The Third Intracellular Loop of α2-Adrenergic Receptors Determines Subtype Specificity of Arrestin Interaction
Journal of Biological Chemistry
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
... 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.