PLCβs are recruited to the plasma membrane in macrophages by both Gβγ and Gαq

PLC{beta} enzymes cleave PIP2 from the plasma membrane, producing IP3 and DAG, which regulate intracellular Ca2+ levels and protein kinase C activity, respectively. They are regulated by GPCR signaling through the G proteins G{beta}{gamma} and Gq and have been shown to function as coincidence detectors for dual stimulation of Gq and Gi-coupled receptors via these G proteins. PLC{beta}s are aqueous-soluble enzymes, but partition onto the membrane surface to access their lipid substrate. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie G{beta}{gamma}-dependent regulation of PLC{beta} enzymes. Using macrophages as a model system, where PLC{beta} signaling is essential for responses to infection and tissue injury, we investigated the contribution of G{beta}{gamma}-dependent regulation and membrane recruitment of PLC{beta} in the context of endogenous signaling. By measuring Ca2+ mobilization, we demonstrate that both Gi and Gq-coupled receptors independently stimulate PLC{beta} activity, illustrating that G{beta}{gamma} alone is sufficient to activate PLC{beta} in certain contexts. Using total internal reflection and stimulated emission depletion microscopy, we demonstrate that most of the PLC{beta}3 in the cell is localized away from the plasma membrane at rest but is rapidly recruited to the plasma membrane upon stimulation by both Gi and Gq-coupled receptors, illustrating that both G{beta}{gamma} and Gq recruit PLC{beta} to the plasma membrane. These results support an updated model for G protein-dependent regulation of PLC{beta} enzymes, where G{beta}{gamma}-induced regulation in the absence of Gq is context dependent and dictated by the local concentration of receptor, G proteins, and PLC{beta}. Significance StatementPLC{beta} enzymes are critical mediators of signal transduction with roles in neuronal, cardiac, and immunological signaling. Despite this importance, many aspects of their function and regulation remain poorly understood. PLC{beta}s are aqueous soluble but must partition onto the membrane surface to access their lipid substrate, which enables regulation at the partitioning step, the catalytic step, or both. We previously demonstrated that membrane recruitment and orientation of the catalytic core on the membrane surface underlie the PLC{beta} regulation by one effector, G{beta}{gamma}. Using macrophages as a model system for physiological signaling, we demonstrate that G{beta}{gamma} is capable of independently activating PLC{beta} via membrane recruitment under the conditions of endogenous signaling.