RESUMO
Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein ßγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gßγ has not been demonstrated. To identify PLCϵ domains that interact with Gßγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gßγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gßγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gßγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gßγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gßγ binding and eliminated Gßγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gßγ directly interacts with PLCϵ and yield insights into the mechanism by which ßγ subunits activate PLCϵ.
