Insights into interactions between the α-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling

Fish-like calcitonins ( CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT( 8-32) containing a single photolabile p-benzo-yl-L- phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa(19)] sCT( 8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys(134)-Lys(141) ( within the amino terminus of the receptor, adjacent to transmembrane 1) ( Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. ( 2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met(49) as the crosslinking site for [ Bpa(8)] sCT( 8-32). This site differed from the previously identified cross-linking site of the agonist [ Bpa(8)] human CT ( Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. ( 2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa8 analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core.