Antagonist-dependent and -independent steps in the mechanism of adrenergic receptor internalization

Epitope tagging and immunocytochemical techniques were used to examine the agonist-regulated internalization of human beta 2-adrenergic receptors in 293 cells. In the absence of agonist, receptors tagged with monoclonal antibody remain in the plasma membrane for > 1 h. In the presence of agonist, tagged receptors are endocytosed within 10 min. Endocytosed receptors are located in endosomes and can be recycled to the plasma membrane. In the prolonged presence of agonist, receptor endocytosis continues even after maximum sequestration of surface receptors (measured by radioligand binding to intact cells) has occurred. The process of receptor endocytosis requires cellular ATP and is temperature-dependent. At 4 degrees C, no agonist-induced redistribution of receptors located in the plasma membrane is observed. At 16 degrees C, agonist causes receptors to cluster in and around coated invaginations of the plasma membrane, but receptor endocytosis does not occur. Agonist treatment of cells at 16 degrees C, but not 4 degrees C, predisposes receptors to agonist-independent endocytosis upon warming to 37 degrees C. These studies suggest that: 1) beta 2-adrenergic receptors reside stably in the plasma membrane of untreated cells, while they continuously cycle between the plasma membrane and endosomes in the presence of agonist; 2) agonist regulates an early step in the endocytosis mechanism, which is associated with the redistribution of adrenergic receptors between distinct microdomains of the plasma membrane; and 3) later steps in the endocytosis mechanism do not require agonist and may utilize the same endocytic machinery that mediates the endocytosis of constitutively recycling receptors.