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J. Biol. Chem., Vol. 282, Issue 26, 19000-19010, June 29, 2007

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Identification and Dissection of Ca²⁺-binding Sites in the Extracellular Domain of Ca²⁺-sensing Receptor^*

Yun Huang¹, Yubin Zhou¹, Wei Yang, Robert Butters, Hsiau-Wei Lee, Shunyi Li, Adriana Castiblanco, Edward M. Brown², and Jenny J. Yang³

From the Department of Chemistry, Center for Biotechnology and Drug Design Georgia State University, Atlanta, Georgia 30303 and Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115

Ca²⁺-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca²⁺ concentration ([Ca²⁺]_o) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca²⁺ in regulating CaSRs is the lack of adequate information about their Ca²⁺-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca²⁺-binding affinities. In this paper, we have reported the identification of three potential Ca²⁺-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials. Mutation of the predicted ligand residues in the full-length CaSR caused abnormal responses to [Ca²⁺]_o, similar to those observed with naturally occurring activating or inactivating mutations of the CaR, supporting the essential role of these predicted Ca²⁺-binding sites in the sensing capability of the CaSR. In addition, to probe the intrinsic Ca²⁺-binding properties of the predicted sequences, we engineered two predicted continuous Ca²⁺-binding sequences individually into a scaffold protein provided by a non-Ca²⁺-binding protein, CD2. We report herein the estimation of the metal-binding affinities of these predicted sites in the CaSR by monitoring aromatic-sensitized Tb³⁺ fluorescence energy transfer. Removing the predicted Ca²⁺-binding ligands resulted in the loss of or significantly weakened cation binding. The potential Ca²⁺-binding residues were shown to be involved in Ca²⁺/Ln³⁺ binding by high resolution NMR and site-directed mutagenesis, further validating our prediction of Ca²⁺-binding sites within the extracellular domain of the CaSR.

Received for publication, February 6, 2007 , and in revised form, April 19, 2007.

^* This work was supported, in part, by National Institutes of Health Grants GM62999-1 and GM070555, National Science Foundation Grant MCB-0092486, American Heart Association Grant 0655168B (to J. J. Y.), and a Predoctoral Fellowship from the Brain and Behavior Program at Georgia State University (to Y. H.). 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.

¹ These authors made equal contributions to this work and should both be considered as first authors.

² Supported by National Institutes of Health Grants DK52005 and DK67155.

³ To whom correspondence should be addressed: Dept. of Chemistry, Georgia State University, University Plaza, Atlanta, GA 30303. Tel.: 404-651-4620; Fax: 404-651-2751; E-mail: chejjy{at}langate.gsu.edu.

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