Biochemistry. 2019 Aug 13;58(32):3454-3467. doi: 10.1021/acs.biochem.9b00441. Epub 2019 Jul 30.
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 Ca 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 Gα allosterically 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, Gα, 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 Gα-dependent activation of PLCβ altered interactions with the model membrane. Gα increased 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 Gα-dependent activation likely includes a spatial component.
磷酯酶 Cβ（PLCβ）酶是正常心血管功能所必需的外周膜蛋白。PLCβ水解磷脂酰肌醇 4,5-二磷酸，产生第二信使，增加细胞内 Ca 水平并激活蛋白激酶 C。在基础条件下，PLCβ 的 C 末端结构域和 X-Y 接头通过自抑制，该接头包含一段保守的酸性残基，是界面激活所必需的。在 G 蛋白偶联受体刺激后，异三聚体 G 蛋白亚基 Gα 变构激活 PLCβ，并帮助将激活的复合物定向到膜上以进行有效的脂质水解。然而，PLCβ X-Y 接头、其 C 末端结构域、Gα 和膜如何协调调节活性的分子基础尚不清楚。使用压缩脂质单层和原子力显微镜，我们发现 X-Y 接头的一个高度保守的酸性区域足以调节吸附。X-Y 接头对吸附和活性的调节也独立于 C 末端结构域发生。我们接下来研究了 Gα 依赖性 PLCβ 激活是否改变了与模型膜的相互作用。Gα 以独立于 PLCβ 调节元件的方式增加 PLCβ 的吸附，并靶向吸附到没有 C 末端结构域的单层的特定区域。因此，Gα 依赖性激活的机制可能包括空间成分。
