Type-specific sorting of G protein-coupled receptors after endocytosis

The beta-adrenergic receptor (B2AR) and delta-opioid receptor (DOR) are str ucturally distinct G protein-coupled receptors (GPCRs) that undergo rapid, agonist-induced internalization by clathrin-coated pits. We have observed t hat these receptors differ substantially in their membrane trafficking afte r endocytosis, B2AR expressed in stably transfected HEK293 cells exhibits n egligible (<10%) down-regulation after continuous incubation of cells with agonist for 3 h, as assessed both by radioligand binding (to detect functio nal receptors) and immunoblotting (to detect total receptor protein), In co ntrast, DOR exhibits substantial (greater than or equal to 50%) agonist-ind uced down-regulation when examined by similar means. Degradation of interna lized DOR is sensitive to inhibitors of lysosomal proteolysis. Flow cytomet ric and surface biotinylation assays indicate that differential sorting of B2AR and DOR between distinct recycling and non-recycling pathways (respect ively) can be detected within similar to 10 min after endocytosis, signific antly before the onset of detectable proteolytic degradation of receptors ( similar to 60 min after endocytosis), Studies using pulsatile application o f agonist suggest that after this sorting event occurs, later steps of memb rane transport leading to lysosomal degradation of receptors do not require the continued presence of agonist in the culture medium. These observation s establish that distinct GPCRs differ significantly in endocytic membrane trafficking after internalization by the same membrane mechanism, and they suggest a mechanism by which brief application of agonist can induce substa ntial down-regulation of receptors.