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.Traditionally, molecular models describing the interaction of G protein-coupled receptors (GPCRs) 1 with their G protein targets have been based on the assumption that the receptors exist as monomers and couple to G proteins in a 1:1 stoichiometry. However, several recent studies suggest that GPCRs are able to form dimeric or oligomeric arrays (1-9), indicating that classical models of receptor/G protein coupling may be oversimplified (for a recent review, see Ref. 10). Hebert et al. (2), for example, provided the first direct evidence that  2 -adrenergic receptors can exist in dimeric forms. These investigators also demonstrated that dimer formation occurred through non-covalent interactions and that receptor dimers were not formed artifactually during processing of samples for immunological studies. Moreover, dimer formation was shown to correlate well with the ability of the  2 -adrenergic receptor to interact productively with G proteins (2). In addition, consistent with the notion that GPCR dimerization may be functionally relevant, a dimerization-defective mutant ␦-op...
To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3 (3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3 (3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3 (3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484 -493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline) 3 , and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [ 35 S]guanosine 5 -3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.G protein-coupled receptors (GPCRs) 1 form one of the largest protein families found in nature, and current estimates are that approximately one thousand different such receptors exist in mammals (1). Despite the remarkable structural diversity of their activating ligands, all GPCRs are predicted to share a common molecular architecture consisting of seven transmembrane helices (TM I-VII) linked by alternating intracellular (i1-i3) and extracellular (o2-o4) loops (Fig. 1). Whereas residues located on the extracellular receptor surface are known to be involved in ligand binding, the cytoplasmic receptor surface is critical for G protein recognition and activation (2-6).At present, high resolution structural information is not a...
The N termini of two G protein ␣ subunits, ␣ q and ␣ 11 , differ from those of other ␣ subunits in that they display a unique, highly conserved six-amino acid extension (MTLESI(M)). We recently showed that an ␣ q deletion mutant lacking these six amino acids (in contrast to wild type ␣ q ) was able to couple to several different G s -and G i/o -coupled receptors, apparently due to promiscuous receptor/G protein coupling (Kostenis, E., Degtyarev, M. Y., Conklin, B. R., and Wess, J. (1997) J. Biol. Chem. 272, 19107-19110). To study which specific amino acids within the N-terminal segment of ␣ q/11 are critical for constraining the receptor coupling selectivity of these subunits, this region of ␣ q was subjected to systematic deletion and alanine scanning mutagenesis. All mutant ␣ q constructs (or wild type ␣ q as a control) were coexpressed (in COS-7 cells) with the m2 muscarinic or the D2 dopamine receptors, two prototypical G i/o -coupled receptors, and ligand-induced increases in inositol phosphate production were determined as a measure of G protein activation. Surprisingly, all 14 mutant G proteins studied (but not wild type ␣ q ) gained the ability to productively interact with the two G i/o -linked receptors. Similar results were obtained when we examined the ability of selected mutant ␣ q subunits to couple to the G s -coupled 2-adrenergic receptor. Additional experiments indicated that the functional promiscuity displayed by all investigated mutant ␣ q constructs was not due to overexpression (as compared with wild type ␣ q ), lack of palmitoylation, or initiation of translation at a downstream ATG codon (codon seven). These data are consistent with the notion that the six-amino acid extension characteristic for ␣ q/11 subunits forms a tightly folded protein subdomain that is critical for regulating the receptor coupling selectivity of these subunits. G protein-coupled receptors (GPCRs)1 regulate the activity of a large variety of effector systems via interaction with specific classes of heterotrimeric G proteins (consisting of ␣, , and ␥ subunits) that are attached to the cytoplasmic side of the plasma membrane (1-6). In most cases, an individual GPCR can only activate a distinct subset of the many structurally similar G proteins present in each cell (7,8). How this selectivity is achieved at a molecular level is currently being explored by a great number of laboratories.A large body of evidence indicates that multiple regions on the G protein ␣ subunits play key roles in receptor binding and dictating the selectivity of receptor/G protein interactions (2,5,6,8,9). Specifically, recent studies have shown that residues at the extreme C terminus of the G protein ␣ subunits are of fundamental importance for regulating receptor/G protein coupling selectivity (10 -13), probably by directly contacting the receptor protein (14, 15). However, several lines of evidence indicate that other regions of G␣ also contribute to receptor binding and the selectivity of receptor recognition (2, 5, 6, 9). Biochemical stu...
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