Role of arrestins in endocytosis and signaling of α2-adrenergic receptor subtypes

We investigated the role of arrestins in the trafficking of human alpha(2)-adrenergic receptors (alpha(2)-ARs) and the effect of receptor trafficking on p42/p44 RAP kinase activation. alpha(2)-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with alpha(2c) > alpha(2b) > alpha(2a). However, upon coexpression of arrestin-2 (beta-arrestin-1) or arrestin-3 (beta-arrestin-2), internalization of the alpha(2b) AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the alpha 2c AR was selectively promoted by coexpression of arrestin-3, while alpha 2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any alpha 2-AR subtype, either in the presence or absence of arrestins. Internalization of the alpha 2b and alpha 2c ARs was inhibited by coexpression of dominant negative dynamin-K44A However, alpha 2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (IMAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/ p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and beta(2)-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the alpha(2b), alpha(2c), and to a lesser extent alpha(2a) ARs, is both arrestin- and dynamin de pendent. However, endocytosis does not appear to be required for alpha(2)-adrenergic, epidermal growth factor, lysophosphatidic acid, or beta(2)-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.