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Multiple Domains Interacting with G_s in the Porcine Calcitonin Receptor

Department of Medicine (P.O., S.M.K., S.R.G., I.N.) Harvard Medical School Cardiovascular Research Center and Arthritis Research Massachusetts General Hospital-East Charlestown, Massachusetts 02129
INSERM U349 (P.O.) Centre Viggo Petersen Hopital Lariboisière 75010 Paris, France
Department of Pharmacology and Neurosciences (H.T., I.N.) KEIO University School of Medicine Tokyo 160, Japan
Department of Medicine (Y.M., T. F.) University of Tokyo School of Medicine Mejirodai, Bunkyo-ku Tokyo 113, Japan
Cancer Research Center (E.O.) Toshima-ku, Tokyo 170, Japan
Department of Medicine (S.R.G.) Beth Israel Deaconess Medical Center and New England Baptist Bone and Joint Institute Harvard Institutes of Medicine Boston, Massachusetts 02215

The molecular basis for G_s activation by the calcitonin (CT) receptor was investigated. Based upon the analysis of conserved regions in G protein-coupled receptors, two nonoverlapping regions in the heptahelical porcine CT receptor (CTR) were selected as candidate G_s-interacting domains: the third intracellular loop residues 327–344 (KLKESQEAESHMYLKAVR, P3 region) and the C-tail residues 404–418 (KRQWNQYQAQRWAGR, P4 region). To assess their G_s-interacting function, we expressed these sequences in hybrid insulin-like growth factor II receptors in which the receptor native G_i-interacting domain was converted to CTR sequences. In COS cells transfected with either P3- or P4-substituted hybrid receptor, membrane adenylyl cyclase activity significantly increased. The up-regulated activity of cAMP was confirmed by measuring the transcriptional activity of the cAMP response element in cells expressing either hybrid receptor. A mutant CTR lacking the P4 region maintained positive cAMP response but with an attenuated maximal capacity to produce cAMP. In contrast, we could not assess the function of the P3 region using a conventional deletion method, as CT bound poorly to cells transfected with either of the two P3-deficient CTRs (one lacking the P3 region and the other lacking P3 but having the P3 sequence in reverse orientation). These data suggest that the third intracellular loop and the C-tail in CTR have domain-specific roles in G_s activation and that the hybrid receptor approach used here, combined with a conventional mutagenesis approach, is useful for intact cell analysis and functional dissection of G protein-coupled receptors.

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