The mechanism of <i>Gα_q</i> regulation of <i>PLCβ3</i>-catalyzed <i>PIP2</i> hydrolysis.

Abstract

<i>PLCβ</i> (<i>Phospholipase Cβ</i>) enzymes cleave phosphatidylinositol 4,5-bisphosphate (<i>PIP2)</i> producing <i>IP3</i> and <i>DAG</i> (diacylglycerol). <i>PIP2</i> modulates the function of many ion channels, while <i>IP3</i> and <i>DAG</i> regulate intracellular Ca²⁺ levels and protein phosphorylation by protein kinase C, respectively. <i>PLCβ</i> enzymes are under the control of G protein coupled receptor signaling through direct interactions with G proteins <i>Gβγ</i> and <i>Gα_q</i> and have been shown to be coincidence detectors for dual stimulation of <i>Gα_q</i> and <i>Gα</i>_<i>i</i>-coupled receptors. <i>PLCβs</i> are aqueous-soluble cytoplasmic enzymes but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that <i>Gβγ</i> activates <i>PLCβ3</i> by recruiting it to the membrane. Using these same methods, here we show that <i>Gα_q</i> increases the catalytic rate constant, <i>k_cat</i>, of <i>PLCβ3</i>. Since stimulation of <i>PLCβ3</i> by <i>Gα_q</i> depends on an autoinhibitory element (the X-Y linker), we propose that <i>Gα_q</i> produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the <i>PLCβ3·Gα_q</i> and <i>PLCβ3·Gβγ</i><i>(2)</i><i>·Gα_q</i> complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate <i>PLCβ3</i> activity. The structures rationalize a finding in the enzyme assay, that costimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of <i>PLCβ3</i> is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.