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J. Biol. Chem., Vol. 279, Issue 50, 51739-51744, December 10, 2004

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The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for L-Amino Acid Sensing^*

Hee-Chang Mun, Alison H. Franks, Emma L. Culverston, Karen Krapcho, Edward F. Nemeth, and Arthur D. Conigrave¶

From the School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia and NPS Pharmaceuticals, Salt Lake City, Utah 84108

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814–4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1–599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.

Received for publication, June 3, 2004 , and in revised form, July 29, 2004.

^* 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. Tel.: 612-9351-3883; Fax: 612-9351-4726; E-mail: a.conigrave{at}mmb.usyd.edu.au.

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