Use of a sandwich enzyme-linked immunosorbent assay strategy to study mechanisms of G protein-coupled receptor assembly

All G protein-coupled receptors are predicted to consist of a bundle of sev en transmembrane helices (I-VII) that are connected by various extracellula r 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 func tional receptor complexes (Schoneberg, T. Liu, J., and Wess, J. (1995) J. B iol. Chem. 270, 18000-18006). As a model system, we used two polypeptides, referred to, m3-trunk and m3-tail, that were generated by "splitting'' the m3 muscarinic receptor within the third intracellular loop. We initially de monstrated, by employing a sandwich enzyme-linked immunosorbent assay strat egy, that the two receptor fragments directly associate with each other whe n coexpressed in COS-7 cells. Additional studies with N- and C terminal fra gments derived from other G protein-coupled receptors showed that fragment association was highly receptor-specific. In subsequent experiments, the sa ndwich enzyme-linked immunosorbent assay system was used to identify amino acids that are required for proper fragment (receptor) assembly. Point muta tions were introduced into m3-trunk or m3-tail, and the ability of these mu tations to interfere with efficient fragment assembly was examined. These s tudies showed that three highly conserved proline residues (located in tran smembrane helices V, VI, and VII) are essential for proper fragment associa tion (receptor assembly). Interestingly, incubation with classical muscarin ic agonists and antagonists or allosteric ligands led to significant increa ses in the efficiency of fragment association (particularly upon substituti on of the conserved proline residues), indicating that all of these ligands can act as "anchors" between the m3-trunk and m3-tail fragments. The appro ach described here should be generally applicable to gain deeper insight in to the molecular mechanisms governing G protein-coupled receptor structure and assembly.