i3 (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 47Hill-Eubanks D. Burstein E.S. Spalding T.A. Bräuner-Osborne H. Brann M.R. J. Biol. Chem. 1996; 271: 3058-3065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). These hydrophobic residues likely fulfill structural roles in forming a G-protein coupling pocket (46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 56Burstein E.S. Spalding T.S. Brann M.R. Biochemistry. 1998; 37: 4052-4058Crossref PubMed Google Scholar). When TM5 and TM6 are aligned such that residues known to be vital for receptor-ligand interactions face into the core of the receptor (10Wess J. Gdula D. Brann M.R. EMBO J. 1991; 10: 3729-3734Crossref PubMed Scopus (125) Google Scholar, 11Wess J. Nanavati S. Vogel Z. Maggio R. EMBO J. 1993; 10: 331-338Crossref Scopus (151) Google Scholar, 12Blüml K. Mutschler E. Wess J. J. Biol. Chem. 1995; 269: 18870-18876Abstract Full Text PDF Google Scholar), the hydrophobic residues in Ni3 and Ci3 converge where they could form a G-protein coupling pocket through protein-protein interactions (56Burstein E.S. Spalding T.S. Brann M.R. Biochemistry. 1998; 37: 4052-4058Crossref PubMed Google Scholar). This model is consistent with the idea that in a soluble protein, hydrophobic residues generally face inward. As TM3 is thought to face TM5 and TM6 (7Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar), it is plausible that the conserved hydrophobic residues identified in i2 interact with those identified in Ni3 and Ci3. Finally, there is functional conservation of basic residues in i2 and i3, in agreement with theories that receptors couple G-proteins through interactions with electrostatically negative regions of the heterotrimeric complex (60Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhl J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1002) Google Scholar). The helical domains of Ni3 and Ci3 each terminate in basic residues (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 47Hill-Eubanks D. Burstein E.S. Spalding T.A. Bräuner-Osborne H. Brann M.R. J. Biol. Chem. 1996; 271: 3058-3065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and mutation of both residues to glutamate abolishes G-protein coupling (56Burstein E.S. Spalding T.S. Brann M.R. Biochemistry. 1998; 37: 4052-4058Crossref PubMed Google Scholar). We have now identified four arginine residues in i2 which are critical for receptor function, consistent with the idea that i2 plays a major role in receptor/G-protein coupling, and that basic residues are crucial to this process. Despite the similarities between i2 and i3 described above, it is clear that these domains also perform distinct roles in receptor/G-protein coupling. Previous work identified Tyr217 in Ni3 (46Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1996; 271: 2882-2885Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and Ala441 in Ci3 (45Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) as the major determinants of G-protein coupling specificity, and mutation of both of these residues to the analogous residues in the Gαi-coupled m2 and m4 muscarinic receptors abolishes signaling by m5 (56Burstein E.S. Spalding T.S. Brann M.R. Biochemistry. 1998; 37: 4052-4058Crossref PubMed Google Scholar). Although four residues in i2 predict G-protein coupling specificity based on sequence conservation (1Hulme E.C. Birdsall H.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1087) Google Scholar), none of these residues was identified as being functionally critical. Thus coupling specificity is governed mainly by the i3 loop, in agreement with earlier findings using chimeric m2/m5 receptors (55Burstein E.S. Spalding T.A. Brann M.R. Biochem. Pharmacol. 1996; 51: 539-544Crossref PubMed Scopus (13) Google Scholar). Ample structural (7Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar, 8Schwartz T.W. Curr. Opin. Biotechnol. 