The adaptor complex 2 directly interacts with the α1b-adrenergic receptor and plays a role in receptor endocytosis

Using the yeast two-hybrid system, we identified the mu(2) subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha(1b)-adrenergic receptor (AR). Direct association between the alpha(1b)-AR and mu(2) was demonstrated using a solid phase overlay assay. The alpha(1b)-AR/mu(2) interaction occurred inside the cells, as shown by the finding that the transfected alpha(1b)-AR and the endogenous mu(2) could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha(1b)-AR revealed that the binding site for mu(2) does not involve canonical YXXPhi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu(2) for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha(1b)-AR specifically interacted with mu(2), but not with the mu(1), mu(3), or mu(4) subunits belonging to other AP complexes. The deletion of the mu(2) binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.