Distinct residues in the carboxyl tail mediate agonist-induced desensitization and internalization of the human dopamine D1 receptor.
We have shown in a previous study that desensitization and internalization of the human dopamine D(1) receptor following short-term agonist exposure are mediated by temporally and biochemically distinct mechanisms. In the present study, we have used site-directed mutagenesis to remove potential phosphorylation sites in the third intracellular loop and carboxyl tail of the dopamine D(1) receptor to study these processes. Mutant D(1) receptors were stably transfected into Chinese hamster ovary cells, and kinetic parameters were measured. Mutations of Ser/Thr residues to alanine in the carboxyl tail demonstrated that the single substitution of Thr-360 abolished agonist-induced phosphorylation and desensitization of the receptor. Isolated mutation of the adjacent glutamic acid Glu-359 also abolished agonist-induced phosphorylation and desensitization of the receptor. These data suggest that Thr-360 in conjunction with Glu-359 may comprise a motif necessary for GRK2-mediated phosphorylation and desensitization. Agonist-induced internalization was not affected with mutation of either the Thr-360 or the Glu-359 residues. However, receptors with Ser/Thr residues mutated in the distal carboxyl tail (Thr-446, Thr-439, and Ser-431) failed to internalize in response to agonist activation, but were able to desensitize normally. These results indicate that agonist-induced desensitization and internalization are regulated by separate and distinct serine and threonine residues within the carboxyl tail of the human dopamine D(1) receptor.
Lamey
Michael
M
Departments of Pharmacology and Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Thompson
Miles
M
Varghese
George
G
Chi
Hong
H
Sawzdargo
Marek
M
George
Susan R
SR
O'Dowd
Brian F
BF
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry; D017447:Receptors, Dopamine D1 / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship; D051552:beta-Adrenergic Receptor Kinases
D018491:Dopamine Agonists; D018343:Receptors, Adrenergic, beta-2; D017447:Receptors, Dopamine D1; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases