Abstract
Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta2AR, 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 beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.
Highlights
Stimulation of G protein-coupled receptors (GPRs)1 leads to adaptive changes that serve to modulate the responsiveness of a cell to further stimulation [1]
Desensitization often occurs within seconds of stimulation and for many GPRs is mediated by phosphorylation of the receptor by a specific G protein-coupled receptor kinase
Since both -arrestin and arrestin 3 can bind to clathrin, the major structural protein of clathrin-coated pits, arrestins can function as adaptors to recruit G protein-coupled receptor kinase-phosphorylated receptors into coated pits where they can be internalized [7]
Summary
Stimulation of G protein-coupled receptors (GPRs)1 leads to adaptive changes that serve to modulate the responsiveness of a cell to further stimulation [1]. Previous studies have established that this effect is likely mediated via a carboxylterminal clathrin binding domain in -arrestin and arrestin 3 [23] that functions to recruit the receptor complex to clathrincoated pits, resulting in receptor internalization [7]. Similar to the internalization results, expression of either -arrestin or arrestin 3, but not visual arrestin, significantly increased isoproterenol-stimulated down-regulation of the 2AR after a 24-h incubation with agonist (Fig. 1B).
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