Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling

Muscarinic acetylcholine receptors (m1-m5) were studied by a combined molecular genetic/pharmacologic approach to elucidate the molecular characteristics of the ligand binding site and of the receptor domains involved in G protein coupling. Site-directed mutagenesis studies of the rat m3 muscarinic receptor suggest that the acetylcholine binding domain is formed by a series of hydrophilic amino acids located in the "upper" half of transmembrane domains (TM) III, V, VI, and VII. Moreover, we showed that mutational modification of a TM VI Asn residue (Asn507 in the rat m3 receptor sequence) which is characteristic for the muscarinic receptor family has little effect on high-affinity acetylcholine binding and receptor activation, but results in dramatic reductions in binding affinities for certain subclasses of muscarinic antagonists. The N-terminal portion of the third intracellular loop (i3) of muscarinic and other G protein-coupled receptors has been shown to play a central role in determining the G protein coupling profile of a given receptor subtype. Insertion mutagenesis studies with the rat m3 muscarinic receptor suggest that this region forms an amphiphilic alpha-helix and that the hydrophobic side of this helix represents an important G protein recognition surface. Further mutational analysis of this receptor segment showed that Tyr254 located at the N-terminus of the i3 loop of the m3 muscarinic receptor plays a key role in muscarinic receptor-induced Gq activation. The studies described here, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.     

Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants.

Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.     

Characterization of the subtype of muscarinic receptor coupled to the stimulation of phosphoinositide hydrolysis in 132-1N1 human astrocytoma cells.

Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.     

Erk1/2- and p38 MAP kinase-dependent phosphorylation and activation of cPLA2 by m3 and m2 receptors

This study examined the upstream signaling pathways initiated by muscarinic m2 and m3 receptors that mediate sustained ERK1/2- and p38 MAP kinase-dependent phosphorylation and activation of the 85-kDa cytosolic phospholipase (cPL)A(2) in smooth muscle. The pathway initiated by m2 receptors involved sequential activation of Gbetagamma(i3), phosphatidylinositol (PI)3-kinase, Cdc42, and Rac1, p21-activated kinase (PAK1), p38 mitogen-activated protein (MAP) kinase, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (61 +/- 5 to 72 +/- 4%) by the m2 antagonist methoctramine, Gbeta antibody, pertussis toxin, the PI3-kinase inhibitor LY 294002, PAK1 antibody, the p38 MAP kinase inhibitor SB-203580, and a Cdc42/Rac1 GEF (Vav2) antibody and by coexpression of dominant-negative Cdc42 and Rac1 mutants. The pathway initiated by m3 receptors involved sequential activation of Galpha(q), PLC-beta1, PKC, ERK1/2, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (35 +/- 3 to 41 +/- 5%) by the m3 antagonist 4-diphenylacetoxy-N-methylpiperdine (4-DAMP), the phosphoinositide hydrolysis inhibitor U-73122, the PKC inhibitor bisindolylmaleimide, and the ERK1/2 inhibitor PD 98059. cPLA(2) activity was not affected in cells coexpressing dominant-negative RhoA and PLC-delta1 mutants, implying that PKC was not derived from phosphatidylcholine hydrolysis. The effects of ERK1/2 and p38 MAP kinase on cPLA(2) activity were additive and accounted fully for activation and phosphorylation of cPLA(2).     

Bacterial expression of human muscarinic receptor fusion proteins and generation of subtype-specific antisera

A family of five muscarinic acetylcholine receptor genes (m1-m5) encode highly related proteins; however, for methodological reasons it has not been possible to detect the gene products individually. To develop antibody probes specific for the receptor subtypes, unique regions of m1-m5 cDNAs, corresponding to the third cytoplasmic (i3) loops, were subcloned into bacterial expression vectors and the fusion proteins expressed in E. coli were used to generate rabbit antisera. These antisera react specifically with the respective fusion proteins on immunoblots and selectively immunoprecipitate each of the native cloned receptors. Since the i3 loops are immunogenic and the epitopes in the cloned receptors are accessible to antibodies, this approach should be valuable for immunological studies of the native receptors.     

