<i>Gβγ</i> activates <i>PIP2</i> hydrolysis by recruiting and orienting <i>PLCβ</i> on the membrane surface.

Abstract

<i>Phospholipase C-βs (PLCβs)</i> catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate [Formula: see text] into [Formula: see text] [Formula: see text] and [Formula: see text]  [Formula: see text]. [Formula: see text] regulates the activity of many membrane proteins, while <i>IP3</i> and <i>DAG</i> lead to increased intracellular Ca²⁺ levels and activate protein kinase C, respectively. <i>PLCβs</i> are regulated by G protein-coupled receptors through direct interaction with [Formula: see text] and [Formula: see text] 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 [Formula: see text] activates <i>PLCβ</i>3. We show that <i>PLCβ</i>3 functions as a slow Michaelis-Menten enzyme ( [Formula: see text] ) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of <i>PLCβ</i>3. Its partition coefficient is such that only a small quantity of <i>PLCβ</i>3 exists in the membrane in the absence of [Formula: see text] . When [Formula: see text] is present, equilibrium binding on the membrane surface increases <i>PLCβ</i>3 in the membrane, increasing [Formula: see text] in proportion. Atomic structures on membrane vesicle surfaces show that two [Formula: see text] anchor <i>PLCβ</i>3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that [Formula: see text] activates <i><i>PLCβ</i></i> by increasing its concentration on the membrane surface and orienting its catalytic core to engage [Formula: see text] . This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by [Formula: see text]<i>, IP3</i>, and <i>DAG</i>.