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J. Biol. Chem., Vol. 279, Issue 8, 6720-6729, February 20, 2004

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Spatial Proximity between a Photolabile Residue in Position 19 of Salmon Calcitonin and the Amino Terminus of the Human Calcitonin Receptor^*

Vi Pham, John D. Wade, Brooke W. Purdue, and Patrick M. Sexton, NHMRC senior research fellow

From the Howard Florey Institute of Experimental Physiology and Medicine, the University of Melbourne, Victoria 3010, Australia

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg^11,18,Bpa¹⁹]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys¹³⁴ and Met¹⁸⁷. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met¹³³ (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys¹³⁴–Lys¹⁴¹. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.

Received for publication, July 7, 2003 , and in revised form, October 23, 2003.

^* This study was supported by National Health and Medical Research Council Project Grant 145703. 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.

NHMRC principal research fellow.

To whom correspondence should be addressed: Howard Florey Institute, University of Melbourne, Gate 11, Royal Parade, Parkville, Victoria 3010, Australia. Fax: 61-3-9348-1707; E-mail: p.sexton{at}hfi.unimelb.edu.au.

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