G‐protein beta‐gamma regulation of Golgi to cell surface transport (802.11)

Abstract

Non‐canonical G‐protein signaling has been shown to regulate intracellular transport and secretion. G‐protein beta‐gamma localizes to the trans‐Golgi network and activates Protein Kinase D (PKD), resulting in fission of vesicles destined for the plasma membrane. While basolaterally‐targeted (BL) or secretory vesicle release is known to be downstream of PKD activation, apically‐targeted (AP) vesicle release from the Golgi occurs via a PKD‐independent pathway. A similar cargo selectivity for beta‐gamma has not been tested. Here, we generate GRK2ct variants that inhibit beta‐gamma in a spatial or temporal manner. We test regulation of AP and BL cargo trafficking via a temperature shift immunofluorescence transport assay, as well as trafficking of other AP and BL candidate cargoes. We find that lipid‐association of the GRK2ct is critical for its inhibitory function on beta‐gamma. We also generate an inducible system of recruitment of GRK2ct variants to distinct subcellular locations, towards the goal of inhibiting solely Golgi‐localized beta‐gamma signaling. Our system can also be used to inhibit beta‐gamma signaling at other endomembrane locations. The results indicate that beta‐gamma is an upstream regulator of vesicle transport from the Golgi to the cell surface.Grant Funding Source: NIH GM056444