Insights into interactions between the alpha-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-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 [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 cross-linking 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 Bpa(8) 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.
Pham
Vi
V
Howard Florey Institute, The University of Melbourne, Victoria 3010, Australia.
Dong
Maoqing
M
Wade
John D
JD
Miller
Laurence J
LJ
Morton
Craig J
CJ
Ng
Hooi-Ling
HL
Parker
Michael W
MW
Sexton
Patrick M
PM
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002116:Calcitonin / Q000037:antagonists & inhibitors* / Q000737:chemistry; D002522:Chlorocebus aethiops; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012474:Salmon; D014162:Transfection
D010446:Peptide Fragments; D018003:Receptors, Calcitonin; D002116:Calcitonin