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 Gβ_1/2 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 Gβ_1/2, and regulates Gβγ effectors to modulate downstream signal transduction.