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.
OBJECTIVE
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.


METHODS
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.


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
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.
Department of Pharmacology, College of Pharmacy, Drug Development Research Institute, Chonnam National University, Gwang-Ju, Korea.
D I
DI
Cho
M
M
Zheng
C
C
Min
K J
KJ
Kwon
C Y
CY
Shin
H K
HK
Choi
K M
KM
Kim
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018491:Dopamine Agonists / Q000494:pharmacology; D059290:Dopaminergic Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D020536:Enzyme Activators / Q000494:pharmacology; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D034622:RNA Interference; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013618:Tachyphylaxis; D013912:Threonine / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; C581293:DRD2 protein, human; D018491:Dopamine Agonists; D020536:Enzyme Activators; D036341:Intercellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D011506:Proteins; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; C424444:WFIKKN2 protein, human; D013912:Threonine; D012694:Serine; D011493:Protein Kinase C; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human