Senarath Kanishka, Fisher Isaac J, Jang Wonjo, Lu Sumin, Inoue Asuka, Kostenis Evi, Lyon Angeline M, Lambert Nevin A
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912.
James Tarpo Jr. and Margaret Tarpo Department of Chemistry, Purdue University, West Lafayette, IN 47907.
Proc Natl Acad Sci U S A. 2025 Apr 22;122(16):e2500318122. doi: 10.1073/pnas.2500318122. Epub 2025 Apr 18.
Phospholipase Cβ (PLCβ) enzymes are the principal effectors activated by G heterotrimers. Both Gα and Gβγ subunits can activate PLCβ, which requires precise positioning of PLCβ at the plasma membrane to relieve structural autoinhibition and give the active site access to the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate. PLCβ enzymes possess a unique distal C-terminal domain (dCTD) that is critical for activation by Gα, but the reason for this is unclear. It is also not known how G protein activation affects the subcellular localization of PLCβ enzymes, some of which are found primarily in the cytosol despite needing to act at the plasma membrane. Here, we use bioluminescence spectroscopy, imaging, and gene editing to study the membrane disposition of PLCβ enzymes in living cells and to define the functional roles of the dCTD. We find that PLCβ translocates to the plasma membrane upon G activation, primarily by binding to Gα subunits. This is rapidly counteracted by PIP2 hydrolysis, which promotes PLCβ translocation back into the cytosol. PLCβ translocation and activation require binding of Gα to the catalytic domain and the dCTD at two distinct interfaces. Gα binding to the dCTD is required for activation even when PLCβ is artificially tethered to the plasma membrane, suggesting that this domain has functions beyond simply recruiting the enzyme to the PIP2 substrate. We propose that in addition to associating PLCβ with the plasma membrane, the dCTD reorders the αN helix of active Gα and thus participates directly in the precise positioning of the catalytic domain.
磷脂酶Cβ（PLCβ）是由G蛋白异源三聚体激活的主要效应物。Gα和Gβγ亚基均可激活PLCβ，这需要PLCβ在质膜上精确定位以解除结构自抑制，并使活性位点能够接触到磷脂酰肌醇4,5-二磷酸（PIP2）底物。PLCβ酶具有一个独特的远端C末端结构域（dCTD），这对于Gα激活至关重要，但其原因尚不清楚。也不清楚G蛋白激活如何影响PLCβ酶的亚细胞定位，其中一些酶尽管需要在质膜上发挥作用，但主要存在于细胞质中。在这里，我们使用生物发光光谱、成像和基因编辑来研究活细胞中PLCβ酶的膜分布，并确定dCTD的功能作用。我们发现，PLCβ在G激活后会转运到质膜，主要是通过与Gα亚基结合。这很快被PIP2水解所抵消，PIP2水解促进PLCβ转运回细胞质。PLCβ的转运和激活需要Gα在两个不同界面与催化结构域和dCTD结合。即使PLCβ被人工拴系在质膜上，Gα与dCTD的结合对于激活也是必需的，这表明该结构域的功能不仅仅是将酶招募到PIP2底物上。我们提出，除了将PLCβ与质膜结合外，dCTD还能重新排列活性Gα的αN螺旋，从而直接参与催化结构域的精确定位。
