Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function

J Biol Chem. 2012 Aug 17;287(34):28966-74. doi: 10.1074/jbc.M112.345629. Epub 2012 Jun 29.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Cysteine / genetics
  • Cysteine / metabolism*
  • Endosomes / genetics
  • Endosomes / metabolism
  • Enzyme Activation / genetics
  • GTP-Binding Protein alpha Subunits, Gq-G11 / genetics
  • GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism
  • HEK293 Cells
  • Humans
  • Lipoylation / physiology*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Mutation, Missense
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Transport / physiology*
  • RGS Proteins / genetics
  • RGS Proteins / metabolism*

Substances

  • Membrane Glycoproteins
  • RGS Proteins
  • TGOLN2 protein, human
  • RGS4 protein
  • GTP-Binding Protein alpha Subunits, Gq-G11
  • Cysteine