Identification of Serine-875 as an Inhibitory Phosphorylation Site in the Calcium-Sensing Receptor

Abstract

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.

International Union of Basic and Clinical Pharmacology. CVIII. Calcium-Sensing Receptor Nomenclature, Pharmacology, and Function

“…In mGlu 5 , the key PKC phosphorylation site, Ser839 (Kim et al, 2005), aligns not with Thr888 in the CaSR but with Ser875, a residue not originally predicted to be phosphorylated by PKC (Garrett et al, 1995a). Intriguingly, current data indicate removal of this putative phosphorylation site from the CaSR (Ser875Ala) also produces a gain of function, similar to that of Thr888Ala, whereas a phosphomimetic mutation at this site (Ser875Asp) produces a loss of function (Binmahfouz et al, 2019). The double Ser875Ala plus Thr888Ala mutant exhibits a greater gain of function than Ser875Ala alone, and concomitant PKC inhibition exerts no further signal enhancement.…”

“…The double Ser875Ala plus Thr888Ala mutant exhibits a greater gain of function than Ser875Ala alone, and concomitant PKC inhibition exerts no further signal enhancement. Thus, Ser875 is most likely the second major inhibitory PKC site in the CaSR (Binmahfouz et al, 2019).…”

Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction

“…As noted above, the choice of CaSR 875 ‐B1 in partnership with CaSR 908 ‐B2 was critical for the delivery of mutant heterodimers to the cell surface that were functionally active in Ca 2+ o ‐stimulated Ca 2+ i mobilization assays. It is pertinent to note that although the two key PKC‐dependent phosphorylation sites at T888 ( 50 ) and S875 ( 51 ) were both intact in one of these constructs, CaSR 908 ‐B2, it seems likely that neither of the phosphorylation sites was active in CaSR 875 ‐B1, in which T888 was deleted and in which S875, although present, may not have been recognized by PKC due to the loss of the immediate C‐terminal peptide, residues 876–879 TAAH. A loss of all four PKC sites in receptor homodimers promotes receptor activity via enhanced Ca 2+ o sensitivity ( 51 ) and T888M is a known activating mutation in humans.…”

Therapeutic Opportunities of Targeting Allosteric Binding Sites on the Calcium-Sensing Receptor

“…The structural integrity of the 7TM is in part maintained by a disulfide bond between C677 in ECL1 and C765 in ECL2 . The 7TM is proceeded by a large C-terminal tail that contributes to cell surface expression, signaling, and binding to accessory proteins. − Further, C-terminal tail residues (S875 and T888) are predicted to be key protein kinase C (PKC) phosphorylation sites, which serve to negatively regulate CaSR activity. , …”

“…35−37 Further, Cterminal tail residues (S875 and T888) are predicted to be key protein kinase C (PKC) phosphorylation sites, which serve to negatively regulate CaSR activity. 38,39 ■ ENDOGENOUS CASR LIGANDS AND THEIR…”