Role of clathrin-mediated endocytosis in agonist-induced down-regulation of the β2-adrenergic receptor

Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta(2)-adrenergic receptor (beta(2)AR), Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta(2)AR, In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta(2)AR, whereas dynamin-K44A, a dominant negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta(2)AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta(2)AR, suggesting that receptor internalization is critical for down-regulation in these cells, Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta(2)AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs, These studies demonstrate that down-regulation of the beta(2)AR is in part due to trafficking of the beta(2)AR via the clathrin-coated pit endosomal pathway to lysosomes.