Non-canonical G protein βγ signaling at the golgi

Heterotrimeric G proteins signal at a variety of endomembrane locations, to transduce external stimuli into changes in the cell status. Localization of activators, G proteins, and downstream effectors is a key mechanism for regulating which signaling pathways are activated. Targeting of G proteins to places such as the Golgi has resulted in identification of βγ signaling upstream of phospholipase C (PLC), Protein Kinase D (PKD), and phosphatidyl inositol 4 kinase IIIβ (PI4KIIIβ). Together activation of this pathway results in vesicle fission from the trans Golgi network (TGN), and over-activation of this pathway results in Golgi fragmentation. Because these components are constantly in flux throughout the cell, and found concurrently at both the plasma membrane (PM) and the Golgi, it has been difficult to identify which subcellular pools of G proteins are responsible for Golgi-localized signaling. To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we modified the widely-used βγ inhibitor GRK2ct, to contain a lipid-association mutation (GRK2ct-KERE). Here, results show that GRK2ct-KERE cannot inhibit function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling, highlighting a dependence of GRK2ct inhibition of βγ on its ability to localize to a membrane. Use of this inducible inhibitor showed that PM-recruited GRK2ct-KERE inhibits LPA-induced phosphorylation of Akt, blunting the cellular response to external stimuli. Golgi-recruited GRK2ct-KERE was shown to inhibit cargo transport from the TGN to the PM, specifically of model basolaterally-targeted but not apically-targeted cargo. This provided the first evidence of selectivity in terms of cargo transport regulated by βγ. GRK2ct-KERE was also used to interrogate the role of βγ on Golgi fragmentation induced by ilimaquinone and nocodazole. We determined that nocodazole only functions through Golgi-localized βγ, while ilimaquinone utilizes both PM- and Golgi-localized βγ. As Gαq subunits can also activate PLC, initial work here sets the stage for determining the contribution of Gαq and βγ on Golgi fragmentation. Lastly, we investigate the role of βγ on cell cycle progression through mitotic G2/M checkpoint, a process known to be dependent on Golgi fragmentation. Together these results further understanding of βγ signaling at the Golgi, and draw attention to wider implications on how subcellular localization can dictate function