Human Multisubunit E3 Ubiquitin Ligase Required for Heterotrimeric G‑Protein β‑Subunit Ubiquitination and Downstream Signaling

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 Gβ1/2 in vitro 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 Gβ1/2, and regulates Gβγ
effectors to modulate downstream signal transduction.

G蛋白偶联受体（GPCRs）通过异源三聚体G蛋白α亚基（Gα）以及βγ亚基二聚体（Gβγ）启动细胞内信号传导事件。在本研究中，我们运用质谱技术识别了人类细胞中Gβγ信号的新型调控因子。此发现促使我们对KCTD2和KCTD5这两种相关钾通道四聚化结构域（KCTD）蛋白进行表征，这两种蛋白特异性识别Gβγ。我们证实这些KCTD蛋白是多亚基CUL3-RING泛素连接酶的底物适配子，其中KCTD2-KCTD5的异源寡聚体通过KCTD5亚基与CUL3结合，并通过G蛋白激活响应招募Gβγ。这些KCTD蛋白在体外和HEK-293细胞中促进Gβ1/2中赖氨酸-23的单泛素化。从癌细胞系中清除这些适配子显著损害下游信号传导。综合来看，我们的研究提出，KCTD2-KCTD5-CUL3-RING E3连接酶在信号传导响应中招募Gβγ，单泛素化Gβ1/2中的赖氨酸-23，并调节Gβγ效应物以调节下游信号转导。