REPETITIVE ENDOCYTOSIS AND RECYCLING OF THE BETA-2-ADRENERGIC RECEPTOR DURING AGONIST-INDUCED STEADY-STATE REDISTRIBUTION

The human beta(2)-adrenergic receptor (beta(2)AR) vapidly internalizes after binding agonist, resulting in a dramatic redistribution of rece ptors from the plasma membrane and into endocytic vesicles, We sought to determine whether intracellular receptors constitute a static pool or represent a fraction of dynamically internalizing and recycling rec eptors. Using cells expressing a beta(2)AR with an epitope tag al its amino-terminal ectodomain, changes in surface receptor levels were mea sured by flow cytometry and radioligand binding assays. The addition o f a saturating level of a strong agonists (isoproterenol) caused the e ndocytosis of receptors with first-order kinetics (k(e) for naive cell s, 0.222 min(-1)). After 10 min, the level of surface receptors remain ed stable at similar to 20% that of untreated cells, even though endoc ytosis continued with similar kinetics (k(e) for pretreated cells, 0.2 58 min(-1)), suggesting that internalized receptors were cycling in st eady state with surface receptors, This prediction was confirmed direc tly by showing that internalized beta(2)ARs recycled to the cell surfa ce in the continued presence of agonist. The calculated transit times (1/k) in the presence of isoproterenol were 3.9 min for endocytosis an d 11.2 min for recycling. The endocytic rate constant and the steady s tate redistribution to the internal pool were much lower after treatme nt with the partial agonist albuterol, suggesting a correlation betwee n the efficiency of endocytosis and that of receptor coupling to the d ownstream signal transduction pathway. These findings indicate that in the presence of agonist, beta(2)ARs are in a dynamic steady state bet ween the plasma membrane and endosomes that is regulated principally b y agonist efficacy.