Protein kinase C (PKC) phosphorylation of the Ca2+o-sensing receptor (CaR) modulates functional interaction of G proteins with the CaR cytoplasmic tail

Abstract

The extracellular calcium (Ca2+o)-sensing receptor (CaR) activates Ca2+ influx independent of the release of intracellular Ca2+ stores. The latter can be negatively regulated by protein kinase C (PKC) through phosphorylation of Thr-888 of the CaR. In this study, we substituted Thr-888 with various amino acid residues or a stop codon to understand how PKC phosphorylation of the CaR inhibits receptor-mediated release of intracellular Ca2+ stores. Substitutions of Thr-888 with hydrophobic and hydrophilic amino acid residues had various effects on CaR-mediated release of intracellular Ca2+ stores as well as activation of Ca2+ influx. Several point mutations, such as T888D, had marked negative effects on CaR-mediated release of intracellular Ca2+ stores but not on phorbol myristate acetate-insensitive activation of Ca2+ influx. Presumably, the negatively charged aspartate mimics phospho-threonine. Interestingly, truncating the receptor at 888 had an even more pronounced negative effect on CaR-elicited release of intracellular Ca2+ stores without significantly affecting CaR-mediated activation of Ca2+ influx. Therefore, truncation at position 888 of the CaR affects the activity of the receptor in a manner that resembles PKC phosphorylation of the CaR. This in turn suggests that PKC phosphorylation of the CaR prevents G protein subtypes from interacting with the region of the receptor critical for releasing Ca2+ stores, which is missing in the truncated receptor.