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J. Biol. Chem., Vol. 282, Issue 13, 9517-9525, March 30, 2007

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Rescue of Calcium-sensing Receptor Mutants by Allosteric Modulators Reveals a Conformational Checkpoint in Receptor Biogenesis^*

Ying Huang and Gerda E. Breitwieser¹

From the Department of Biology, Syracuse University, Syracuse, New York 13244 and the Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822

The calcium-sensing receptor (CaR), a member of G protein-coupled receptor family C, regulates systemic calcium homeostasis by activating G_q- and G_i-linked signaling in the parathyroid, kidney, and intestine. CaR is ubiquitinated by the E3 ligase dorfin and degraded via the endoplasmic reticulum-associated degradation pathway (Huang, Y., Niwa, J., Sobue, G., and Breitwieser, G. E. (2006) J. Biol. Chem. 281, 11610–11617). Here we provide evidence for a conformational or functional checkpoint in CaR biogenesis using two complementary approaches. First we characterized the sensitivity of loss- or gain-of-function CaR mutants to proteasome inhibition by MG132. The stabilization of loss-of-function mutants and insensitivity of gain-of-function mutants to MG132 suggests that receptor sensitivity to calcium influences susceptibility to proteasomal degradation. Second, we used the allosteric activator NPS R-568 and antagonist NPS 2143 to promote the active and inactive conformations of wild type CaR, respectively. Overnight culture in NPS R-568 increased expression of CaR, whereas NPS 2143 had the opposite effect. NPS R-568 and NPS 2143 differentially regulated maturation and cell surface expression of wild type CaR, directly affecting maximal signaling responses. NPS R-568 rescued expression of loss-of-function CaR mutants, increasing plasma membrane expression and ERK1/2 phosphorylation in response to 5 mM Ca²⁺. Disorders of calcium homeostasis caused by CaR mutations may therefore result from altered receptor biogenesis independent of receptor function, i.e. a protein folding disorder. The allosteric modulators NPS R-568 and NPS 2143 not only alter CaR sensitivity to calcium and hence signaling but also modulate receptor expression.

Received for publication, September 25, 2006 , and in revised form, February 5, 2007.

^* This work was supported by National Institutes of Health GM077563, the Weis Center for Research, and Syracuse University. 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 To whom correspondence should be addressed: Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822-2604. Tel.: 570-271-6675; Fax: 570-271-5886; E-mail: gebreitwieser{at}geisinger.edu.

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