Kavli Affiliate: Roderick MacKinnon
| Authors: Maria Elizabeth Falzone and Roderick MacKinnon
| Summary:
PLCβ 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. PLCb enzymes are under the control of GPCR signaling through direct interactions with G proteins Gbg and Gaq and have been shown to be coincidence detectors for dual stimulation of Gaq and Gai coupled receptors. PLCbs 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 Gbg activates PLCb3 by recruiting it to the membrane. Using these same methods, here we show that Gaq increases the catalytic rate constant, kcat, of PLCb3. Since stimulation of PLCb3 by Gaq depends on an autoinhibitory element (the X-Y linker), we propose that Gaq produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the PLCb3-Gaq, and PLCb3-Gbg(2)-Gaq complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate PLCb3 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 PLCb3 is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.