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 alpha-subunits (G alpha) and the beta gamma-subunit dimer (G beta gamma). In this study, we utilized mass spectrometry to identify novel regulators of G beta gamma signaling in human cells. This prompted our characterization of KCTD2 and KCTD5, two related potassium channel tetramerization domain (KCTD) proteins that specifically recognize G beta gamma. 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 G beta gamma through both KCTD proteins in response to Gprotein activation. These KCTD proteins promote monoubiquitination of lysine-23 within G beta(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 G beta gamma in response to signaling, monoubiquitinates lysine-23 within G beta(1/2), and regulates G beta gamma effectors to modulate downstream signal transduction.