Pharmacological sequestration of a chimeric beta 3/beta 2 adrenergic receptor occurs without a corresponding amount of receptor internalization.

Many G-protein-coupled receptors undergo sequestration (rapid loss of cell surface receptor binding sites) following exposure to agonists. This process has been assayed traditionally by the use of membrane-impermeant radioligands to measure cell-surface binding sites before and after exposure of cells to agonists. Pharmacological sequestration of the beta 2 adrenergic receptor is associated with internalization of the receptor protein, although it is not known whether receptor internalization is the only mechanism by which ligand-binding sites can be sequestered from the cell surface. Here we show that a chimeric mutant adrenergic receptor, constructed by attaching the carboxyl-terminal cytoplasmic domain from the beta 2 receptor to the beta 3 receptor sequence, exhibits agonist-induced sequestration (measured by 3H-CGP-12177 binding to intact human embryonal kidney [HEK] 293 cells) that is in significant excess to the small amount of receptor internalization measured in the same cells by quantitative flow cytometry. Furthermore, sequestration of the chimeric mutant receptor is reversible at 13 degrees C, a condition that blocks internalization and recycling of adrenergic receptors and has no effect on the sequestration of beta 2 receptors. These data suggest the operation of two distinguishable mechanisms of receptor sequestration in the same cells: agonist-induced internalization and an alternative, biochemically distinguishable mechanism that occurs without a corresponding amount of internalization of the receptor protein.