The mechanism of regulation of -catalyzed hydrolysis.

UNLABELLED

enzymes cleave producing IP3 and DAG. modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca levels and protein phosphorylation by protein kinase C, respectively. enzymes are under the control of GPCR signaling through direct interactions with G proteins and and have been shown to be coincidence detectors for dual stimulation of and G coupled receptors. 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 activates by recruiting it to the membrane. Using these same methods, here we show that increases the catalytic rate constant, , of . Since stimulation of by depends on an autoinhibitory element (the X-Y linker), we propose that produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the and complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate activity. The structures rationalize a finding in the enzyme assay, that co-stimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.

SIGNIFICANCE STATEMENT

For certain cellular signaling processes, the background activity of signaling enzymes must be minimal and stimulus-dependent activation robust. Nowhere is this truer than in signaling by , whose activity regulates intracellular Ca , phosphorylation by Protein Kinase C, and the activity of numerous ion channels and membrane receptors. In this study we show how enzymes are regulated by two kinds of G proteins, and . Enzyme activity studies and structures on membranes show how these G proteins act by separate, independent mechanisms, leading to a product rule of co-stimulation when they act together. The findings explain how cells achieve robust stimulation of in the setting of very low background activity, properties essential to cell health and survival.