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

Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the ␣ 2b -and ␣ 2c -adrenergic receptors (ARs) while having no effect on the ␣ 2a AR, here we used ␣ 2 ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the ␣ 2a AR, ␣ 2b AR, or ␣ 2c AR. These studies revealed that arrestin-3 directly binds to the ␣ 2b AR and ␣ 2c AR but not the ␣ 2a AR, whereas arrestin-2 only binds to the ␣ 2b AR. Truncation mutagenesis of the ␣ 2b AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194 -214) and the other at the C-terminal end (residues 344 -368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the ␣ 2b AR third intracellular loop. Mutation of these residues in the holo-␣ 2b AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant ␣ 2b AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the ␣ 2b AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.