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 ( P I P 2 ) into inositoltriphosphate ( IP3 ) and diacylglycerol   ( DAG ) . P I P 2 regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca 2+ levels and activate protein kinase C, respectively. PLCβs are regulated by G protein–coupled receptors through direct interaction with G α q and G β γ 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 G β γ activates PLCβ 3. We show that PLCβ 3 functions as a slow Michaelis–Menten enzyme (  k cat ∼ 2   s - 1 ,   K M ∼ 0.43   m o l %  ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of PLCβ 3. Its partition coefficient is such that only a small quantity of  PLCβ 3 exists in the membrane in the absence of G β γ  . When G β γ is present, equilibrium binding on the membrane surface increases PLCβ 3 in the membrane, increasing V max in proportion. Atomic structures on membrane vesicle surfaces show that two G β γ anchor  PLCβ 3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that G β γ activates PLCβ by increasing its concentration on the membrane surface and orienting its catalytic core to engage P I P 2  . This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by P I P 2 , IP3 , and DAG .