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The Functional Topography of Transmembrane Domain 3 of the M1 Muscarinic Acetylcholine Receptor, Revealed by Scanning Mutagenesis

1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês

10.1074/jbc.274.11.7309

1083-351X

Zhi-Liang Lu, Edward C. Hulme,

Neuroscience and Neuropharmacology Research

Alanine-scanning mutagenesis has been applied to residues 100–121 in transmembrane domain 3 of the M1muscarinic acetylcholine receptor. This study complements a previous investigation of the triad Asp122-Arg123-Tyr124 (Lu, Z-L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234–241). The results demonstrate the α-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val113, Leu116, and Ser120, which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second. Alanine-scanning mutagenesis has been applied to residues 100–121 in transmembrane domain 3 of the M1muscarinic acetylcholine receptor. This study complements a previous investigation of the triad Asp122-Arg123-Tyr124 (Lu, Z-L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234–241). The results demonstrate the α-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val113, Leu116, and Ser120, which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second. Models of the rhodopsin-like superfamily of G protein-coupled receptors suggest that transmembrane domain 3 (TMD 3) 1The abbreviations used are: TMD, transmembrane domain; ACh, acetylcholine; mAChR, muscarinic acetylcholine receptor; NMS, (−)-N-methyl scopolamine; QNB, (−)-3-quinuclidinyl benzilate; PI, phosphoinositide; GTPγS, guanosine 5′-3-O-(thio)triphosphate forms the core of the seven-membered helical bundle that traverses the phospholipid bilayer (1Baldwin J.M. Schertler G.F.X. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (635) Google Scholar, 2Donnelly D. Findlay J.B.C. Blundell T.L. Receptors Channels. 1994; 2: 61-78PubMed Google Scholar). Spin labeling and cross-linking studies on Cys-substituted rhodopsin mutants have shown that a reorganization of the contacts between TMD 3 and surrounding transmembrane domains probably accompanies receptor activation (3Farabakhsh Z.T. Ridge K.D. Khorana H.G. Hubbell W.L. Biochemistry. 1995; 34: 8812-8819Crossref PubMed Scopus (186) Google Scholar, 4Farrens D.L. Altenbach C. Yang K. Hubbell W. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1117) Google Scholar, 5Sheikh S.P. Zvyaga T.A. Lichtarge O. Sakmar T.P. Bourne H.R. Nature. 1996; 383: 347-350Crossref PubMed Scopus (399) Google Scholar). Substituted cysteine accessibility mutagenesis of TMD 3 of the D2 dopamine receptor has supported its α-helical character (6Javitch J.A. Fu D. Chen J. Karlin A. Neuron. 1995; 14: 825-831Abstract Full Text PDF PubMed Scopus (169) Google Scholar). TMD 3 of the M1 muscarinic acetylcholine receptor (mAChR) is constrained at its N terminus by the conserved disulfide-bonded residue Cys98 (7Kurtenbach 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, 8Savarese T.M. Wang C.-D. Fraser C.M. J. Biol. Chem. 1992; 267: 11439-11448Abstract Full Text PDF PubMed Google Scholar). It carries the acetylcholine (ACh) binding side chain of Asp105 (9Spalding 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, 10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar) and, near its C terminus, Arg123, which is completely conserved in the rhodopsin-like G protein-coupled receptors and is important in G protein binding and activation (11Zhu S.Z. Wang S.Z. Hu J. El-Fakahany E.E. Mol. Pharmacol. 1994; 45: 517-523PubMed Google Scholar, 12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar). In the M1 mAChR, the highly conserved residues Asp122 and Tyr124appear to make intramolecular contacts whose integrity is important for receptor expression (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Other residues within TMD 3 may also make inter- or intramolecular contacts, and these may be important determinants of receptor structure and activation. Scanning mutagenesis can help to establish the secondary structure of receptor domains (6Javitch J.A. Fu D. Chen J. Karlin A. Neuron. 