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

Br J Pharmacol. 2013 Mar;168(6):1355-74. doi: 10.1111/bph.12025.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factors / antagonists & inhibitors
  • ADP-Ribosylation Factors / genetics
  • ADP-Ribosylation Factors / metabolism*
  • Cell Line
  • Dopamine Agonists / pharmacology
  • Dopaminergic Neurons / cytology
  • Dopaminergic Neurons / drug effects
  • Dopaminergic Neurons / metabolism*
  • Endosomes / drug effects
  • Endosomes / metabolism*
  • Enzyme Activators / pharmacology
  • G-Protein-Coupled Receptor Kinase 2 / chemistry
  • G-Protein-Coupled Receptor Kinase 2 / genetics
  • G-Protein-Coupled Receptor Kinase 2 / metabolism*
  • HEK293 Cells
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Phosphorylation / drug effects
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / chemistry
  • Protein Kinase C / metabolism*
  • Protein Kinase Inhibitors / pharmacology
  • Protein Processing, Post-Translational / drug effects
  • Protein Transport / drug effects
  • Proteins / metabolism*
  • RNA Interference
  • Receptors, Dopamine D2 / chemistry
  • Receptors, Dopamine D2 / genetics
  • Receptors, Dopamine D2 / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Serine / metabolism
  • Tachyphylaxis
  • Threonine / metabolism

Substances

  • DRD2 protein, human
  • Dopamine Agonists
  • Enzyme Activators
  • Intercellular Signaling Peptides and Proteins
  • Mutant Proteins
  • Protein Kinase Inhibitors
  • Proteins
  • Receptors, Dopamine D2
  • Recombinant Proteins
  • WFIKKN2 protein, human
  • Threonine
  • Serine
  • Protein Kinase C
  • GRK2 protein, human
  • G-Protein-Coupled Receptor Kinase 2
  • ADP-Ribosylation Factors
  • ADP-ribosylation factor 6