title: Use of a sandwich enzyme-linked immunosorbent assay strategy to study mechanisms of G protein-coupled receptor assembly.
abstract: All G protein-coupled receptors are predicted to consist of a bundle of seven transmembrane helices (I-VII) that are connected by various extracellular and intracellular loops. At present, little is known about the molecular interactions that are critical for the proper assembly of the transmembrane receptor core. To address this issue, we took advantage of the ability of coexpressed N- and C-terminal m3 muscarinic receptor fragments to form functional receptor complexes (Schöneberg, T., Liu, J., and Wess, J. (1995) J. Biol. Chem. 270, 18000-18006). As a model system, we used two polypeptides, referred to as m3-trunk and m3-tail, that were generated by "splitting" the m3 muscarinic receptor within the third intracellular loop. We initially demonstrated, by employing a sandwich enzyme-linked immunosorbent assay strategy, that the two receptor fragments directly associate with each other when coexpressed in COS-7 cells. Additional studies with N- and C-terminal fragments derived from other G protein-coupled receptors showed that fragment association was highly receptor-specific. In subsequent experiments, the sandwich enzyme-linked immunosorbent assay system was used to identify amino acids that are required for proper fragment (receptor) assembly. Point mutations were introduced into m3-trunk or m3-tail, and the ability of these mutations to interfere with efficient fragment assembly was examined. These studies showed that three highly conserved proline residues (located in transmembrane helices V, VI, and VII) are essential for proper fragment association (receptor assembly). Interestingly, incubation with classical muscarinic agonists and antagonists or allosteric ligands led to significant increases in the efficiency of fragment association (particularly upon substitution of the conserved proline residues), indicating that all of these ligands can act as "anchors" between the m3-trunk and m3-tail fragments. The approach described here should be generally applicable to gain deeper insight into the molecular mechanisms governing G protein-coupled receptor structure and assembly.
authors.affiliation: Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: J
authors.initials: J
authors.lastname: Jakubik
authors.forename: J
authors.initials: J
authors.lastname: Wess
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011392:Proline / Q000378:metabolism; D051381:Rats; D043587:Receptor, Muscarinic M3; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017483:Receptors, Vasopressin / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D010446:Peptide Fragments; D043587:Receptor, Muscarinic M3; D011956:Receptors, Cell Surface; D011976:Receptors, Muscarinic; D017483:Receptors, Vasopressin; D004247:DNA; D011392:Proline; D019204:GTP-Binding Proteins