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

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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

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salmon calcitonin
Calcitonin Receptors
Salmon
Calcitonin
Labeling
Peptides
Peptide Fragments
Molecular modeling
G-Protein-Coupled Receptors
Fish
Bone
Fishes
Bone and Bones
salmon calcitonin (8-32)

ASJC Scopus subject areas

  • Biochemistry

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Insights into interactions between the α-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling. / Pham, Vi; Dong, Maoqing; Wade, John D.; Miller, Laurence J; Morton, Craij J.; Ng, Hooi Ling; Parker, Michael W.; Sexton, Patrick M.

In: Journal of Biological Chemistry, Vol. 280, No. 31, 05.08.2005, p. 28610-28622.

Research output: Contribution to journalArticle

Pham, Vi ; Dong, Maoqing ; Wade, John D. ; Miller, Laurence J ; Morton, Craij J. ; Ng, Hooi Ling ; Parker, Michael W. ; Sexton, Patrick M. / Insights into interactions between the α-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 31. pp. 28610-28622.
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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.",
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AU - Miller, Laurence J

AU - Morton, Craij J.

AU - Ng, Hooi Ling

AU - Parker, Michael W.

AU - Sexton, Patrick M.

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