Dynamin and rab5 regulate GRK2-dependent internalization of dopamine D2 receptors.
Dopamine D2 receptors (D2Rs; short form, which is one of the alternative splicing variants) expressed in COS-7 cells are internalized in an agonist-dependent manner only when G protein-coupled receptor kinase 2 (GRK2) is coexpressed [Ito, K., Haga, T., Lameh, J. & Sadée, W., (1999) Eur. J. Biochem. 260, 112-119]. We have examined the effects of coexpression of dynamin, a small molecular mass GTP-binding protein, rab5A, and their mutants on the internalization of D2Rs in the presence of both dopamine (10 or 100 microM) and GRK2. The rate and extent of D2R internalization was increased or decreased by coexpression of dynamin I or a dominant-negative form of dynamin I (dynamin I K44E), respectively. The effects of coexpressing these two dynamins were more prominent at 10 microM dopamine than at 100 microM. In the presence of 10 microM dopamine, internalization of D2R was completely suppressed when dynamin I K44E was coexpressed, and the half-life (t 1/2) of D2R internalization decreased relative to cells not expressing dynamin from 82 to 29 min when dynamin I was coexpressed. Internalization of D2Rs was facilitated or suppressed by coexpression of a constitutively active form of rab5A (rab5A Q79L) or a dominant-negative form of rab5A (rab5A S34N), respectively. The t 1/2 of D2R internalization at 10 microM dopamine decreased from 82 to 16 min in cells coexpressing rab5A Q79L. The effect of coexpression of rab5A S34N was more apparent at 100 microM dopamine than at 10 microM; the t 1/2 of D2R internalization at 100 microM dopamine increased from 20 to 56 min and the proportion of internalized D2Rs after 120 min decreased from 53 to 28%. These results indicate that the internalization of D2Rs is dependent on the action of dynamin as well as GRK2, and is regulated by the action of rab5A.
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Japan. keniwata@m.u-tokyo.ac.jp
K
K
Iwata
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Ito
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A
Fukuzaki
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Inaki
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Haga
D000818:Animals; D019556:COS Cells; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D034284:Dynamin I; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D017448:Receptors, Dopamine D2 / Q000819:agonists / Q000502:physiology*; D013997:Time Factors; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases; D020696:rab5 GTP-Binding Proteins
D017448:Receptors, Dopamine D2; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins