Heterotrimeric G-protein α subunit (Gα) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Gα palmitoylating enzymes. DHHC3 and 7 robustly palmitoylated Gαq, Gαs, and Gαi2 in HEK293T cells. Knockdown of DHHC3 and 7 decreased Gαq⁄11 palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Gαq rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and 7 are necessary for this continuous Gαq shuttling. Furthermore, DHHC3 and 7 knockdown blocked the α1A-adrenergic receptor⁄Gαq/11-mediated signaling pathway. Together, our findings revealed that DHHC3 and 7 regulate GPCR-mediated signal transduction by controlling Gα localization to the plasma membrane.