1994; 5: 434-444Crossref PubMed Scopus (280) Google Scholar, 9Unger V.M. Hargrave P.A. Baldwin J.M. Schertler G.F.X. Nature. 1997; 389: 203-206Crossref PubMed Scopus (479) Google Scholar) and functional (14Fraser C.M. Wang C. Robinson D.A. Gocayne J.D. Venter J.C. Mol. Pharmacol. 1989; 36: 840-847PubMed Google Scholar, 15Kurtenbach E. Curtis C.A.M. Pedder E.K. Aitken A. Harris A.C.M. Hulme E.C. J. Biol. Chem. 1990; 265: 13702-13708Abstract Full Text PDF PubMed Google Scholar, 16Spalding T.A. Birdsall N.J.M. Curtis C.A.M. Hulme E.C. J. Biol. Chem. 1994; 269: 4092-4097Abstract Full Text PDF PubMed Google Scholar) data point to a central role for TM3 in receptor-ligand interactions. Conformational changes occurring due to ligand interactions with TM3 are likely to be propagated into i2 to enable receptor/G-protein activation. Experiments on rhodopsin show that i2 moves as a rigid body following changes in chromophore interactions with TM3 (61Ridge K.D. Zhang C. Khorana H.G. Biochemistry. 1995; 34: 8804-8811Crossref PubMed Scopus (40) Google Scholar, 62Farahbakhsh Z.T. Ridge K.D. Khorana H.G. Hubbell W.L. Biochemistry. 1995; 34: 8812-8819Crossref PubMed Scopus (184) Google Scholar, 63Resek J.F. Farahbakhsh Z.T. Hubbell W.L. Khorana H.G. Biochemistry. 1993; 32: 12025-12032Crossref PubMed Scopus (145) Google Scholar). We have reported that a series of mutations at Ser465 (located at the extracellular end of TM6) constitutively activate m5, probably by causing movement of TM6 relative to the other transmembrane domains (30Spalding T.A. Burstein E.S. Wells J.W. Brann M.R. Biochemistry. 1997; 36: 10109-10116Crossref PubMed Scopus (55) Google Scholar, 40Spalding T.A. Burstein E.S. Bräuner-Osborne H. Hill-Eubanks D. Brann M.R. J. Pharmacol. Exp. Ther. 1995; 275: 1274-1279PubMed Google Scholar). More recently we have observed that m5 is activated by mutations in multiple positions across TM6, similar to results for i2, and thus TM6 may function as a ligand-operated switch region of m5. (67Spalding T.A. Burstein E.S. Henderson S.C. Ducote K.R. Brann M.R. J. Biol. Chem. 1998; 273: 21563-21568Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Recent reports on rhodopsin (64Sheikh S.P. Zvyaga T.A. Lichtarge O. Sakmar T.P. Bourne H.R. Nature. 1996; 383: 347-350Crossref PubMed Scopus (395) Google Scholar, 65Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1102) Google Scholar) and the β2 adrenoreceptor (66Gether U. Lin S. Ghanouni P. Ballesteros J.A. Weinstein H. Kobilka B.K. EMBO J. 1997; 16: 6737-6747Crossref PubMed Google Scholar) indicate that TM3/i2 and TM6 move relative to each other upon receptor activation. Overall our results indicate that the ultimate goal of these changes is to reposition i2, exposing the catalytic surface of the receptor, enabling G-protein activation. We thank K. Ducote for technical assistance." @default.
• W2013289991 created "2016-06-24" @default.
• W2013289991 creator A5053279288 @default.
• W2013289991 creator A5072392251 @default.
• W2013289991 creator A5087373728 @default.
• W2013289991 date "1998-09-01" @default.
• W2013289991 modified "2023-10-17" @default.
• W2013289991 title "The Second Intracellular Loop of the m5 Muscarinic Receptor Is the Switch Which Enables G-protein Coupling" @default.
• W2013289991 cites W1485119817 @default.
• W2013289991 cites W151716105 @default.
• W2013289991 cites W1528146981 @default.
• W2013289991 cites W1536524213 @default.
• W2013289991 cites W1543368646 @default.
• W2013289991 cites W1544899066 @default.
• W2013289991 cites W1558365920 @default.
• W2013289991 cites W1569349971 @default.
• W2013289991 cites W1595295964 @default.
• W2013289991 cites W1604118064 @default.
• W2013289991 cites W1641874896 @default.
• W2013289991 cites W1677997440 @default.
• W2013289991 cites W1916235480 @default.