1995; 14: 825-831Abstract Full Text PDF PubMed Scopus (169) Google Scholar) and to locate and define functional epitopes within them (14Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1793) Google Scholar). Replacement of individual amino acids by alanine, which deletes the side chain beyond the β-carbon (in this study, Ala residues themselves were replaced by Gly), is normally well tolerated and allows the functions of individual side chains to be probed and analyzed (15Ward W.H.J. Timms D. Fersht A.R. Trends Pharmacol. Sci. 1990; 11: 280-284Abstract Full Text PDF PubMed Scopus (45) Google Scholar). We have applied this systematic approach to residues 100–121 of TMD 3 of the M1 mAChR. The results show that different, topographically distinct, groups of amino acids are responsible for the selective stabilization of the ground and activated states of the receptor. We propose that ACh binding causes TMD 3 to switch from one set of contacts to the other. A preliminary account of some of these results has been given (16Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar). Experimental procedures were performed as described previously (10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar, 12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar, 13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Briefly, residues 100–121 of the rat M1 mAChR were individually mutated to Ala (or, if they were Ala in the parent sequence, to Gly), using a polymerase chain reaction method. After verification by dideoxy sequencing, mutant receptors were subcloned into the pCD expression vector and expressed transiently in COS-7 cells by electroporation using a Bio-Rad Gene Pulser at 180 V and 960 microfarads with 10 μg of DNA/0.4-cm cuvette (4 × 107 cells, 0.8 ml). Membrane preparations were made after 72 h as described previously (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Binding of (−)-N-[3H]methylscopolamine ([3H]NMS), and (−)-[3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations was measured at 30 °C in a buffer containing 20 mm Na-HEPES, 100 mm NaCl and 1 mm MgCl2 using an incubation time of 60 min to 3 h, as described previously (10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar). The binding of ACh and other unlabeled ligands was measured by inhibition of the binding of the tritiated antagonist. For the study of guanine nucleotide shifts, MgCl2 was replaced by 2 mmMnCl2 (12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar). Phosphoinositide (PI) turnover was assayed as described previously (12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar,13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Briefly, transfected COS-7 cells were prelabeled with [3H] inositol for 48 h, followed by washing and preincubation with Krebs bicarbonate solution containing 10 mm LiCl for 30 min, and then ACh or other agonists were added for 30 min. When the effects of antagonists on the basal phosphoinositide response were to be studied, the antagonist was added at different times up to 48 h before the performance of the PI assay. In some instances, the antagonist was washed out (three changes of Krebs bicarbonate solution without Li+ over a period of about 15 min) before adding Li+ for 30 min to initiate the PI assay. Expression of receptor protein was assessed 72 h after transfection by incubation of fixed, permeabilized cells with a 1:100 dilution of an immunoaffinity-purified antiserum raised against the C-terminal 13 amino acids of the M1 mAChR sequence (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). This was followed by incubation with a 1:5000 dilution of an alkaline phosphatase-labeled second antibody and visualization as described (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). (−)-N-[3H]methylscopolamine (85 Ci/mmol), (−)-[3H]quinuclidinyl benzilate (50 Ci/mmol), and myo-d-[3H]inositol (80 Ci/mmol) were obtained from Amersham Pharmacia Biotech. Guanosine 5′-O-(3-thiotriphosphate) tetralithium salt, was from Boehringer Mannheim. Binding curves were analyzed (10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar, 12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar, 13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar) using SigmaPlot (SPSS Software Inc.). Ligand saturation curves were fitted to a one-site model of binding to yield an affinity constant,KBin, and a total concentration of binding sites, RT. Competition curves were fitted to the Hill equation, or to one-site or two-site models of binding. Binding constants were corrected for the Cheng-Prusoff shift, as necessary (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Unless otherwise stated, the agonist KBinis taken to be the reciprocal of the corrected IC50 value. PI dose-response curves were fitted to a four-parameter logistic function, yielding a basal activity (PIbas), a maximum response (PImax), an EC50 value, and a slope factor. The slopes of the PI dose-response curves were, on average, not significantly different from 1.0. The KAct value is defined as 1/EC50. Emax is the maximum receptor-induced signal, defined as (PImax,mut − PIbas,wt)/(PImax,wt − PIbas,wt). For two constitutively active mutants, the basal activity parameter, denoted Basal, was calculated relative to the wild-type receptor as (PIbas,mut − PIbas,wt)/(PImax,mut − PIbas,wt). With these two exceptions,Basal = 0. Agonist signaling efficacy values were calculated by fitting the ratio ([RG] + [ARG])/[GT] calculated from the ternary complex model (17DeLean A. Stadel J.M. Lefkowitz R.J. J. Biol. Chem. 1980; 255: 7108-7117Abstract Full Text PDF PubMed Google Scholar) to the dose-response data, where [RG] is the concentration of the receptor-G protein binary complex, [ARG] is the concentration of the agonist-receptor-G protein ternary complex, and [GT] is the receptor-accessible concentration of G protein. Unless the basal activity is raised, the contribution of [RG] can be ignored. The calculation uses the agonist binding constant, KBin, and an estimate of the effective ratio of total receptor concentration to receptor-accessible G protein, [RT]/[GT], denoted RT. In performing these calculations, we have used the value of 20 forRT for the wild-type receptor estimated in a previous study in which the effect of an irreversible blocking agent on the ACh dose-response curve was studied (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Values of RT for the different mutants were calculated from the expression of [3H]NMS binding sites. For the L116A mutant, the calculatedRT value of 0.4 is consistent with the experimentally determined Emax of 40% of wild type (legend to Fig. 3). The agonist signaling efficacy parameter isKG·[GT] (denotedKG), where KGis the apparent affinity constant of the G protein for the ensemble of agonist-receptor complexes. KG was computed to reproduce the pEC50 of the PI dose-response curve. For values of RT > 1, the efficacy parameter, KG, was calculated as follows. K¯G=(KAct/(KBin(1−Basal))−1)/R¯T(whenEmax∼1)(Eq. 1) or K¯G=(Emax/(1−Emax))/R¯T(whenEmax<1)(Eq. 2) These equations are extensions of those derived by Whaleyet al. (18Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar). Other details are given by Hulme and Lu (16Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar). When RT was <1, the efficacy was calculated from a fit of the ternary complex model to the dose-response data, as described previously (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Several Ala substitution mutations in the N-terminal half of TMD 3 strongly reduced the affinity of the quaternary antagonist NMS (Fig. 1a). D105A and Y106A gave 100-fold reductions, measured by direct radioligand saturation or by competition binding assays (10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar). 10-Fold reductions followed mutations of Trp101, Leu102, Asn110, and Val113. Large decreases were also seen in the affinity of the tertiary antagonist QNB (legend to Fig. 1), 6000-fold for D105A (measured by competition with [3H]NMS), and 31-fold for Y106A. In contrast, mutations in the C-terminal part of TMD 3 had little effect on NMS affinity. The largest change was a 2-fold reduction, from I119A (Fig. 1a). Ala substitutions in the C-terminal part of the domain had the largest effects on the expression of [3H]NMS binding sites. The L116A mutant was expressed at 2% of the wild-type level. Mutations of Val113, Asn115, and Leu117 also caused greater than 3-fold reductions (Fig. 1b). In the N-terminal region, only the mutation of Asp105 decreased receptor expression, to 10% of the wild-type level. Unusually, mutation of Asn110 somewhat enhanced receptor expression. Alterations in the expression of the receptor protein were also visualized by immunocytochemical staining of transfected cells using an antibody directed against the C terminus of the receptor (Fig. 2). As noted previously, the apparent variation in staining intensity was less than might be expected from the variation in expression of binding sites, suggesting that, particularly in the case of poorly expressing mutants such as D105A (Fig. 2e), part of the receptor protein was misfolded or was inappropriately localized (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Acetylcholine binding was assayed by inhibition of the binding of [3H]NMS. ACh binding to the wild-type receptor expressed in COS-7 