Papers of the Week

Phospholipase C β (PLCβ) plays an important role in cardiovascular diseases and opioid analgesia. PLCβ catalyzes the hydrolysis of the inner membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP ) to inositol-1,4,5-triphosphate (IP ) and diacylglycerol (DAG). IP and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control responses to extracellular signals. PLCβ is a downstream effector of G-protein coupled receptors (GPCRs) and is activated by both the Gα and Gβγ subunits. Activation by Gα requires a unique 400 amino acid C-terminal region of the lipase, which is subdivided into the proximal and distal C-terminal domains (CTDs). These domains are required for allosteric activation by Gα and for membrane binding. Our lab and others have reported that PLCβ is more flexible in solution, as compared to crystal structures of the protein. In small angle X-ray scattering (SAXS) experiments, the solution structure of PLCβ had additional density that cannot be accounted by the crystal structure. Additionally, crosslinking the PH and EF hand domains decreased this density and the maximum particle diameter. We propose that this additional density corresponds to an open conformation, wherein the PH and EF hand domains are extended from the core of the lipase, and that this conformation of PLCβ represents an autoinhibited state. To test this hypothesis, we mutated residues in PLCβ3 at the interface between the PH and EF hand domains to disrupt their interaction, and then measured changes in PLCβ basal activity and Gβγ-stimulated activity. These mutations had no significant impact on basal activity, however in many of these mutants, there was a decrease in apparent Gβγ activation. Since these mutations should stabilize an open conformation of PLCβ, this suggests the open conformation has less ability to bind to or be activated by Gβγ. We are currently working to solve the structure of PLCβ3 in solution using single particle cryo-electron microscopy (cryo-EM), which will allow us to determine a solution structure of PLCβ to a higher resolution than SAXS studies. We hypothesize that PLCβ3 will exist in the open, autoinhibited conformation, and will also shed light on possible allosteric interactions between the distal CTD and the core.