• W2013289991 cites W1924799369 @default.
• W2013289991 cites W1963667944 @default.
• W2013289991 cites W1964191104 @default.
• W2013289991 cites W1965835906 @default.
• W2013289991 cites W1972961295 @default.
• W2013289991 cites W1973024435 @default.
• W2013289991 cites W1973197643 @default.
• W2013289991 cites W1978745809 @default.
• W2013289991 cites W1984924360 @default.
• W2013289991 cites W1987339017 @default.
• W2013289991 cites W1990110274 @default.
• W2013289991 cites W1992650668 @default.
• W2013289991 cites W1994451460 @default.
• W2013289991 cites W1996057736 @default.
• W2013289991 cites W1996699433 @default.
• W2013289991 cites W1999767086 @default.
• W2013289991 cites W2002523841 @default.
• W2013289991 cites W2002915668 @default.
• W2013289991 cites W2003513113 @default.
• W2013289991 cites W2013592625 @default.
• W2013289991 cites W2019742616 @default.
• W2013289991 cites W2020339143 @default.
• W2013289991 cites W2032677663 @default.
• W2013289991 cites W2044835337 @default.
• W2013289991 cites W2046378079 @default.
• W2013289991 cites W2046826628 @default.
• W2013289991 cites W2050274797 @default.
• W2013289991 cites W2054444073 @default.
• W2013289991 cites W2062778364 @default.
• W2013289991 cites W2066315102 @default.
• W2013289991 cites W2069828003 @default.
• W2013289991 cites W2076225147 @default.
• W2013289991 cites W2078122870 @default.
• W2013289991 cites W2082968554 @default.
• W2013289991 cites W2083637735 @default.
• W2013289991 cites W2083754747 @default.
• W2013289991 cites W2108696499 @default.
• W2013289991 cites W2111339077 @default.
• W2013289991 cites W2113630547 @default.
• W2013289991 cites W2116261904 @default.
• W2013289991 cites W2123446516 @default.
• W2013289991 cites W2136906322 @default.
• W2013289991 cites W2160380609 @default.
• W2013289991 cites W2161685942 @default.
• W2013289991 cites W22250383 @default.
• W2013289991 cites W236137996 @default.
• W2013289991 cites W2414461651 @default.
• W2013289991 doi "https://doi.org/10.1074/jbc.273.38.24322" @default.
• W2013289991 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9733718" @default.
• W2013289991 hasPublicationYear "1998" @default.
• W2013289991 type Work @default.
• W2013289991 sameAs 2013289991 @default.
• W2013289991 citedByCount "116" @default.
• W2013289991 countsByYear W20132899912012 @default.
• W2013289991 countsByYear W20132899912013 @default.
• W2013289991 countsByYear W20132899912014 @default.
• W2013289991 countsByYear W20132899912015 @default.
• W2013289991 countsByYear W20132899912016 @default.
• W2013289991 countsByYear W20132899912017 @default.
• W2013289991 countsByYear W20132899912018 @default.
• W2013289991 countsByYear W20132899912019 @default.
• W2013289991 countsByYear W20132899912020 @default.
• W2013289991 countsByYear W20132899912022 @default.
• W2013289991 countsByYear W20132899912023 @default.
• W2013289991 crossrefType "journal-article" @default.
• W2013289991 hasAuthorship W2013289991A5053279288 @default.
• W2013289991 hasAuthorship W2013289991A5072392251 @default.
• W2013289991 hasAuthorship W2013289991A5087373728 @default.
• W2013289991 hasBestOaLocation W20132899911 @default.
• W2013289991 hasConcept C114614502 @default.
• W2013289991 hasConcept C12554922 @default.
• W2013289991 hasConcept C131584629 @default.
• W2013289991 hasConcept C170493617 @default.
• W2013289991 hasConcept C184670325 @default.
• W2013289991 hasConcept C185592680 @default.
• W2013289991 hasConcept C191897082 @default.
• W2013289991 hasConcept C192562407 @default.

• next