Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States.
Molecular Biology Institute, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States.
J Proteome Res. 2021 Sep 3;20(9):4318-4330. doi: 10.1021/acs.jproteome.1c00292. Epub 2021 Aug 3.
G-protein-coupled receptors (GPCRs) initiate intracellular signaling events through heterotrimeric G-protein α-subunits (Gα) and the βγ-subunit dimer (Gβγ). In this study, we utilized mass spectrometry to identify novel regulators of Gβγ signaling in human cells. This prompted our characterization of KCTD2 and KCTD5, two related potassium channel tetramerization domain (KCTD) proteins that specifically recognize Gβγ. We demonstrated that these KCTD proteins are substrate adaptors for a multisubunit CUL3-RING ubiquitin ligase, in which a KCTD2-KCTD5 hetero-oligomer associates with CUL3 through KCTD5 subunits and recruits Gβγ through both KCTD proteins in response to G-protein activation. These KCTD proteins promote monoubiquitination of lysine-23 within Gβ and in HEK-293 cells. Depletion of these adaptors from cancer cell lines sharply impairs downstream signaling. Together, our studies suggest that a KCTD2-KCTD5-CUL3-RING E3 ligase recruits Gβγ in response to signaling, monoubiquitinates lysine-23 within Gβ, and regulates Gβγ effectors to modulate downstream signal transduction.
G 蛋白偶联受体（GPCRs）通过异三聚体 G 蛋白α亚基（Gα）和βγ亚基二聚体（Gβγ）启动细胞内信号事件。在这项研究中，我们利用质谱法鉴定了人细胞中 Gβγ 信号的新型调节因子。这促使我们对 KCTD2 和 KCTD5 进行了表征，这两种相关的钾通道四聚化结构域（KCTD）蛋白特异性识别 Gβγ。我们证明这些 KCTD 蛋白是一种多亚基 CUL3-RING 泛素连接酶的底物衔接子，其中 KCTD2-KCTD5 异寡聚体通过 KCTD5 亚基与 CUL3 结合，并通过两种 KCTD 蛋白响应 G 蛋白激活而募集 Gβγ。这些 KCTD 蛋白促进 Gβ 中赖氨酸-23 的单泛素化，在 HEK-293 细胞中也是如此。从癌细胞系中耗尽这些衔接子会严重损害下游信号。总之，我们的研究表明，KCTD2-KCTD5-CUL3-RING E3 连接酶在信号响应时募集 Gβγ，对 Gβ 中的赖氨酸-23 进行单泛素化，并调节 Gβγ 效应物以调节下游信号转导。
