TY - JOUR
T1 - Insights into interactions between the α-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling
AU - Pham, Vi
AU - Dong, Maoqing
AU - Wade, John D.
AU - Miller, Laurence J.
AU - Morton, Craij J.
AU - Ng, Hooi Ling
AU - Parker, Michael W.
AU - Sexton, Patrick M.
PY - 2005/8/5
Y1 - 2005/8/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=23344453698&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=23344453698&partnerID=8YFLogxK
U2 - 10.1074/jbc.M503272200
DO - 10.1074/jbc.M503272200
M3 - Article
C2 - 15929987
AN - SCOPUS:23344453698
SN - 0021-9258
VL - 280
SP - 28610
EP - 28622
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 31
ER -