Binding of the beta 2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling

Following agonist stimulation, most G protein-coupled receptors become dese nsitized and are internalized, either to be degraded or recycled back to th e cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recy cling beta2 adrenergic receptor (beta 2AR) binds via a determinant includin g the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitatio ns. Receptors bearing mutations in any of the last three residues fail to i nteract with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta 2AR but not with a tail-mutated beta 2AR. The beta 2AR-NSF interaction is requi red for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta 2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation . Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors rem ain sequestered. Thus, the direct binding of the beta 2AR to NSF demonstrat es how, after internalization, the fate of a receptor is reliant on a speci fic interaction with a component of the cellular membrane-trafficking machi nery.