cells gave a slightly flattened isotherm (nH = 0.9) with aKBin of 1.26 × 105m−1 (10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar). Mutations that diminished NMS affinity caused reductions in the affinity of ACh (Fig. 3a). Mutation of Ser109, which was without effect on NMS binding, gave a 10-fold reduction in the affinity of ACh. Three mutations located between the center and the C terminus of TMD 3 increased the affinity of ACh by between 4- and 30-fold. They were V113A, L116A, and S120A. In COS-7 cells transfected with the wild-type M1 mAChR, ACh elicits a robust phosphoinositide response, with an EC50 of 70 nm and a maximum stimulation of 3 times the basal activity (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Qualitatively, the effects of the Ala substitution mutations on the PI response were parallel to their effects on ACh binding. Mutations that decreased ACh affinity decreased the KAct (defined as 1/EC50), while mutations that increased ACh affinity increased the KAct (Fig. 3b). However, S112A decreased ACh potency without affecting its affinity. The quantitative effects of the D105A, Y106A, and I119A mutations on ACh potency in the PI response were much greater than their effects on ACh binding. In particular, the D105A mutant completely failed to give a PI response to ACh stimulation, and ACh potency was decreased by more than 1000-fold by both the Y106A and I119A mutations. The basal PI activities of most of the mutant receptors were between 92% (I119A) and 112% of the wild-type basal, but that of the S120A mutant was significantly raised (122 ± 6%; p < 0.01;Basal = 0.07). Receptor-transducer models (19Black J.W. Leff P. Proc. R. Soc. Lond. B Biol. Sci. 1983; 220: 141-162Crossref PubMed Scopus (959) Google Scholar), such as the ternary complex model (17DeLean A. Stadel J.M. Lefkowitz R.J. J. Biol. Chem. 1980; 255: 7108-7117Abstract Full Text PDF PubMed Google Scholar), can be used to quantitate the effects of mutations on signaling efficacy. Provided that signal generation is directly proportional to the sum of the concentrations of the RG binary and ARG ternary complexes, there exists a quantitative relationship between the maximum response,Emax (as a fraction of the maximum obtainable signal); the KAct; the basal activity parameter,Basal; and the agonist binding constant,KBin (parameter definitions are given under “Experimental Procedures”). When the ratio of receptor concentration to accessible G protein concentration is greater than 1, the relationship is as follows. Emax=1−(1−Basal)·KBin/KAct(Eq. 3) For the present set of mutants, as well as R123A, R123K (12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar), and D105E (10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar), this relationship is reasonably well obeyed (Fig. 4a). In only one case, N115A, was the deviation of the measured from the predictedEmax greater than 20% of the total signal, suggesting a reduction in the signaling activity of the ternary complex in this case. A measure of agonist signaling efficacy, adjusted for differences in receptor expression level, is provided by the affinity of the G protein for the ensemble of agonist-receptor complexes,KG, multiplied by the concentration of receptor-accessible G protein, [GT] (Refs. 13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar,16Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar, 18Whaley B.S. Yuan N. Birnbaumer L. Clark R.B. Barber R. Mol. Pharmacol. 1994; 45: 481-489PubMed Google Scholar; see “Experimental Procedures”). The efficacy values obtained for ACh activation of the TMD 3 mutants are illustrated in Fig. 4b. Deleting the side chains of Tyr106 and Ile119caused reductions in the calculated signaling efficacy of ACh of 100-fold or greater. The D105A mutant had zero efficacy. There were also 10-fold effects from Trp101, Ser109, and Ser112. In contrast, the mutations of Leu116and Ser120 caused 2.2- and 1.4-fold increases in the ACh signaling efficacy, respectively. The effects of the nonhydrolyzable GTP analogue GTPγS (10−5m) were investigated on the mutants that showed enhanced ACh affinity and efficacy. Binding studies were performed in the presence of 2 mm Mn2+ions to promote the stability of the agonist-receptor G protein complex (12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar). Mn2+ increased the affinity of ACh for the wild-type receptor by 1.5-fold relative to the standard Mg2+-containing buffer, but no significant GTPγS shift resulted. The