Abstract
Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.
Highlights
Intracellular trafficking of Regulator of G-protein signaling (RGS) proteins is an important determinant of their function as G-protein inhibitors
Cys-2 is largely dispensable for plasma membrane targeting, it does appear to play a role in the trafficking of RGS4 through different intracellular membrane compartments, and its palmitoylation status may be important for regulating its normal trafficking and recycling within the cell
Very little is known about the intracellular trafficking of RGS4 between plasma membrane and endosomal or cytosolic pools
Summary
Intracellular trafficking of RGS proteins is an important determinant of their function as G-protein inhibitors. Previous work demonstrated that cysteine palmitoylation on residues in the aminoterminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4 These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and Gq inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization. Cys-2 is largely dispensable for plasma membrane targeting, it does appear to play a role in the trafficking of RGS4 through different intracellular membrane compartments, and its palmitoylation status may be important for regulating its normal trafficking and recycling within the cell
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