title: Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.
abstract: 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 βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ 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 βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Klayman
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107. Electronic address: Philip.Wedegaertner@jefferson.edu.
authors.forename: Philip B
authors.initials: PB
authors.lastname: Wedegaertner
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D044387:GTP-Binding Protein beta Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D021381:Protein Transport / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D021601:trans-Golgi Network / Q000235:genetics / Q000378:metabolism*
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D051057:Proto-Oncogene Proteins c-akt
keywords: G protein; Golgi; cell biology; intracellular trafficking; protein trafficking (Golgi); signal transduction