| Abstrakt: |
Phospholipase Cβ (PLCβ) enzymes are peripheral membrane proteins required for normal cardiovascular function. PLCβ hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers that increase intracellular Ca2+level and activate protein kinase C. Under basal conditions, PLCβ 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 Gαqallosterically activates PLCβ and helps orient the activated complex at the membrane for efficient lipid hydrolysis. However, the molecular basis for how the PLCβ 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 PLCβ altered interactions with the model membrane. Gαqincreased PLCβ adsorption in a manner that was independent of the PLCβ 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. |