V113A, and L116A mutants also showed no GTP shift. Mn2+ enhanced the affinity of ACh for the S120A mutant by 3-fold; in this case, the binding data could be described by a two-site model of binding (pKdL = 5.74 ± 0.01 (60% of total sites); pKdH = 7.33 ± 0.06 (40% of total sites)). GTPγS gave a significant decrease in the ACh affinity for this mutant. Analysis showed a 3.5-fold decrease in the high affinity binding constant (pKdH = 6.79 ± 0.11; p < 0.05) without an effect on the low affinity binding constant. The partial nature of this effect indicates that only a fraction of the high affinity sites were affected. The mutants that showed increased ACh affinity and efficacy were probed with the partial agonist pilocarpine. Pilocarpine gave 78% of the maximum PI response elicited by ACh at the wild-type receptor (Fig. 5). With respect to the wild-type receptor, V113A and L116A gave approximately 10-fold increases in affinity for pilocarpine, while S120A gave less than a 2-fold enhancement (Fig. 5). The potencies of pilocarpine in the PI response were increased by all three mutations. At the V113A and S120A mutants, pilocarpine became a full agonist. At the L116A mutant, its maximum effect was maintained with respect to that of ACh, despite the very low expression of the mutant receptor. Calculation of the efficacy of pilocarpine gave a value equal to 1.4% of the efficacy of ACh at the wild-type receptor. This was increased to 5% (3.6-fold) for the S120A and V113A mutants and to 30% (21-fold) for the L116A mutant. The mutants that showed enhanced ACh and pilocarpine efficacies were examined for inverse agonist effects. Preincubation of transfected cells with NMS for 24–48 h before the addition of LiCl and the isolation of phosphoinositides reduced the basal activity of the S120A mutant (Basal = 7% of the maximum PI signal) to the wild-type basal level with an IC50 of 10−8m (Fig. 6a). There was no detectable effect of NMS on the basal activity of the wild-type receptor, but in some experiments a reduction of about 3% in the V113A Basalparameter was seen (not shown). Prolonged incubations of cells transfected with the L116A mutant with NMS (Fig. 6a) or atropine increased the basal PI signal. To see whether this effect might be partly mediated by an ability to stabilize, and so up-regulate, the receptor, we examined the effect of atropine on the level of expression of the L116A mutant by immunocytochemistry and by [3H]NMS binding. A 48-h incubation of the cells with atropine increased the accumulation of the L116A protein (Fig. 2d) but not the wild-type receptor (Fig. 2c). Measurement of [3H]NMS saturation curves after atropine pretreatment of the cells showed no effect on the wild-type receptor, but the expression of the L116A mutant was increased by up to 50-fold, close to the wild-type level, without changing the NMS affinity (Fig. 6b) or the ACh binding properties (data not shown). Preincubation with atropine (10−4m) also doubled the expression of the D105A mutant (data not shown). Atropine pretreatment of the transfected cells, followed by washout, before measurement of the PI response increased the basal activity of the L116A mutant to a level equivalent to 70–120% of the maximum signal obtained in cells transfected with the wild-type receptor. The enhanced basal activity was partially inhibited by the addition of atropine, with an IC50 of 10−8m(Fig. 6c). The PI signal was stimulated by 40% by the addition of carbachol (Fig. 6c) or ACh (not shown). The Basal parameter was equivalent to 70% of the maximum signal in these cells. In contrast, pretreatment with atropine had no effect on the PI response of the wild-type receptor. One category of residues is those whose mutation had minimal effect, defined as less than a 3-fold change in the level of receptor expression, in NMS and ACh affinities and less than a 6-fold change in ACh functional potency. Five of these (Leu100, Ala103, Leu104, Val107, and Ala108) occur in the N-terminal half of TMD 3, and three (Met114, Leu118, and Phe121) occur in the C-terminal half. In a helical wheel plot, they are clustered in one sector (Fig. 7a), strongly supporting an α-helical conformation for TMD 3. Cys substitution mutants of the equivalent positions in the D2 receptor failed to react with a polar sulfhydryl reagent (6Javitch J.A. Fu D. Chen J. Karlin A. Neuron. 