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bioRxiv · 10.1101/2023.08.29.555394

The mechanism of Gαq regulation of PLCβ3-catalyzed PIP2 hydrolysis.

Falzone, M. E.·MacKinnon, R.
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Abstract

PLC{beta} enzymes cleave PIP2 producing IP3 and DAG. PIP2 modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca2+ levels and protein phosphorylation by protein kinase C, respectively. PLC{beta} enzymes are under the control of GPCR signaling through direct interactions with G proteins G{beta}{gamma} and Gq and have been shown to be coincidence detectors for dual stimulation of Gq and Gi coupled receptors. PLC{beta}s 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 G{beta}{gamma} activates PLC{beta}3 by recruiting it to the membrane. Using these same methods, here we show that Gq increases the catalytic rate constant, kcat, of PLC{beta}3. Since stimulation of PLC{beta}3 by Gq depends on an autoinhibitory element (the X-Y linker), we propose that Gq produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the PLC{beta}3-Gq, and PLC{beta}3-G{beta}{gamma}(2)-Gq complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate PLC{beta}3 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 PLC{beta}3 is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation. Significance StatementFor 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 PLC{beta}3, whose activity regulates intracellular Ca2+, phosphorylation by Protein Kinase C, and the activity of numerous ion channels and membrane receptors. In this study we show how PLC{beta}3 enzymes are regulated by two kinds of G proteins, G{beta}{gamma} and Gq. 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 PLC{beta}3 in the setting of very low background activity, properties essential to cell health and survival.

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Falzone, M. E., MacKinnon, R.. 2023-08-30. The mechanism of Gαq regulation of PLCβ3-catalyzed PIP2 hydrolysis.. https://doi.org/10.1101/2023.08.29.555394

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