Palmitoylation of RGS20 affects Gαo-mediated signaling independent of its GAP activity

Abstract

Regulator of protein signaling (RGS20) is a member of the RGS protein superfamily, which serve as key negative regulators of G protein-mediated signal transduction. Through their GTPase accelerating protein (GAP) activity, RGS proteins deactivate α-subunits of heterotrimeric G proteins. In addition, the majority of RGS proteins also have the ability to act through other, non-GAP related, functions. RGS20 is one of three members of the RZ subfamily, which all show selective GAP activity towards Gα_z, however emerging data suggest that RGS20 can also regulate Gi/o-mediated signaling. While increased RGS20 expression is associated with the progression of multiple cancers, a large gap still exists relating to the mechanisms of RGS20 regulation and function. RGS20 contains a poly-cysteine string motif and a conserved cysteine in RGS domain, which are assumed to be palmitoylated. Palmitoylation, an important post-translational modification, plays an important role in cells by changing cellular functions of proteins. Consequently, the aim of this study was to confirm that RGS20 is palmitoylated and determine how palmitoylation affects its inhibition of Gα_o-mediated signaling. We found a significant positive correlation between RGS20 palmitoylation and its association with active Gα_o. We also showed that a conserved cysteine residue in the RGS domain is a critical site for its palmitoylation, with large impact on its association with Gα_o. Palmitoylation on this site did not affect its GAP activity, however, it increased the inhibition of Gα_o-mediated cAMP signaling. Altogether these data suggest that palmitoylation is a regulatory mechanism controlling RGS20 function, and that RGS20 can inhibit Gα_o signaling through both GAP activity and non-GAP mechanisms.

Keywords: G protein-mediated signaling; Palmitoylation; Posttranslational modifications; Regulator of G protein signaling (RGS).