Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor

• Published in: The Journal of biological chemistry Vol. 279; no. 30; pp. 31177 - 31182
• Main Authors: Dong, Maoqing, Pinon, Delia I, Cox, Richard F, Miller, Laurence J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 23.07.2004
• Subjects: Affinity Labels; Amino Acid Sequence; Binding Sites - genetics; Calcitonin - chemistry; Calcitonin - genetics; Calcitonin - metabolism; Cell Line; Cyclic AMP - metabolism; Humans; In Vitro Techniques; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Tertiary; Receptors, Calcitonin - chemistry; Receptors, Calcitonin - genetics; Receptors, Calcitonin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M404113200

The calcitonin receptor is a member of the class B family of G protein-coupled receptors, which contains numerous potentially important drug targets. Delineation of themes for agonist binding and activation of these receptors will facilitate the rational design of receptor-active drugs. We reported previously that a photolabile residue within the carboxyl-terminal half (residue 26) and mid-region (residue 16) of calcitonin covalently label the extracellular amino-terminal domain of this receptor (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 1167–1175). Chimeric receptor studies support the importance of this region and suggest important contributions of extracellular loop domains. To examine whether other parts of the ligand may contact those loops, we developed another probe that has its photolabile site of labeling within the amino-terminal half in position 8 of the ligand. This probe was a full agonist (EC 50 = 563 ± 67 p m ), stimulating cAMP accumulation in receptor-bearing human embryonic kidney 293 cells in a concentration-dependent manner. It bound specifically and saturably ( K i = 14.3 ± 1.9 n m ) and was able to efficiently label the calcitonin receptor. By purification, specific cleavage, and sequencing of labeled wild-type and mutant calcitonin receptors, the site of attachment was identified as residue Leu 368 within the third extracellular loop of the receptor, a domain distinct from that labeled by previous probes. These data are consistent with a common ligand binding mechanism for receptors in this important family.