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J. Biol. Chem., Vol. 280, Issue 32, 29067-29072, August 12, 2005

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A Double Mutation in the Extracellular Ca²⁺-sensing Receptor's Venus Flytrap Domain That Selectively Disables L-Amino Acid Sensing^*

Hee-Chang Mun, Emma L. Culverston, Alison H. Franks, Charles A. Collyer, Roderick J. Clifton-Bligh, and Arthur D. Conigrave¶

From the School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia and Department of Endocrinology, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

The extracellular Ca²⁺-sensing receptor is activated allosterically by L-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca²⁺-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca²⁺ sensing intact, as determined by receptor-dependent activation of intracellular Ca²⁺ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to L-Phe, and T145A also exhibited markedly impaired L/D selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca²⁺ but was resistant to L-Phe at concentrations up to 100 mM. We conclude that T145A/S170T selectively disables L-amino acid sensing and that the Ca²⁺ and L-amino acid-sensing functions of the CaR can be dissociated.

Received for publication, January 1, 2005 , and in revised form, April 21, 2005.

^* This work was supported by the National Health and Medical Research Council of Australia. 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.

^¶ To whom correspondence should be addressed: School of Molecular and Microbial Biosciences (G08), University of Sydney, NSW 2006, Australia. Tel.: 61-2-9351-3883; Fax: 61-2-9351-4726; E-mail: a.conigrave{at}mmb.usyd.edu.au.

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