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

Vi Pham, Maoqing Dong, John D. Wade, Laurence J. Miller, Craij J. Morton, Hooi Ling Ng, Michael W. Parker, Patrick M. Sexton

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

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-benzoyl-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 [Bpa19]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 Cys134-Lys141 (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 Met49 as the cross-linking site for [Bpa8]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa8]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.

Original languageEnglish (US)
Pages (from-to)28610-28622
Number of pages13
JournalJournal of Biological Chemistry
Volume280
Issue number31
DOIs
StatePublished - Aug 5 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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