Kinetic analysis of M2 muscarinic receptor activation of Gi in Sf9 insect cell membranes.

A steady-state kinetic mechanism describing the interaction of M(2) muscarinic acetylcholine receptors and the guanine nucleotide-binding protein G(i)alpha(2)beta(1)gamma(3) are presented. Data are consistent with two parallel pathways of agonist-promoted GTPase activity arising from receptor coupled to a single or multiple guanine nucleotide-binding proteins. An aspartate 103 to asparagine receptor mutation resulted in a receptor lacking the ability to catalyze the binding of guanosine-5'-O-(3-thiotriphosphate) or guanosine triphosphate hydrolysis by the G protein. An aspartate 69 to asparagine receptor mutant was able to catalyze agonist-specific guanine nucleotide exchange and GTPase activity. A threonine 187 to alanine receptor mutation resulted in a receptor that catalyzed guanine nucleotide exchange comparable with wild-type receptors but had reduced ability to stimulate GTP hydrolysis. A tyrosine 403 to phenylalanine receptor mutation resulted in an increase in agonist-promoted GTPAse activity compared with wild type. The observation that the threonine 187 and tyrosine 403 mutants promote guanine nucleotide exchange similarly to wild type but alter GTPase activity compared with wild type suggests that the effects of the mutations arise downstream from guanine nucleotide exchange and may result from changes in receptor-G protein dissociation.     

Mutation screening of the muscarinic m2 and m3 receptor genes in asthmatics,outgrow subjects,and normal controls

Muscarinic receptors are important in the development of airway hyperresponsiveness. In some patients with asthma and in animal models of hyperreactivity, functional abnormalities in these receptors are suggested to contribute to disease. Here, we have screened for single nucleotide polymorphisms in the coding region of human muscarinic m2 and m3 receptor genes using direct fluorescence sequencing. DNA samples from 102 current asthmatics and 58 who had outgrown asthma ("outgrow" patients) were compared with 70 random non-asthmatic controls. A mutation characterized by a single base substitution (A1050G, Ser350Ser) was identified in the muscarinic m2 receptor gene. This polymorphism was common and was represented in all three groups studied. In contrast, in the m3 receptor coding region examined, we found a very rare nucleotide variant (C261T, Ile87Ile), identified in only one of the 230 samples genotyped. Therefore, neither A1050G in the m2 receptor nor C261T in the m3 receptor is likely to be functionally significant for airway hyperresponsiveness in asthma. Our data suggest that both the m2 and m3 receptor genes are highly conserved, and no significant genetic mutations are related to their possible functional changes in human asthma.     

Deletion analysis of the mouse m1 muscarinic acetylcholine receptor: effects on phosphoinositide metabolism and down-regulation

Deletions have been constructed in the putative third cytoplasmic loop of the mouse m1 muscarinic acetylcholine receptor (mAChR) gene, and the effects of these mutations on mAChR coupling to phosphoinositide metabolism and agonist-induced down-regulation have been examined following expression in Y1 adrenal carcinoma cells. Deletion of up to 123 of the 156 amino acids in this loop has no effect on antagonist or agonist binding, or on coupling to stimulation of phosphoinositide metabolism. These results suggest that the membrane proximal portions of this loop are involved in determining the specificity of functional coupling of the receptor. Deletion of 75% of the loop has no effect on short-term agonist-induced internalization but does cause a significant decrease in the magnitude of agonist-induced down-regulation of receptor number. Thus, this portion of the receptor may be involved in mediating the response to long-term agonist exposure.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Identification and molecular characterization of m3 muscarinic receptor dimers.

Several studies suggest, but do not prove directly, that muscarinic receptors may be able to form dimeric or oligomeric arrays. To address this issue in a more direct fashion, we designed a series of biochemical experiments using a modified version of the rat m3 muscarinic receptor (referred to as m3') as a model system. When membrane lysates prepared from m3' receptor-expressing COS-7 cells were subjected to Western blot analysis under non-reducing conditions, several immunoreactive species were observed corresponding in size to putative receptor monomers, dimers, and oligomers. However, under reducing conditions, the monomeric receptor species represented the only detectable immunoreactive protein, consistent with the presence of disulfide-linked m3 receptor complexes. Similar results were obtained when native m3 muscarinic receptors present in rat brain membranes were analyzed. Control experiments carried out in the presence of high concentrations of the SH group alkylating agent, N-ethylmaleimide, suggested that disulfide bond formation did not occur artifactually during the preparation of cell lysates. The formation of m3' receptor dimers/multimers was confirmed in coimmunoprecipitation studies using differentially epitope-tagged m3' receptor constructs. In addition, these studies showed that m3' receptors were also able to form non-covalently associated receptor dimers and that m3' receptor dimer formation was receptor subtype-specific. Immunological studies also demonstrated that m3' receptor dimers/multimers were abundantly expressed on the cell surface. Site-directed mutagenesis studies indicated that two conserved extracellular Cys residues (Cys-140 and Cys-220) play key roles in the formation of disulfide-linked m3' receptor dimers. These results provide the first direct evidence for the existence of muscarinic receptor dimers and highlight the specificity and molecular diversity of G protein-coupled receptor dimerization/oligomerization.     

Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes

The human and rat genes for a fifth muscarinic receptor have been cloned and expressed in mammalian cells. The 532 amino acid human protein has 89% sequence identity to the 531 amino acid rat protein and is most closely related to the m3 receptor. Both proteins are encoded by single exons. The receptor has intermediate affinity for pirenzepine and low affinity for AF-DX 116, and it increases metabolism of phosphatidylinositol when stimulated with carbachol. Expression of mRNA has yet to be observed in brain or selected peripheral tissues, suggesting that either it is substantially less abundant than m1-m4 or its distribution is quite different.     

Functional analysis of transmembrane domain 2 of the M1 muscarinic acetylcholine receptor

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M1 muscarinic acetylcholine receptor, and of the highly conserved Asn43 in TM1. The mutation of Asn43, Asn61, and Leu64 caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro415 and Tyr418 in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp71 and Asn414 may be a critical feature stabilizing the activated state of the M1 receptor. Mutation of Leu67, Ala70, and Ile74 also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp71 and Asn414. Mutation of Leu72, Gly75, and Met79 in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met79 as well as Asp71.     

The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity.

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.     

Kinetic analysis of the interleukin-13 receptor complex

Interleukin (IL)-13 is a key cytokine associated with the asthmatic phenotype. It signals via its cognate receptor, a complex of IL-13 receptor alpha1 chain (IL-13Ralpha1) with IL-4Ralpha; however, a second protein, IL-13Ralpha2, also binds IL-13. To determine the binding contributions of the individual components of the IL-13 receptor to IL-13, we have employed surface plasmon resonance and equilibrium binding assays to investigate the ligand binding characteristics of shIL-13Ralpha1, shIL-13Ralpha2, and IL-4Ralpha. shIL-13Ralpha1 bound IL-13 with moderate affinity (K(D) = 37.8 +/- 1.8 nm, n = 10), whereas no binding was observed for hIL-4Ralpha. In contrast, shIL-13Ralpha2 produced a high affinity interaction with IL-13 (K(D) = 2.49 +/- 0.94 nm n = 10). IL-13Ralpha2 exhibited the binding characteristics of a negative regulator with a fast association rate and an exceptional slow dissociation rate. Although IL-13 interacted weakly with IL-4Ralpha on its own (K(D) > 50 microm), the presence of hIL-4Ralpha significantly increased the affinity of shIL-13Ralpha1 for IL-13 but had no effect on the binding affinity of IL-13Ralpha2. Detailed kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in which IL-13 first binds to IL-13Ralpha1 and this then recruits IL-4Ralpha to stabilize a high affinity interaction.     

Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.     

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.     

Presence of antibodies against the third intracellular loop of the m2 muscarinic receptor in the sera of chronic chagasic patients.

Patients in the chronic phase of Chagas' disease suffer from a slowly evolving inflammatory cardiomyopathy that can lead to severe cardiac dilatation, congestive heart failure, and death. This process appears to be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after infection with the parasite Trypanosoma cruzi. Recent evidence suggests that there are circulating antibodies in chronic chagasic patients that alter the physiological behavior of the heart on binding to G-protein-coupled cardiovascular receptors, including beta1-adrenergic and m2 muscarinic receptors. A 42 kDa fusion protein was constructed that contains the central part of the third intracellular loop (i3; Arg(267)-Arg(381)) of the human m2 muscarinic receptor, linked to glutathione S-transferase. This fusion protein was overexpressed in Escherichia coli and subsequently purified by affinity chromatography. Based on Western blots, the i3 loop is specifically recognized by the sera of chronic chagasic patients who have reached advanced stages of cardiac failure (according to the Los Andes classification). Analysis of the prevalence and distribution of these antibodies shows a strong association between seropositive patients and moderate (group II) to severe (group III) heart dysfunction.     

Kinetic analysis of chemotactic peptide receptor modulation

The dynamics of the chemotactic peptide receptor on rabbit peritoneal polymorphonuclear leucocytes were followed using the tritiated peptide N-formylnorleucylleucylphenylalanine (FNLLP). We have used a kinetic analysis to examine the possible interrelationships between receptor loss (down-regulation), receptor-mediated peptide uptake, and receptor recycling. We have previously demonstrated that cells incubated with FNLLP show a dose-dependent reduction in the number of receptors available on the surface. This receptor down-regulation is complete within 20 min and then the number of receptors available for binding remains at a plateau level. Peptide continues to be taken up in a receptor-mediated manner even after down-regulation is complete. If peptide is removed, receptor recovery occurs and does not require protein synthesis. In these studies we have investigated the kinetics of these processes. On the basis of this analysis, we propose that the plateau receptor level is a steady-state in which receptor internalization and return occur continuously. We demonstrate that the rate of receptor-mediated peptide uptake is approximately equal to the rate of receptor recovery measured after peptide removal. In addition, the rate of receptor recovery is proportional to the number of receptors missing from the surface, suggesting receptor recycling may be occurring.