Homologous regulation of the heptahelical D1A receptor responsiveness: specific cytoplasmic tail regions mediate dopamine-induced phosphorylation, desensitization and endocytosis.
In the present study, we investigate the role of specific cytoplasmic tail (CT) regions of the D1A receptor in mediating dopamine (DA)-induced phosphorylation, desensitization and endocytosis. Results obtained in human embryonic kidney (HEK) cells expressing the wild-type (WT) or truncation forms (Delta425, Delta379 and Delta351) of the D1A receptor show that sequences located downstream of Gly379 regulate DA-mediated phosphorylation-dependent desensitization of D1A receptors. However, the longer truncation mutant Delta351 failed to undergo detectable DA-induced phosphorylation while exhibiting DA-induced desensitization features similar to the shorter truncation mutant Delta379. These data potentially suggest a novel role for a receptor phosphorylation-independent process in the DA-promoted D1A subtype desensitization. Our immunofluorescence data also suggest that sequences located between Cys351 and Gly379 play an important role in DA-mediated receptor endocytosis. Additionally, time-course studies were done in intact cells expressing WT or truncation receptors to measure the observed rate constant for adenylyl cyclase (AC) activation or k(obs), a parameter linked to the receptor-G protein coupling status. In agreement with the desensitization data, Delta425- and Delta379-expressing cells exhibit an increase of kobs in comparison with WT-expressing cells. Nevertheless, Delta351-expressing cells, which harbor similar desensitization features of Delta379-expressing cells, display no change in k(obs) when compared with WT-expressing cells. Our results suggest that a defective DA-induced endocytosis may hamper Delta351 resensitization and concomitant increase in k(obs). Thus, our study showing that specific D1A receptor CT sequences regulate DA-induced phosphorylation, desensitization, and endocytosis highlights the underlying molecular complexity of signaling at dopaminergic synapses.
Jackson
Adele
A
Ottawa Health Research Institute, Department of Medicine/Cellular, University of Ottawa, Ontario, Canada.
Iwasiow
Rafal M
RM
Chaar
Ziad Y
ZY
Nantel
Marie-France
MF
Tiberi
Mario
M
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004298:Dopamine / Q000494:pharmacology*; D004351:Drug Resistance; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D051381:Rats; D017447:Receptors, Dopamine D1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
D017447:Receptors, Dopamine D1; C098593:dopamine D1A receptor; D000262:Adenylyl Cyclases; D004298:Dopamine