Rescue of Calcium-sensing Receptor Mutants by Allosteric Modulators Reveals a Conformational Checkpoint in Receptor Biogenesis
The calcium-sensing receptor (CaR), a member of G protein-coupled receptor family C, regulates systemic calcium homeostasis by activating Gq- and Gi-linked signaling in the parathyroid, kidney, and intestine. CaR is ubiquitinated by the E3 ligase dorfin and degraded via the endoplasmic reticulum-ass...
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Published in | The Journal of biological chemistry Vol. 282; no. 13; pp. 9517 - 9525 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
30.03.2007
American Society for Biochemistry and Molecular Biology |
Subjects | |
Online Access | Get full text |
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Summary: | The calcium-sensing receptor (CaR), a member of G protein-coupled receptor family C, regulates systemic calcium homeostasis by activating Gq- and Gi-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. |
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Bibliography: | http://www.jbc.org/ ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M609045200 |