Gβγ activates PIP2 hydrolysis by recruiting and orienting PLCβ on the membrane surface

Abstract

Phospholipase C-βs (PLCβs) catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositoltriphosphate (IP3) and diacylglycerol (DAG). PIP2 regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca2+ levels and activate protein kinase C, respectively. PLCβs are regulated by G protein-coupled receptors through direct interaction with Gαq and Gβγ and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate. This study addresses the mechanism by which Gβγ activates PLCβ3. We show that PLCβ3 functions as a slow Michaelis-Menten enzyme ( kcat ∼2 s-1, KM ∼ 0.43 mol % ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of PLCβ3. Its partition coefficient is such that only a small quantity of PLCβ3 exists in the membrane in the absence of Gβγ. When Gβγ is present, equilibrium binding on the membrane surface increases PLCβ3 in the membrane, increasing Vmax in proportion. Atomic structures on membrane vesicle surfaces show that two Gβγ anchor PLCβ3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that Gβγ activates PLCβ by increasing its concentration on the membrane surface and orienting its catalytic core to engage PIP2. This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by PIP2, IP3, and DAG.