G-protein-coupled Receptor Activation Induces the Membrane Translocation and Activation of Phosphatidylinositol-4-phosphate 5-Kinase Iα by a Rac- and Rho-dependent Pathway

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K Iα activation following the addition of thrombin. Under resting condition expressed PIP5K Iα was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1 , or overexpression of a constitutively active variant of Gαq, PIP5K Iα translocated to the plasma membrane. Movement of PIP5K Iα to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active Gαq, 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K Iα translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K Iα. In addition, a dominant negative variant of Rho blocked the PIP5K Iα membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K Iα membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K Iα by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K Iα, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K Iα correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K Iα with either constitutively active Gαq, Rac, or Rho led to a 5- to 7-fold increase in PIP5K Iα activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of Gαq, Rac, Rho, and PIP5K Iα. Once activated and translocated to the cell membrane, PIP5K Iα becomes available to phosphorylate PI4P to generate PI4,5P2 on the plasma membrane.