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12/13 to activate phospholipase D in Madin-Darby canine kidney cells
1 Medicine, Case Western Reserve University, Cleveland, Ohio, USA
* To whom correspondence should be addressed. E-mail: cxh87{at}po.cwru.edu.
The Ca-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G
i, Gq , and G
to stimulate phosphatidylinositol 3-kinase. In order to determine if the receptor also couples to G12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G12/13 target. We established MDCK cell lines that stably express the wild type CaR (CaRWT) or the non-functional mutant CaRR796W as a negative control, prelabeled these cells with [3H]palmitic acid and measured CaR-stimulated PLD activity as the formation of [3H]phosphatidylethanol (PEt). The formation of [3H]PEt increased in a time-dependent manner in the cells that express the CaRWT but not the CaRR796W. Treatment of the cells with C3 exoenzyme inhibited PLD activity, which suggested that the CaR activates the Rho family of small G proteins, targets of G12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate Gi, or co-expressed Regulators of G protein Signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gi and Gq, and p115RhoGEF construct containing the RGS domain for G12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWT inhibited extracellular Ca2+ (Cao2+)-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C (PKC), ADP-ribosylation factor (Arf) and phosphatidylinositol bisphosphate (PIP2) was excluded. These findings demonstrate that the CaR couples to G12/13 to regulate PLD via Rho-dependent, and does so independently of Gi and Gq. This suggests that the CaR potentially regulates cytoskeleton via G12/13, Rho and PLD.
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