Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor.
Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.
Gicquiaux
Hervé
H
Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Institut Fédératif Gilbert Laustriat, IFR 85, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, BP 24, 67401 Illkirch, France.
Lecat
Sandra
S
Gaire
Mireille
M
Dieterlen
Alain
A
Mély
Yves
Y
Takeda
Kenneth
K
Bucher
Bernard
B
Galzi
Jean-Luc
JL
D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D006982:Hypertonic Solutions; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D009478:Neuropeptide Y / Q000494:pharmacology; D010766:Phosphorylation; D021381:Protein Transport; D017476:Receptors, Neuropeptide Y / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods; D013314:Stress, Mechanical; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection
C092926:BIBP 3226; D006982:Hypertonic Solutions; D008164:Luminescent Proteins; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; C097212:neuropeptide Y-Y1 receptor; D003208:Concanavalin A; D049452:Green Fluorescent Proteins; D013395:Sucrose; D001120:Arginine; D019204:GTP-Binding Proteins; D002118:Calcium