Inducible Inhibition of Gßγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on ßγ signaling at the Golgi, where ßγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous ßγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used ßγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit ßγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit ßγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by ßγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by ßγ inhibition, demonstrating that ßγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate ßγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for ßγ regulation of TGN to PM transport and a novel role for ßγ in mediating Golgi fragmentation.