MODELING OF SEQUESTRATION AND DOWN-REGULATION IN CELLS CONTAINING BETA(2)-ADRENERGIC RECEPTORS

Desensitization of G-protein coupled receptors following agonist occup ancy is accompanied by two temporally distinguishable cellular traffic king phenomena of the receptors referred to as sequestration and down regulation. For the beta(2)-adrenergic receptor, sequestration occurs within minutes of agonist binding and results in a reversible internal ization and loss of cell surface receptor binding. With longer occupan cy, 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 (I-125-pindolol) with a hydroph ilic ligand (CGP-12177) and flow cytometry quantification of immunolog ically tagged beta(2)-adrenergic receptor. While both methods give rel iable results, we show that because of a 1:500 partitioning of the hyd rophilic ligand into cells, slightly different conditions should be us ed to assess basally or agonist stimulated sequestered receptor levels . Using a sequestration defective beta(2)-adrenergic receptor mutant w e demonstrate that even though sequestration and down regulation behav e as independent processes, sequestration can significantly affect the rate at which receptors are lost by the down regulatory process by re moving receptors from the pool of down regulating receptors. A mathema tical model expressing these relationships is provided.