1995; 14: 825-831Abstract Full Text PDF PubMed Scopus (169) Google Scholar). In both receptor types, these residues probably face toward the lipid bilayer. A helical net representation (Fig. 7b) shows that the sector of TMD 3 in which mutations can be made without major effect narrows toward the cytoplasmic end of the helix, in agreement with models based on the structure of rhodopsin, which suggest that this part of the helix is mostly surrounded by the other TMDs (1Baldwin J.M. Schertler G.F.X. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (635) Google Scholar). The residues whose mutation lowered receptor expression levels have the opposite distribution. They are concentrated in the C-terminal half of TMD3 (Fig. 7b) and are spread over 220° of arc. We have argued that the side chains of such residues make intramolecular contacts that contribute to the stability of the transmembrane structure of the ground state of the receptor (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar). Their removal reduces the free energy of the folded state with respect to inactive intermediates on the folding pathway. Besides Asp122 and Tyr124 (13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar), we have now found a third position, Leu116, at which a side chain deletion strongly reduced the level of expression. Three other mutations in the C-terminal part of the helix also had significant effects. Only one such sensitive residue occurs in the N-terminal half of the helix. Interestingly, it is the binding site aspartate itself. The distribution of these mutants suggests that the side chains of residues in the C-terminal half of TMD 3 make more frequent intramolecular contacts than those in the N-terminal half. The free energy difference between the folded and unfolded states was restored by incubation of the expressing cells with a high affinity antagonist. This counteracted the effect of the L116A mutation on expression and doubled the expression of the D105A mutant. At three of the six positions in the C-terminal half of the domain (Val113, Leu116, and, as reported in Ref. 13Lu Z.-L. Curtis C.A. Jones P.G. Pavia J. Hulme E.C. Mol. Pharmacol. 1997; 51: 234-241Crossref PubMed Scopus (88) Google Scholar, Asp122), mutations that decreased expression levels increased ACh affinity; in two cases (Val113 and Leu116), signaling efficacy and/or basal activity was also increased. Mutations of acidic residues homologous to Asp122 in rhodopsin (20Cohen G.B. Yang T. Robinson P.R. Oprian D.D. Biochemistry. 1993; 32: 6111-6115Crossref PubMed Scopus (218) Google Scholar, 21Fahmy K. Sakmar T.P. Biochemistry. 1993; 32: 7229-7236Crossref PubMed Scopus (135) Google Scholar), the α1 receptor (22Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar), and the β2 receptor (23Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar) increase basal activity, but we have not observed this for mutations of Asp122 in the M1mAChR, even after antagonist preincubation to increase the expression levels. 2Z-L. Lu, unpublished observations. In contrast, S120A behaved like a classical constitutively active mutant, showing raised basal activity, increased agonist affinity, enhanced efficacy of a partial agonist, and an increased GTP shift (24Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar, 25Spalding T.A. Burstein E.S. Wells J.W. Brann M.R. Biochemistry. 1997; 36: 10109-10116Crossref PubMed Scopus (55) Google Scholar). Together, Val113, Leu116, and Ser120 form a compact patch of residues located on the inward facing surface of the cytoplasmic half of TMD 3 (Fig. 7b). In the extended ternary complex model of agonist-receptor-G protein interaction, a conformational equilibrium is postulated to exist between preexisting ground and active states of the receptor, governed by an isomerization constant, K (24Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar). Agonists bind to the ground state conformation with low affinity, Ka, but to the activated state with a higher affinity, α·Ka. In the simplest case, the G-protein binds selectively to the activated conformation. The extended ternary complex model offers three routes to mutational effects on efficacy: a change in the isomerization constant, K; a change in the agonist cooperativity, α; or a change in the affinity of the G protein for the activated receptor, KG. A selective increase in K provides the most economical explanation for the simultaneously increased basal activity, agonist affinity, and signaling efficacy of the group of constitutively active mutants (24Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar), although it is not possible entirely to