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J. Biol. Chem., Vol. 277, Issue 8, 6645-6655, February 22, 2002

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Rapid Internalization and Recycling of the Human Neuropeptide Y Y₁ Receptor^*

Hervé Gicquiaux, Sandra Lecat^§, Mireille Gaire, Alain Dieterlen^¶, Yves Mély, Kenneth Takeda, Bernard Bucher, and Jean-Luc Galzi

From the 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, the  Département Récepteurs et Protéines Membranaires, UPR CNRS 9050, Institut Fédératif Gilbert Laustriat, IFR 85, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67401 Illkirch, France, and the ^¶ Laboratoire MIPS-Groupe LABEL, Université de Haute-Alsace, IUT de Mulhouse, 61 rue Albert Camus, 68093 Mulhouse, France

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) Y₁ receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y₁ receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y₁ receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y₁ 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 Y₁ receptor antagonist, or by pertussis toxin, in agreement with Y₁ receptor activation. Internalization of EGFP-hNPY Y₁ 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 Y₁ receptors.

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