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J. Biol. Chem., Vol. 280, Issue 31, 28610-28622, August 5, 2005

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Insights into Interactions between the -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, Craig J. Morton||, Hooi-ling Ng||, Michael W. Parker||, and Patrick M. Sexton**

From the Howard Florey Institute, The University of Melbourne, Victoria 3010, Australia, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, and ^||St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia

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.

Received for publication, March 24, 2005 , and in revised form, June 1, 2005.

^* This work was funded by National Health and Medical Research Council Project Grant 145703, the Potter Foundation Neuropeptide laboratory, and National Institutes of Health Grant DK46577 (to L. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ A National Health and Medical Research Council Principal Research Fellow.

^** A National Health and Medical Research Council Senior Research Fellow. To whom correspondence should be addressed: Howard Florey Institute, The University of Melbourne, Gate 11, Royal Parade, Parkville, Victoria 3010, Australia. Tel.: 61-3-8344-1954; Fax: 61-3-9347-0446; E-mail: p.sexton{at}hfi.unimelb.edu.au.

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