Higazy-Mreih Sabreen, Avital-Shacham Meirav, LeGouill Christian, Bouvier Michel, Kosloff Mickey
Department of Human Biology, Faculty of Natural Science, University of Haifa, Israel.
Department of Biochemistry and Molecular Medicine & Institute for Research in Immunology and Cancer, Université de Montréal, Canada.
FEBS J. 2025 Oct;292(19):5244-5254. doi: 10.1111/febs.70149. Epub 2025 May 28.
Heterotrimeric (αβγ) G proteins are molecular switches that are activated by G protein-coupled receptors (GPCRs) and regulate numerous intracellular signaling cascades. Most active Gα subunits are inactivated by regulators of G protein signaling (RGS) proteins, which determine the duration of G protein-mediated signaling by accelerating the catalytic turn-off of the Gα subunit. However, the G protein Gα does not interact with known RGS proteins. To understand the molecular basis for this divergent phenomenon, we combined a comparative structural analysis of experimental and modeled structures with functional biochemical assays. This analysis showed that Gα contains unique structural elements in both the helical and the GTPase domains. Modeling suggested that helical domain insertions, which were missing in experimental structures, might project toward the interface with RGS proteins. Alternatively, residues in the Gα GTPase domain might lead to direct interference with RGS binding. Mutagenesis of Gα and measurements of RGS GTPase-activating protein (GAP) activity showed that three residues in the Gα GTPase domain are both necessary and sufficient to prevent Gα inactivation by RGSs. Indeed, substitution of all three Gα residues with the corresponding residues from Gα enabled efficient inactivation by RGS proteins. These results shed new light on the mechanistic bases for G protein specificity toward RGS proteins.
异源三聚体（αβγ）G蛋白是分子开关，由G蛋白偶联受体（GPCR）激活，并调节众多细胞内信号级联反应。大多数活性Gα亚基被G蛋白信号调节（RGS）蛋白失活，RGS蛋白通过加速Gα亚基的催化失活来决定G蛋白介导的信号传导持续时间。然而，G蛋白Gα不与已知的RGS蛋白相互作用。为了理解这种不同现象的分子基础，我们将实验结构和模型结构的比较结构分析与功能性生化测定相结合。该分析表明，Gα在螺旋结构域和GTPase结构域中均包含独特的结构元件。模型表明，实验结构中缺失的螺旋结构域插入可能伸向与RGS蛋白的界面。或者，Gα GTPase结构域中的残基可能直接干扰RGS结合。对Gα进行诱变并测量RGS GTPase激活蛋白（GAP）活性表明，Gα GTPase结构域中的三个残基对于防止Gα被RGS失活既必要又充分。事实上，用来自Gα的相应残基替换Gα的所有三个残基可使RGS蛋白有效失活。这些结果为G蛋白对RGS蛋白的特异性作用机制提供了新的线索。
