Gβγ TRANSLOCATION TO THE GOLGI APPARATUS MEDIATES MAPK ACTIVATION BY GPCRS VIA PI3Kγ AND ARF1

Abstract

G protein-coupled receptors (GPCRs) transduce signals mainly by activating heterotrimeric G proteins consisting of Gα, Gβ, and Gγ subunits. Gβ and Gγ subunits bind tightly to form Gβγ dimers. Previous studies have shown that after the activation of GPCRs, Gβγ may translocate from the plasma membrane (PM) to intracellular compartments, including the Golgi apparatus (GA). However, the exact functions of this translocation are poorly defined. Here, we demonstrate that Gβγ dimers containing Gγ9 translocate robustly to the GA in response to chemokine receptor CXCR4 activation by stromal cell-derived factor 1α (SDF1α). CRISPR-Cas9-mediated knockout of Gγ9, but not the least translocatable Gγ3, abolishes the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) by SDF1α. Chemically-induced recruitment of different Gγ subunits, in combination with Gβ1, to the GA activates ERK1/2, whereas recruitment to the PM is ineffective. We also show that the pharmacological inhibition of phosphoinositide 3-kinase γ (PI3Kγ) and depletion of its subunits p110γ and p101 abrogate ERK1/2 activation by CXCR4 and Gβγ recruitment to the GA. Knockout of Gγ9 or p110γ significantly suppresses PC3 cell migration and invasion in vitro, and metastasis in vivo. We further demonstrate that, similar to ERK1/2 activation, CXCR4 and the constitutive targeting of Gβγ to the GA activate ADP-ribosylation factor 1 (ARF1) which can be blocked by Gγ9 knockout and PI3Kγ inhibition and depletion. CXCR4 activation and forced Gβγ recruitment to the GA also enhance ARF1 translocation from the cytoplasm to the GA. Depletion of ARF1 by siRNA and CRISPR-Cas9 and inhibition of GA-localized ARF1 abolish ERK1/2 activation by CXCR4 and Gβγ translocation to the GA and suppress in vitro prostate cancer PC3 cell migration and invasion. Collectively, our data reveal a novel function for Gβγ translocation to the GA to activate ERK1/2 through PI3Kγ and ARF1, and strongly suggest that the GA functions as a spatial station to coordinate Gβγ translocation and sequential activation of PI3Kγ, ARF1, and ERK1/2. These data provide important insights into spatiotemporal regulation of GPCR signaling to the mitogen-activated protein kinase pathway.