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Originally published In Press as doi:10.1074/jbc.M909613199 on April 7, 2000

J. Biol. Chem., Vol. 275, Issue 26, 19955-19963, June 30, 2000
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Amino Acids in the Second and Third Intracellular Loops of the Parathyroid Ca2+-sensing Receptor Mediate Efficient Coupling to Phospholipase C*

Wenhan Chang, Tsui-Hua Chen, Stacy Pratt, and Dolores ShobackDagger

From the Endocrine Research Unit, Department of Veteran Affairs Medical Center, Department of Medicine, University of California, San Francisco, California 94121

To determine the role of amino acids in the second and third intracellular (IC) loops of the Ca2+-sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [Ca2+] ([Ca2+]o)-induced inositol phosphate accumulation and protein expression by immunoblotting and immunocytochemistry in human embryonic kidney 293 cells. Two CaR constructs in the second IC loop, F707A CaR and to a lesser extent L704A CaR, demonstrated reduced activation of PLC, despite levels of protein expression comparable with the wild-type (wt) CaR. Substitution of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially restored the ability of high [Ca2+]o to activate PLC. Eight residues in the third IC loop were involved in PLC signaling. The responses to high [Ca2+]o in cells expressing CaRs with Ala substitutions at these sites were <35% of the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired in their responses to [Ca2+]o even up to 30 mM. Substitutions of Leu-798 with other hydrophobic residues (Ile, Val, or Phe), but not with acidic, basic, or polar residues, produced reduced responses compared with wt. Phe-802 could be replaced with either Tyr or Trp with partial retention of the ability to activate PLC. Glu-804 could only be substituted with Asp or Gln and maintain its signaling capacity. Cell surface expression of the CaRs mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR. Cell surface CaR expression was, however, reduced substantially in cells expressing several mutants at position Glu-804 by confocal microscopy. These studies strongly implicate specific hydrophobic and acidic residues in the second and third IC loops of the parathyroid CaR (and potentially larger stretches of the third loop) in mediating efficient high [Ca2+]o-induced PLC activation and or CaR expression.

* This work was supported by Department of Veterans Affairs Merit Review and National Institutes of Health Grants DK 43400 and DK 55846.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Endocrine Research Unit, 111N, San Francisco VA Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2089; Fax: 415-750-6929; E-mail: dolores@itsa.ucsf.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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