Discrete amino acid sequences of the alpha 1-adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis.

We have constructed a variety of chimeric beta 2/alpha 1 adrenergic receptors (AR) in which selected portions of the third intracellular loop of the alpha (1B)AR were substituted into the corresponding regions of the beta 2AR. The mutant receptors were both transiently and permanently expressed in COS-7 or L-cells, respectively, and tested for their ability to mediate epinephrine-induced activation of polyphosphoinositide (PI) hydrolysis and adenylylcyclase. We have determined that 27 amino acids of the alpha (1B)AR (residues 233-259) derived from the N-terminal portion of the third intracellular loop represent the structural determinant conferring to the beta 2AR the ability to activate PI hydrolysis. This finding suggests that in the alpha (1B)AR the N-terminal portion of the third intracellular loop plays a major role in determining the selectivity of receptor-G protein coupling. However, replacement of alpha 1B sequences in the third intracellular loop of the beta 2AR did not abolish the latter receptor's coupling to activation of adenylylcyclase, thus resulting in chimeric adrenergic receptors which activated both PI hydrolysis and adenylylcyclase. These results indicate that, even if the N-terminal portion of the third intracellular loop is a major determinant of the selectivity of receptor-G protein coupling, other structural domains of the receptors also modulate this property. The comparison of the amino acid sequences which determine the selectivity of G protein coupling in functionally similar receptors may help to elucidate the structural basis for activation of specific G protein-effector systems.     

Cloning and Expression of a G Protein-Linked Acetylcholine Receptor from Caenorhabditis elegans

Abstract : We have isolated a cDNA clone from the nematode Caenorhabditis elegans that encodes a protein of greatest sequence similarity to muscarinic acetylcholine receptors. This gene codes for a polypeptide of 682 amino acids containing seven putative transmembrane domains. The amino acid identities, excluding a highly variable middle portion of the third intracellular loop, to the human m1-m5 receptors are 28-34%. When this cloned receptor was coexpressed with a G protein-gated inwardly rectifying K⁺ channel (GIRK1) in Xenopus oocyte, acetylcholine was able to elicit the GIRK current. This acetylcholine-induced current was substantially inhibited by the muscarinic antagonist atropine in a reversible manner. However, another muscarinic agonist oxotremorine and antagonists scopolamine and pirenzepine had little or negligible effects on this receptor. Taken together, these results suggest that the cloned gene encodes a G protein-linked acetylcholine receptor that is most similar to but pharmacologically distinct from muscarinic acetylcholine receptors.     

Molecular cloning and expression of a fifth muscarinic acetylcholine receptor

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1-516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.     

Combined phosphoinositide and Ca2+ signals mediating receptor specificity toward neuronal Ca2+ channels

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates Ca(2+) (I(Ca)) and M-type K(+) currents in superior cervical ganglion sympathetic neurons. In those cells, M(1) muscarinic and AT(1) angiotensin types do not elicit Ca(2+)(i) signals and suppress both currents via depletion of PIP(2), whereas the B(2) bradykinin and P2Y purinergic types elicit robust IP(3)-mediated [Ca(2+)](i) rises and neither deplete PIP(2) nor inhibit I(Ca). We have suggested that this specificity arises from differential Ca(2+)(i) signals underlying receptor-specific stimulation of PIP(2) synthesis by phosphatidylinositol (PI) 4-kinase. Here, we investigate which PI 4-kinase isoform underlies this signal, whether stimulation of PI 4-phosphate 5-kinase is also required, and the origin of receptor-specific Ca(2+)(i) signals. Recordings of I(Ca) were used as a PIP(2) "biosensor." In control, stimulation of M(1), but not B(2) or P2Y, receptors robustly suppressed I(Ca). However, when PI 4-kinase IIIβ, diacylglycerol kinase, Rho, or Rho-kinase was blocked, agonists of all three receptors robustly suppressed I(Ca). Overexpression of exogenous M(1) receptors yielded large [Ca(2+)](i) rises by muscarinic agonist, and transfection of wild-type IRBIT decreased Ca(2+)(i) signals, whereas dominant negative IRBIT-S68A had little effect on B(2) or P2Y responses but greatly increased muscarinic responses. We conclude that overlaid on microdomain organization is IRBIT, setting a "threshold" for [IP(3)], assisting in fidelity of receptor specificity.     

Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, we derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of [3H]-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing m1-m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for m1 receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at m1 receptors, and a 4-fold greater potency at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.     

Cholinergic Deafferentation of Dorsal Hippocampus by Fimbria-Fornix Lesioning Differentially Regulates Subtypes (m1-m5) of Muscarinic Receptors

Abstract: Unilateral aspiration lesions of the rostral supracallosal stria/cingulum bundle and fimbria-fornix were performed on adult female rats. Ten and 24 days post lesioning, an elevation (17%; p<0.01) of total muscarinic receptors was observed in lesioned versus control hippocampi. By using antisera selective for each of the five molecularly defined subtypes (m1-m5) of muscarinic receptors, significant changes were observed in the levels of expression for at least four receptor proteins. Three receptor subtypes increased in density: m1 by 14% (from 943 to 1,078 fmol/mg); m3 by 77% (from 150 to 268 fmol/ mg); and m4 by 29% (from 220 to 285 fmol/mg). In contrast, a 22% decrease in the density of m2 receptors was found (from 220 to 173 fmol/mg). Detectable levels of m5 receptors were low in the hippocampus (∼1% of total receptors), and reliable measurements were not obtained. The directions of these changes are likely to be related to the pre- or postsynaptic localization of these receptor subtypes.     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

Heterogeneity of muscarinic receptors coupled to phosphoinositide breakdown in guinea pig brain and peripheral tissues

Muscarinic receptors coupled to phosphoinositide hydrolysis (PI) are present in guinea pig bladder and colon. Compared to rat cerebral cortex, an extensively studied muscarinic/PI turnover system, all agonists were more potent and efficacious in both bladder and colon. The "M1-selective antagonists", pirenzepine and dicyclomine, were much more potent (Ki = 1-5 nM) and selective (300 to 500-fold) at both rat and guinea pig brain and guinea pig colon receptors, compared to PI-coupled receptors in guinea pig bladder. In contrast, "M2-selective antagonists", AF-DX 116 and HHSiD, were 2-6 fold more potent in bladder than in brain, while HHSiD was very potent in the colon (50 times more potent than in brain). These results suggest a pharmacological heterogeneity of PI-linked muscarinic receptors. If muscarinic receptors with a low affinity for pirenzepine are defined as M2, these results show that the guinea pig bladder contains PI-linked M2 muscarinic receptors, whereas the guinea pig colon contains PI-linked M1 receptors.     

Structural aspects of M? muscarinic acetylcholine receptor dimer formation and activation

To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (～50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.     

Distinct structural changes in a G protein-coupled receptor caused by different classes of agonist ligands

The activity of G protein-coupled receptors can be modulated by different classes of ligands, including agonists that promote receptor signaling and inverse agonists that reduce basal receptor activity. The conformational changes in receptor structure induced by different agonist ligands are not well understood at present. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced conformational changes in a series of cysteine-substituted mutant M(3) muscarinic acetylcholine receptors. The observed disulfide cross-linking patterns indicated that muscarinic agonists trigger a separation of the N-terminal segment of the cytoplasmic tail (helix 8) from the cytoplasmic end of transmembrane domain I. In contrast, inverse muscarinic agonists were found to increase the proximity between these two receptor regions. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most G protein-coupled receptors, our findings should be of broad general relevance.