Parallel activation of phosphatidylinositol 4-kinase and phospholipase C by the extracellular calcium-sensing receptor

The calcium-sensing receptor (Call) is a G protein-coupled receptor that regulates physiological processes including Ca2+ metabolism, Na+, Cl-, K+, and H2O balance, and the growth of some epithelial cells through diverse signaling pathways. Although many effects of CaR are mediated by the heterotrimeric G proteins Galpha(q) and Galpha(i), not all signaling pathways regulated by CaR have been identified. We used human embryonic kidney (HEK)-293 cells that stably express human CaR to study the regulation of inositol lipid metabolism by CaR. The nonfunctional mutant CaRR796W was used as a negative control. We found that CaR regulates phosphatidylinositol (PI) 4-kinase, the first step in inositol lipid biosynthesis. In cells pretreated with U73122 to inhibit phospholipase C activation and to block the degradation of PI 4,5-bisphosphate to form [H-3]inositol trisphosphate (IP3), CaR stimulated the accumulation of [3H]PI monophosphate (PIP). Additionally, wortmannin, an inhibitor of both PI 3-kinase and type III PI 4-kinase, blocked Call-stimulated accumulation of [H-3]PIP and inhibited [H-3]IP3 production. CaR-stimulated inositol lipid synthesis was attributable to PI 4-kinase and not PI 3-kinase because CaR did not activate Akt, a downstream target of PI 3-kinase. CaR associates with PI 4-kinase based on the findings that CaR and the 110-kDa PI 4-kinase 13 can be co-immunoprecipitated with antibodies against either CaR or PI 4-kinase. The PI-4 kinase in co-immunoprecipitates with anti-Call antibody was activated in Ca2+-stimulated HEK-293 cells, which stably express the wild type CaR. Pertussis toxin did not affect the formation of [H-3]IP3 or the rise in intracellular Ca2+ (Handlogten, M. E., Huang, C. F., Shiraishi, N., Awata, H., and Miller, R. T. (2001) J. Biol. Chem. 276, 1394113948). RGS4, an accelerator of GTPase activity of members of the Gai and Gaq families, attenuated the CaR-stimulated PLC activation and IP3 accumulation, which is mediated by Gaq, but did not inhibit CaR-stimulated [H-3]PIP formation. In HEK-293 cells, which express wild type Call, Rho was enriched in immune complexes co-immunoprecipitated with the anti-Call antibody. C-3 toxin, an inhibitor of Rho, also inhibited the CaR-stimulated [H-3]IP3 production but did not lead to CaR-stimulated [H-3]PIP formation, reflecting inhibition of PI 4-kinase. Taken together, our data demonstrate that CaR stimulates PI 4-kinase, the first step in inositol lipid biosynthesis conversion of PI to PI 4-P by Rho-dependent and Galpha(q)- and Galpha(i)-independent pathways.