Kavli Affiliate: Roderick MacKinnon
| Authors: Maria Falzone, Priyam Banerjee and Roderick MacKinnon
| Summary:
PLCβ 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 Gβγ and Gαq and have been shown to function as coincidence detectors for dual stimulation of Gαq and Gαi-coupled receptors via these G proteins. PLCβs are aqueous-soluble enzymes, but partition PLCβ 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 Gβγ and Gαq and have been shown to function as coincidence detectors for dual stimulation of Gαq and Gαi-coupled receptors via these G proteins. PLCβs 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 Gβγ-dependent regulation of PLCβ enzymes. Using macrophages as a model system, where PLCβ signaling is essential for responses to infection and tissue injury, we investigated the contribution of Gβγ-dependent regulation and membrane recruitment of PLCβ in the context of endogenous signaling. By measuring Ca2+ mobilization, we demonstrate that both Gαi and Gαq-coupled receptors independently stimulate PLCβ activity, illustrating that Gβγ alone is sufficient to activate PLCβ in certain contexts. Using total internal reflection and stimulated emission depletion microscopy, we demonstrate that most of the PLCβ3 in the cell is localized away from the plasma membrane at rest but is rapidly recruited to the plasma membrane upon stimulation by both Gαi and Gαq-coupled receptors, illustrating that both Gβγ and Gαq recruit PLCβ to the plasma membrane. These results support an updated model for G protein-dependent regulation of PLCβ enzymes, where Gβγ-induced regulation in the absence of Gαq is context dependent and dictated by the local concentration of receptor, G proteins, and PLCβ. Significance Statement PLCβ 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. PLCβs 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 PLCβ regulation by one effector, Gβγ. Using macrophages as a model system for physiological signaling, we demonstrate that Gβγ is capable of independently activating PLCβ via membrane recruitment under the conditions of endogenous signaling.