Rapid internalization and recycling of the human neuropeptide YY1 receptor

被引:100
作者
Gicquiaux, H
Lecat, S
Gaire, M
Dieterlen, A
Mély, Y
Takeda, K
Bucher, B
Galzi, JL
机构
[1] Univ Louis Pasteur Strasbourg 1, Fac Pharm, Inst Fed Gilbert Laustriat, IFR 85,UMR CNRS 7034, F-67401 Illkirch Graffenstaden, France
[2] Ecole Super Biotecnol Strasbourg, Inst Federatif Gilbert Laustriat, UPR CNRS 9050, Dept Recepteurs & Prot Membrainaires, F-67401 Illkirch Graffenstaden, France
[3] Univ Haute Alsace, IUT Mulhouse, Lab MIPS, GRP LABEL, F-68093 Mulhouse, France
关键词
D O I
10.1074/jbc.M107224200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:6645 / 6655
页数:11
相关论文
共 58 条
[1]   Neuropeptide Y family of hormones: Receptor subtypes and antagonists [J].
Balasubramaniam, A .
PEPTIDES, 1997, 18 (03) :445-457
[2]   β-Arrestins regulate interleukin-8-induced CXCR1 internalization [J].
Barlic, J ;
Khandaker, MH ;
Mahon, E ;
Andrews, J ;
DeVries, ME ;
Mitchell, GB ;
Rahimpour, R ;
Tan, CM ;
Ferguson, SSG ;
Kelvin, DJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (23) :16287-16294
[3]   Somatostatin receptor desensitization in NG108-15 cells [J].
Beaumont, V ;
Hepworth, MB ;
Luty, JS ;
Kelly, E ;
Henderson, G .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (50) :33174-33183
[4]   Y-receptor subtypes - How many more? [J].
Blomqvist, AG ;
Herzog, H .
TRENDS IN NEUROSCIENCES, 1997, 20 (07) :294-298
[5]  
Bohm SK, 1997, BIOCHEM J, V322, P1
[6]   Sequential steps in clathrin-mediated synaptic vesicle endocytosis [J].
Brodin, L ;
Löw, P ;
Shupliakov, O .
CURRENT OPINION IN NEUROBIOLOGY, 2000, 10 (03) :312-320
[7]   ADENOSINE 3',5' CYCLIC MONOPHOSPHATE ASSAY AT 10-15 MOLE LEVEL [J].
CAILLA, HL ;
RACINEWE.MS ;
DELAAGE, MA .
ANALYTICAL BIOCHEMISTRY, 1973, 56 (02) :394-407
[8]   HIGH-EFFICIENCY TRANSFORMATION OF MAMMALIAN-CELLS BY PLASMID DNA [J].
CHEN, C ;
OKAYAMA, H .
MOLECULAR AND CELLULAR BIOLOGY, 1987, 7 (08) :2745-2752
[9]   FUNCTIONAL-CHARACTERIZATION OF RECEPTORS WITH AFFINITY FOR PYY, NPY, [LEU(31),PRO(34)]NPY AND PP IN A HUMAN COLONIC EPITHELIAL-CELL LINE [J].
COX, HM ;
TOUGH, IR .
BRITISH JOURNAL OF PHARMACOLOGY, 1995, 116 (06) :2673-2678
[10]   INHIBITION OF RECEPTOR-MEDIATED BUT NOT FLUID-PHASE ENDOCYTOSIS IN POLYMORPHONUCLEAR LEUKOCYTES [J].
DAUKAS, G ;
ZIGMOND, SH .
JOURNAL OF CELL BIOLOGY, 1985, 101 (05) :1673-1679