Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling.
Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including 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 coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR 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 remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.
Cong
M
M
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Perry
S J
SJ
Hu
L A
LA
Hanson
P I
PI
Claing
A
A
Lefkowitz
R J
RJ
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D005033:Ethylmaleimide / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007545:Isoproterenol / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D009154:Mutation; D052066:N-Ethylmaleimide-Sensitive Proteins; D011233:Precipitin Tests; D011433:Propranolol / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D011433:Propranolol; D005982:Glutathione Transferase; D052066:N-Ethylmaleimide-Sensitive Proteins; D007545:Isoproterenol; D005033:Ethylmaleimide