Protein Kinase C-mediated Phosphorylation of the Calcium-sensing Receptor Is Stimulated by Receptor Activation and Attenuated by Calyculin-sensitive Phosphatase Activity

Abstract

The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr 888 contributing significantly to this effect. To determine whether CaR T888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaR pT888 mobilization rather than oscillations (9). Whereas this suggests that PKC-mediated CaR phosphorylation regulates CaR function, no reagents have existed previously for investigating CaR phosphorylation directly. Thus, we have developed a phospho-specific antibody that recognizes the phosphorylated form of CaR T888 , this residue being chosen because its mutation elicited the greatest effect on CaR function of the putative PKC sites. EXPERIMENTAL PROCEDURESMaterials-The calcimimetics NPS R-467 and S-467 and the calcilytic NPS-89636 were a kind gift of Dr. E. F. Nemeth and NPS Pharmaceuticals, Inc. (Toronto, Canada). Fura-2/AM was from Invitrogen and Gö6976, PKC␤-selective inhibitor (anilinomonoindolylmaleimide), and endothall thioanhydride (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid) were from Calbiochem (EMD Biosciences, Inc., San Diego, CA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from DakoCytomation (Ely, Cambridgeshire, UK). Unless stated otherwise, all other chemicals were purchased from Sigma.Cell Culture-HEK293 cells, stably transfected with human parathyroid CaR (6), were a gift from Dr. E. F. Nemeth (NPS Pharmaceuticals). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal * This work was supported in part by Biotechnology and Biological Sciences Research Council Grant BBSB04986. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Increased Receptor Stimulation Elicits Differential Calcium-sensing ReceptorT888 Dephosphorylation

“…Phosphomimetic substitution of this site (CaR T888D ) lowers the Ca 2ϩ o sensitivity of the receptor (13) while nonphosphorylatable CaR T888A is more sensitive to Ca 2ϩ o and promotes sustained rather than oscillatory Ca 2ϩ i mobilization (14,15). This is the basis for the hypothesis that dynamic CaR T888 phosphorylation is required for oscillatory Ca 2ϩ i mobilization, an effect that had been proposed previously for other class C G proteincoupled receptors (16 -18).…”

Calmodulin Regulates Ca2+-sensing Receptor-mediated Ca2+ Signaling and Its Cell Surface Expression

“…Given that the mutants 3E2 and 3E3, which similarly failed to interact with CaM, still preserve reduced Ca 2ϩ responses, it is likely that deletion of this region has adverse effects on the structural integrity and functionality of other regions of the cytoplasmic tail of CaSR, which also contribute to the Ca 2ϩ response. It has been reported that increases of extracellular Ca 2ϩ to 2-3 mM can induce intracellular Ca 2ϩ oscillations in HEK293 cells expressing CaSR (31)(32)(33). We then examined the [Ca 2ϩ ] i oscillatory frequencies in HEK293 cells transfected with CaSR and its mutants.…”

A novel mutation of the primary protein kinase C phosphorylation site in the calcium-sensing receptor causes autosomal dominant hypocalcemia

“…2; wt, 3.0G0.1 mM; T888M, 2.5G0.2 mM; P!0.05 by unpaired t-test of log EC 50 values). Western blotting revealed similar expression of protein bands consistent with the wellrecognized 140 kDa core-glycosylated and 160 kDa mature receptors (7,(12)(13)(14)(15)20) in both sets of transfected cells (Fig. 2C).…”

“…This mutation was predicted by PolyPhen (prediction of functional effect of human nsSNPs; http://genetics. bwh.harvard.edu/pph/) to have a position-specific independent counts (PSIC) score of 1.711, and modeling of the effect on the amino acid sequence of the CASR protein predicted a substitution of methionine for threonine at position 888 (p.T888M) that would abolish a major intracellular PKC phosphorylation site (12).…”

“…CASR-induced changes in intracellular calcium ðCa 2C i Þ concentration were measured by epifluorescence microscopy as described previously (11,12) using cells loaded with Fura-2 (Invitrogen). CASR-transfected cells were then incubated at room temperature in Experimental Buffer (20 mM HEPES (pH 7.4), 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , and 5.5 mM glucose) and exposed to increasing …”