Specificity and structural requirements of phospholipase C-β stimulation by Rho GTPases versus G protein βγ dimers

Abstract

Phospholipase C-β2 is activated both by heterotrimeric G protein α- and βγ- subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLCβ isozymes with the rank order of PLCβ2 > PLCβ3 ≥ PLCβ1. The sensitivity of PLCβ isozymes to Rho GTPases was clearly different from that observed for G protein βγ dimers, which decreased in the following order: PLCβ3 > PLCβ2 > PLCβ1 for β1γ1/2 and PLCβ2 > PLCβ1 ⋙ PLCβ3 for β5γ2. Rac1 and Rac2 were found to be more potent and efficacious activators of PLCβ2 than was Cdc42Hs. The stimulation of PLCβ2 by Rho GTPases and G protein βγ dimers was additive, suggesting that PLCβ2 activation can be augmented by independent regulation of the enzyme by the two stimuli. Using chimeric PLCβ1-PLCβ2 enzymes, βγ dimers, and Rho GTPases are shown to require different regions of PLCβ2 to mediate efficient stimulation of the enzyme. Although the catalytic subdomains X and Y of PLCβ2 were sufficient for efficient stimulation by βγ, the presence of the putative pleckstrin homology domain of PLCβ2 was absolutely required for the stimulation of the enzyme by Rho GTPases. Taken together, these results identify Rho GTPases as novel PLCβ regulators, which mediate PLCβ isozyme-specific stimulation and are potentially involved in coordinating the activation of PLCβ2 by extracellular mediators in intact cells.