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.
Highlights
HEK293 cells transiently transfected with equivalent amounts of cDNA for FLAGtagged with FLAG-CaR (WT) CaR, the loss-of-function mutant R795W (22), or the gain-of-function mutant A843E (22), were treated without or with 10 M MG132 for 12 h
MG132 increased the amount of the loss-of-function mutant R795W (from 80.2 Ϯ 7.0% to 134.9 Ϯ 6.0% (MG132)), but had no effect on the gain-of-function mutant A843E (117.5.2 Ϯ 2.9% versus 119.1 Ϯ 2.7% (MG132)) (Fig. 1, A, D, and E)
To determine whether the differential sensitivity to degradation is generalizable to additional CaR mutants, the effect of MG132 was quantified for loss-of-function CaR mutants identified in patients with FHH or NSHPT, having decreased sensitivity to extracellular Ca2ϩ (R66C, R185Q, R680C, and V817I) and gain-of-function CaR mutants from patients with ADH having increased sensitivity to extracellular Ca2ϩ (F128L, E191K, Q681H, and F788C) (Table 1) (22)
Summary
231, antagonize the stimulatory effects of Ca2ϩ and are in clinical trials as a treatment for osteoporosis (11). Treatment of loss-of-function mutants with NPS R-568 increases processing of the 130-kDa form to the mature 150-kDa form, resulting in increased plasma membrane localization and robust signaling in response to 5 mM Ca2ϩ, suggesting that some CaR mutations cause a protein folding/processing defect (17, 18) that can be ameliorated by pharmacochaperones (18). These results suggest that long-term treatment with calcimimetics and calcilytics can regulate turnover of CaR, providing unique possibilities for interventions in Ca2ϩ handling diseases
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