title: Rescue of calcium-sensing receptor mutants by allosteric modulators reveals a conformational checkpoint in receptor biogenesis.
abstract: 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 Ca(2+). 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.
authors.affiliation: Department of Biology, Syracuse University, Syracuse, New York 13244; Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822. Electronic address: gebreitwieser@geisinger.edu.
authors.forename: Gerda E
authors.initials: GE
authors.lastname: Breitwieser
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism / Q000502:physiology; D011487:Protein Conformation; D017510:Protein Folding; D044169:Receptors, Calcium-Sensing / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D044169:Receptors, Calcium-Sensing; D046988:Proteasome Endopeptidase Complex