The third intracellular loop of α2-adrenergic receptors determines subtype specificity of arrestin interaction

Abstract

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 α2b- and α2c-adrenergic receptors (ARs) while having no effect on the εa2aAR, here we used α2ARs 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 α2aAR, α2bAR, or α2cAR. These studies revealed that arrestin-3 directly binds to the α2bAR and α2cAR but not the α2aAR, whereas arrestin-2 only binds to the α2bAR. Truncation mutagenesis of the α2bAR 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 α2bAR third intracellular loop. Mutation of these residues in the holo-α2bAR 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 α2bAR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the α2bAR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.