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J. Biol. Chem., Vol. 281, Issue 43, 32485-32495, October 27, 2006

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Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor^*

Thomas Dean, Ashok Khatri, Zhanna Potetinova, Gordon E. Willick, and Thomas J. Gardella¹

From the Endocrine Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114 and the Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada

The principal receptor-binding domain (Ser¹⁷–Val³¹) of parathyroid hormone (PTH) is predicted to form an amphiphilic -helix and to interact primarily with the N-terminal extracellular domain (N domain) of the PTH receptor (PTHR). We explored these hypotheses by introducing a variety of substitutions in region 17–31 of PTH-(1–31) and assessing, via competition assays, their effects on binding to the wild-type PTHR and to PTHR-delNt, which lacks most of the N domain. Substitutions at Arg²⁰ reduced affinity for the intact PTHR by 200-fold or more, but altered affinity for PTHR-delNt by 4-fold or less. Similar effects were observed for Glu substitutions at Trp²³, Leu²⁴, and Leu²⁸, which together form the hydrophobic face of the predicted amphiphilic -helix. Glu substitutions at Arg²⁵, Lys²⁶, and Lys²⁷ (which forms the hydrophilic face of the helix) caused 4–10-fold reductions in affinity for both receptors. Thus, the side chains of Arg²⁰, together with those composing the hydrophobic face of the ligand's putative amphiphilic -helix, contribute strongly to PTHR-binding affinity by interacting specifically with the N domain of the receptor. The side chains projecting from the opposite helical face contribute weakly to binding affinity by different mechanisms, possibly involving interactions with the extracellular loop/transmembrane domain region of the receptor. The data help define the roles that side chains in the binding domain of PTH play in the PTH-PTHR interaction process and provide new clues for understanding the overall topology of the bimolecular complex.

Received for publication, June 28, 2006 , and in revised form, August 9, 2006.

^* This work was supported in part by National Institutes of Health Grant DK-11794 (to T. J. G.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

¹ To whom correspondence should be addressed: Endocrine Unit, Massachusetts General Hospital, Wellman Bldg., Rm. 503C, 50 Blossom St., Boston, MA 02114. Tel.: 617-726-3966; Fax: 617-726-7543; E-mail: gardella{at}helix.mgh.harvard.edu.

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