exclude an increase in KG, combined with a simultaneous increase in Ka or in the cooperativity factor α. A priori, an increase in KG seems unlikely to result from a side chain deletion. Detailed calculations based on the assumption of a changed isomerization constant (described in Ref. 16Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar) have suggested a value of about 2 × 10−4 for K for the wild-type M1 receptor and increases of about 2- and 40-fold in this parameter for the S120A and L116A mutants. Particularly in the case of L116A, this may reflect a selective destabilization of the ground state of the receptor, because the expression of the mutant was decreased by approximately the same ratio. These changes are enough to account for the observed increases in the ACh binding affinity, as well as in the ACh signaling efficacy, without an increase in the ground state binding constant, Ka (16Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar). However, in the case of the L116A mutant, Ka for pilocarpine may be increased. The pharmacology of this mutant would repay further investigation, particularly as it retains some signaling activity even in the presence of NMS and atropine (Fig. 6, a and c). This may imply a partial uncoupling of the ligand binding site from the G protein binding site. For V113A, there is a 30% increase in the signaling efficacy of ACh (16Hulme E.C. Lu Z.-L. J. Physiol. (Paris). 1998; 92: 269-274Crossref PubMed Scopus (13) Google Scholar), and a 3-fold increase for pilocarpine. However, the 10-fold increases observed in ACh and pilocarpine binding affinity and a significant decrease in antagonist affinity imply an additional perturbation of the ground state conformation of the receptor by this mutation. The residues whose mutation decreased the signaling efficacy of ACh form a strip extending the full length of TMD 3 (Fig. 7b). In the outer, N-terminal, part of the helix, they lie on the opposite face to the sector of null residues. In the inner, C-terminal, part, they are displaced by one residue (100° of arc) from the group of residues whose mutations enhanced signaling. The mutation of Trp101, Asp105, Tyr106, and Ser109 strongly reduced ACh signaling efficacy. Mutation of these residues also reduced the ground state binding of ACh and, with the exception of Ser109, decreased the affinities of NMS and QNB. Mutation of the two more peripheral residues, Leu102 and Asn110, inhibited ground state binding of ACh but had little effect on receptor activation. Our studies suggest that Tyr106 contributes almost as much free energy as Asp105 to the binding of NMS. This may indicate an amino-aromatic interaction (26Dougherty D.A. Science. 1996; 271: 163-168Crossref PubMed Scopus (2366) Google Scholar), since mutation of the equivalent Tyr to Phe in the M3 mAChR had little effect on the binding of NMS, although it reduced the affinity of ACh (27Wess J. Gdula D. Brann M.R. EMBO J. 1991; 10: 3729-3734Crossref PubMed Scopus (125) Google Scholar). In contrast, Asp105 plays a much larger part in the binding of the tertiary amine QNB, consistent with the formation of a charge-reinforced hydrogen bond. In the case of Trp101, the aromaticity of the side chain has been reported to be important for both NMS and ACh binding (28Matsui H. Lazareno S. Birdsall N.J.M. Mol. Pharmacol. 1995; 47: 88-98PubMed Google Scholar). In general, the data support the conjecture of Hibert et al. that aromatic as well as polar interactions are important for the binding of muscarinic ligands (29Hibert M.F. Trumpp-Kallmeyer S. Bruinvels A. Hoflack J. Mol. Pharmacol. 1991; 40: 8-15PubMed Google Scholar). These findings underline the primary role of Asp105, supported by Tyr106 in ACh binding and signal transduction (9Spalding 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, 10Page K.M. Curtis C.A.M. Jones P.G. Hulme E.C. Eur. J. Pharmacol. 1995; 289: 429-437Crossref PubMed Scopus (50) Google Scholar, 27Wess J. Gdula D. Brann M.R. EMBO J. 1991; 10: 3729-3734Crossref PubMed Scopus (125) Google Scholar). Notably, Asp105 is the only residue in this set whose mutation completely abolished signaling. However, Asp105 appears to have a complex function. It may contribute to the stability of the receptor's ground state structure, as well as being vital for ligand contact. This may imply that its side chain also forms an intramolecular interaction, as appears to be the case for Glu113 in rhodopsin (30Robinson P.R. Cohen G.B. Zhukovsky E.A. Oprian D.D. Neuron. 