UCLA

GPCRs are stimulated by extracellular ligands and initiate a range of intracellular signaling events through heterotrimeric G-proteins. Upon activation, G-protein α-subunits (Gα) and the stable βγ-subunit dimer (Gβγ) bind and alter the activity of diverse effectors. These signaling events are fundamental and subject to multiple layers of regulation. In this study, we used an unbiased proteomic mass spectrometry approach to uncover novel regulators of Gβγ. We identified a subfamily of potassium channel tetramerization domain (KCTD) proteins that specifically bind Gβγ. Several KCTD proteins are substrate adaptor proteins for CUL3–RING E3 ubiquitin ligases. Our studies revealed that a KCTD2-KCTD5 hetero-oligomer associates with CUL3 through KCTD5 subunits and recruits Gβγ through both subunits. Using in vitro ubiquitination reactions, we demonstrated that these KCTD proteins promote monoubiquitination of lysine-23 within Gβ1/2. This ubiquitin modification of Gβ1/2 is also observed in human cells and is dependent on these substrate adaptor proteins. Because these KCTD proteins bind Gβγ in response to G-protein activation, we investigated their role in GPCR signaling. Their deletion strongly impairs cAMP generation and downstream signaling pathways in response to signaling activation. Consistent with these results, depletion of CUL3, the component of the E3 ligase that binds KCTD proteins, causes similar defects. Together, our studies suggest that a KCTD2/KCTD5–CUL3–RING E3 ubiquitin ligase recruits Gβγ in response to signaling, monoubiquitinates lysine-23 within Gβ, and stimulates adenylyl cyclases—Gβγ effectors—to positively regulate cAMP signaling.