Modeling of sequestration and down regulation in cells containing beta2-adrenergic receptors.

ABSTRACT

Desensitization of G-protein coupled receptors following agonist occupancy is accompanied by two temporally distinguishable cellular trafficking phenomena of the receptors referred to as sequestration and down regulation. For the beta2-adrenergic receptor, sequestration occurs within minutes of agonist binding and results in a reversible internalization and loss of cell surface receptor binding. With longer occupancy, greater than 1 hour, down regulation results in a variable loss of the complement of cellular receptors. Here we compare the two methods that have been used to monitor these receptor changes, competition of whole cell hydrophobic ligand binding (125I-pindolol) with a hydrophilic ligand (CGP-12177) and flow cytometry quantification of immunologically tagged beta2-adrenergic receptor. While both methods give reliable results, we show that because of a 1500 partitioning of the hydrophilic ligand into cells, slightly different conditions should be used to assess basally or agonist stimulated sequestered receptor levels. Using a sequestration defective beta2-adrenergic receptor mutant we demonstrate that even though sequestration and down regulation behave as independent processes, sequestration can significantly affect the rate at which receptors are lost by the down regulatory process by removing receptors from the pool of down regulating receptors. A mathematical model expressing these relationships is provided.