ARF6 and GASP‐1 are post‐endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D₂ receptors mediated by GRK and PKC in transfected cells

Abstract

Background and Purpose GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC‐mediated heterologous internalization pathways for the dopamine D2 receptor were investigated. Experimental Approach All of the S/T residues located within the intracellular loops of D2 receptor were mutated, and the residues responsible for GRK‐ and PKC‐mediated internalization were determined in HEK‐293 cells and SH‐SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post‐endocytic fate of internalized D2 receptors were investigated in the transfected cells. Key Results T134, T225/S228/S229 and S325 were involved in PKC‐mediated D2 receptor desensitization. S229 and adjacent S/T residues mediated the PKC‐dependent internalization of D2 receptors, which induced down‐regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK‐mediated internalization of D2 receptors, which induced receptor resensitization. ARF6 mediated the recycling of D2 receptors internalized in response to agonist stimulation. In contrast, GASP‐1 mediated the down‐regulation of D2 receptors internalized in a PKC‐dependent manner. Conclusions and Implications GRK‐ and PKC‐mediated internalizations of D2 receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D2 receptors and different sorting proteins are involved in the dissimilar regulation of D2 receptors by GRK2 and PKC.

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor

“…Knockdown of either GRK2 or arrestin3 significantly reduces the amount of agonist-induced D 2 receptor internalization (10,38), and overexpression of either protein enhances the amount of internalization seen in response to agonist (8,20,39,40). Furthermore, this response is potentiated when GRK2 and arrestin3 are overexpressed together (8,20,39).…”

“…Furthermore, this response is potentiated when GRK2 and arrestin3 are overexpressed together (8,20,39). Thus, it is possible that the reduced internalization of D 2 -A3 simply reflects its diminished GRK2 and arrestin3 recruitment, with a partial recruitment deficit somehow translating into a more severe internalization deficit.…”

Endo‐lysosomal sorting of G‐protein‐coupled receptors by ubiquitin: Diverse pathways for G‐protein‐coupled receptor destruction and beyond

“…ALIX directs receptors into budding intraluminal vesicles and couples YPX 3 L‐motif GPCRs to the ESCRT‐III complex. Similarly, lysosomal sorting of the DOR and the D2 dopamine receptor is mediated by the adaptor protein GASP‐1, which couples these proteins to the ESCRT‐0 complex independent of receptor ubiquitination. Recent findings have demonstrated that the DOR can recycle from the limiting membrane of the late endosome .…”

PKCβII inhibits the ubiquitination of β‐arrestin2 in an autophosphorylation‐dependent manner

“…T250A-PKCbII, Flagtagged T250A-PKCbII, K11/12R-b-arrestin2, and Flag-tagged K11/12R-b-arrestin2 were prepared by site-directed mutagenesis. The generation of PKCa, PKCbI, PKCbII, PKCe, and PKCf conjugated to GFP, along with PKCa, PKCbII, PKC c, and PKC d fused to HA has been described previously [20,21]. The shRNAs for GRK2, MDM2, and PKCbII were described previously [22][23][24].…”