Gαq and the Phospholipase Cß3 X-Y Linker Regulate Adsorption and Activity on Compressed Lipid Monolayers.

Phospholipase Cß (PLCß) enzymes are peripheral membrane proteins required for normal cardiovascular function. PLCß hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers that increase intracellular Ca2+ level and activate protein kinase C. Under basal conditions, PLCß is autoinhibited by its C-terminal domains and by the X-Y linker, which contains a stretch of conserved acidic residues required for interfacial activation. Following stimulation of G protein-coupled receptors, the heterotrimeric G protein subunit Gαq allosterically activates PLCß and helps orient the activated complex at the membrane for efficient lipid hydrolysis. However, the molecular basis for how the PLCß X-Y linker, its C-terminal domains, Gαq, and the membrane coordinately regulate activity is not well understood. Using compressed lipid monolayers and atomic force microscopy, we found that a highly conserved acidic region of the X-Y linker is sufficient to regulate adsorption. Regulation of adsorption and activity by the X-Y linker also occurs independently of the C-terminal domains. We next investigated whether Gαq-dependent activation of PLCß altered interactions with the model membrane. Gαq increased PLCß adsorption in a manner that was independent of the PLCß regulatory elements and targeted adsorption to specific regions of the monolayer in the absence of the C-terminal domains. Thus, the mechanism of Gαq-dependent activation likely includes a spatial component.

Intramolecular electrostatic interactions contribute to phospholipase Cß3 autoinhibition.

Phospholipase Cß (PLCß) enzymes regulate second messenger production following the activation of G protein-coupled receptors (GPCRs). Under basal conditions, these enzymes are maintained in an autoinhibited state by multiple elements, including an insertion within the catalytic domain known as the X-Y linker. Although the PLCß X-Y linker is variable in sequence and length, its C-terminus is conserved and features an acidic stretch, followed by a short helix. This helix interacts with residues near the active site, acting as a lid to sterically prevent substrate binding. However, deletions that remove the acidic stretch of the X-Y linker increase basal activity to the same extent as deletion of the entire X-Y linker. Thus, the acidic stretch may be the linchpin in autoinhibition mediated by the X-Y linker. We used site-directed mutagenesis and biochemical assays to investigate the importance of this acidic charge in mediating PLCß3 autoinhibition. Loss of the acidic charge in the X-Y linker increases basal activity and decreases stability, consistent with loss of autoinhibition. However, introduction of compensatory electrostatic mutations on the surface of the PLCß3 catalytic domain restore activity to basal levels. Thus, intramolecular electrostatics modulate autoinhibition by the X-Y linker.

Phospholipase Cß3 Membrane Adsorption and Activation Are Regulated by Its C-Terminal Domains and Phosphatidylinositol 4,5-Bisphosphate.

Phospholipase Cß (PLCß) enzymes hydrolyze phosphatidylinositol 4,5-bisphosphate to produce second messengers that regulate intracellular Ca2+, cell proliferation, and survival. Their activity is dependent upon interfacial activation that occurs upon localization to cell membranes. However, the molecular basis for how these enzymes productively interact with the membrane is poorly understood. Herein, atomic force microscopy demonstrates that the ∼300-residue C-terminal domain promotes adsorption to monolayers and is required for spatial organization of the protein on the monolayer surface. PLCß variants lacking this C-terminal domain display differences in their distribution on the surface. In addition, a previously identified autoinhibitory helix that binds to the PLCß catalytic core negatively impacts membrane binding, providing an additional level of regulation for membrane adsorption. Lastly, defects in phosphatidylinositol 4,5-bisphosphate hydrolysis also alter monolayer adsorption, reflecting a role for the active site in this process. Together, these findings support a model in which multiple elements of PLCß modulate adsorption, distribution, and catalysis at the cell membrane.