Dynamin and Rab5 regulate GRK2-dependent internalization of dopamine D2 receptors

Dopamine D2 receptors (D2Rs; short form, which is one of the alternative sp licing variants) expressed in COS-7 cells are internalized in an agonist-de pendent manner only when G protein-coupled receptor kinase 2 (GRK2) is coex pressed [Ito, K., Haga, T., Lameh, J. & Sadee, W., (1999) fur. J. Biochem. 260, 112-119]. We have examined the effects of coexpression of dynamin, a s mall molecular mass GTP-binding protein, rab5A, and their mutants on the in ternalization of D2Rs in the presence of both dopamine (10 or 100 mu M) and GRK2. The rate and extent of D2R internalization was increased or decrease d by coexpression of dynamin I or a dominant-negative form of dynamin I (dy namin I K44E), respectively. The effects of coexpressing these two dynamins were more prominent at 10 mu M dopamine than at 100 mu M. In the presence of 10 mu M dopamine, internalization of D2R was completely suppressed when dynamin I K44E was coexpressed, and the half-life (t 1/2) of D2R internaliz ation 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 mu M dopamine decreased from 82 to 16 mi n in cells coexpressing rab5A Q79L. The effect of coexpression of rab5A S34 N was more apparent at 100 mu M dopamine than at 10 mu M; the t 1/2 of D2R internalization at 100 mu M dopamine increased from 20 to 56 min and the pr oportion of internalized D2Rs after 120 min decreased from 53 to 28%. These results indicate that the internalization of D2Rs is dependent on the acti on of dynamin as well as GRK2, and is regulated by the action of rab5A.