1992; 9: 719-725Abstract Full Text PDF PubMed Scopus (442) Google Scholar). The tightly delimited strip of residues whose mutation strongly diminished ACh signaling efficacy extends to the cytoplasmic end of the helix. At this point, it becomes continuous with residues homologous to the functionally conserved residues recently identified by random saturation mutagenesis in the second intracellular loop of the M5 mAChR (31Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1998; 273: 24322-24327Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), which provides a cytoplasmic extension of TMD 3. Mutation of Ser112 and particularly Ile119 as well as Arg123 (12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar) had large effects on ACh signaling efficacy. However, these mutations did not strongly affect receptor expression levels, suggesting that these residues make limited contributions to the stability of the ground state. The mutation S112A caused a small but significant enhancement of NMS affinity, while I119A decreased NMS as well as ACh affinity. We suggest that the side chains of these two residues make weak intramolecular contacts in the ground state and that these become stronger in the active conformation of the receptor. An interdomain contact has also been postulated for the residue homologous to Ile119 in the GnRH receptor (32Ballesteros J. Kitanovic S. Guarnieri F. Davies P. Fromme B.J. Konvicka K. Chi L. Millar R.P. Davidson J.S. Weinstein H. Sealfon S.C. J. Biol. Chem. 1998; 273: 10445-10453Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). If this is true, the main effect of the deletion of these side chains should be to decrease the isomerization constant, K. However, it is not possible to exclude the possibility of a more direct role for them in the formation of the G protein binding pocket, leading to effects on KG; the basal activity of the I119A mutant is decreased,2 showing that there are reductions in the product K·KG, which governs basal activity. Arg123 also has the potential for direct binding to the G protein as well as for intrareceptor contacts (12Jones P.G. Curtis C.A.M. Hulme E.C. Eur. J. Pharmacol. 1995; 288: 251-257Crossref PubMed Scopus (68) Google Scholar). With the possible exception of Asn115, there is no evidence that these residues influence the catalytic steps within the ARG ternary complex that occur subsequent to its formation. In summary, scanning mutagenesis has revealed strong functional differentiation of the surface of TMD 3 of the M1 mAChR. It has demonstrated the α-helical secondary structure of the domain and suggested its orientation with respect to the other transmembrane elements and the lipid bilayer. The results suggest that the C-terminal half of the helix is deeply buried within the receptor structure. On the buried surface, we propose that a distinct group of amino acid side-chains make intramolecular bonds that selectively stabilize the ground state of the receptor. Mutagenic disruption of these characteristically decreases receptor expression and increases agonist affinity and signal transduction. Running parallel to the helix axis and spanning the whole transmembrane region, a topologically distinct strip of residues on one face of TMD 3 forms intermolecular (ACh-receptor, receptor-G protein) or intramolecular bonds that are necessary for the stability or activity of the ARG ternary complex. Mutation of these decreases signal transduction. The binding of ACh may disrupt the first set of contacts while promoting the second. This may trigger the rotational movement of TMD 3 deduced from spectroscopic studies of rhodopsin (4Farrens D.L. Altenbach C. Yang K. Hubbell W. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1117) Google Scholar) and the β2 receptor (23Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar), and could mobilize the cytoplasmic ends of the surrounding TMDs. We propose that TMD 3 and its cytoplasmic extension (31Burstein E.S. Spalding T.A. Brann M.R. J. Biol. Chem. 1998; 273: 24322-24327Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) may act as an intramolecular rotary switch in receptor activation. Several of the key amino acids, particularly Ser112, Leu116, Ile119, Ser120, Asp122, Arg123, and Tyr124, are very highly conserved in the G protein-coupled receptor superfamily (1Baldwin J.M. Schertler G.F.X. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (635) Google Scholar), suggesting the potential generality of such a mechanism.