Extracellular phosphate regulates its own renal excretion by eliciting concentration-dependent secretion of parathyroid hormone (PTH). However, the phosphate-sensing mechanism remains unknown and requires elucidation for understanding the aetiology of secondary hyperparathyroidism in chronic kidney disease (CKD). The calcium-sensing receptor (CaSR) is the main controller of PTH secretion and here we show that raising phosphate concentration within the pathophysiologic range for CKD significantly inhibits CaSR activity via non-competitive antagonism. Mutation of residue R62 in anion binding site-1 abolishes phosphate-induced inhibition of CaSR. Further, pathophysiologic phosphate concentrations elicit rapid and reversible increases in PTH secretion from freshly-isolated human parathyroid cells consistent with a receptor-mediated action. The same effect is seen in wild-type murine parathyroid glands, but not in CaSR knockout glands. By sensing moderate changes in extracellular phosphate concentration, the CaSR represents a phosphate sensor in the parathyroid gland, explaining the stimulatory effect of phosphate on PTH secretion.
The calcium-sensing receptor (CaS) is the principal controller of extracellular calcium (Ca 21 o) homeostasis and is inhibited in vitro and in vivo by protein kinase C (PKC)-mediated phosphorylation at CaS T888. However, PKC inhibition enhances signaling even in CaSs lacking Thr-888, suggesting that an additional inhibitory site exists. An apparently equivalent PKC regulatory site in metabotropic glutamate receptor 5 (Ser-839) aligns not with CaS T888 but instead with CaS S875 , which was not previously considered to be a PKC site. CaS S875A (nonphosphorylatable) exhibited significantly enhanced Ca 21 o sensitivity of both intracellular Ca 21 mobilization and extracellular signal-regulated kinase 1/2 activation, whereas the phosphomimetic CaS S875D mutant exhibited a loss of function. The CaS S875A/T888A double mutant exhibited even greater Ca 21 o sensitivity than CaS T888A alone, a response no longer enhanced by PKC inhibition. Finally, when expressed in CaS lacking its extracellular domain, the CaS S875A/T888A double mutation elicited maximal activation even under control conditions, but remained sensitive to negative allosteric modulation [N-(2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl)-1,1-dimethyl-2-(2-nephthyl)ethylamine] or Ca 21 o removal. Therefore, we have now identified CaS S875 as the missing PKC phosphorylation site that, together with CaS T888 , shapes the CaS signaling that underpins Ca 21 o homeostasis. Together with the inactive form of the CaS extracellular domain, these sites attenuate Ca 21 o sensitivity to attain appropriate physiologic Ca 21 o sensing. SIGNIFICANCE STATEMENT Serine-875 represents the missing inhibitory PKC phosphorlyation site in CaS that in tandem with Thr-888 controls receptor activity. s This article has supplemental material available at molpharm.aspetjournals.org.
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