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Recycling ability of the mouse and the human neurotensin type 2 receptors depends on a single tyrosine residue

Stéphane Martin, Jean-Pierre Vincent and Jean Mazella*

Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Unité Mixte de Recherche 6097, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France



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  		Fig. 1. Chimeric NT receptors and modeling of internalization-recycling processes. (A) Modeling of internalization-recycling processes showing the effects of drugs used in this study. (B) Putative seven-transmembrane topology of chimeric receptors indicating i3 portions of rat NTR1 (black line) and of mouse NTR2 (grey line). (C) Sequence alignment of the third intracellular loop (i3) of the rat NTR1 (rNTR1), the mouse and human NTR2 (mNTR2 and hNTR2, respectively). Invariant residues for all three NT receptors are highlighted in black. Identical amino acids of the two NTR2 are boxed. The location of residue 237 is emphasized by an asterisk. TM, transmembrane domain.

 


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  		Fig. 2. Time course of receptor recycling. After induction of receptor sequestration for 15 minutes with 100 nM NT, the peptide was removed by acid washes and the amount of cell surface receptor was determined at indicated times of incubation at 37°C as described in Materials and Methods. (A) Recycling of the wild-type (WT) rNTR1 (n=4) (closed squares), mNTR2-WT (n=6) (closed circles) and effect of the recycling inhibitor monensin on the mNTR2-WT (n=3) (open circles). (B) Recycling of the mNTR2-WT in the absence of drug (closed circles) (n=6) or in the presence of genistein (closed inverted triangles) (n=2) or brefeldin A (BFA) (open diamonds) (n=2), and of the Y237A-mNTR2 mutant (inverse open triangles) (n=4).

 


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  		Fig. 3. Effect of various drugs on the amount of internalized 125I-Tyr3-NT measured by kinetics experiments performed in COS-7 cells transfected with the wild-type mNTR2 (A,C) or the Y237A-mNTR2 mutant (B,D). In cells expressing the wild-type mNTR2 (A,C), the amount of internalized 125I-Tyr3-NT was measured in the absence of drug (closed circles) or in the presence of monensin (open circles), brefeldin A (closed inverted triangles), genistein (open inverted triangles) after acid-NaCl wash and expressed as the percent of total cell-associated ligand at each time. The residual amount of sequestrated-NT was determined in the presence of PheAsO (open diamonds) or sucrose (closed diamonds). In cells expressing the Y237A-mNTR2 mutant (B,D), the internalization of 125I-Tyr3-NT was measured in the absence of drug (closed squares) or in the presence of monensin (open squares), brefeldin A (closed triangles), genistein (open triangles). Each point represents the mean±s.e.m. of at least three different experiments determined in duplicate.

 


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  		Fig. 4. Time course of receptor phosphorylation. (A and B) The amount of 33P incorporated into proteins in response to various incubation times with 100 nM NT was quantified after immunoprecipitation of the WT HA-tagged-mNTR2 and the HA-Y237A-mNTR2 mutant. (A) Autoradiography and western Blot (WB) of phosphorylated WT HA-mNTR2 (lanes a-c) and HA-Y237A-mNTR2 mutant (lanes d-f) receptors from a representative experiment. (B) Summary of the quantification of receptor phosphorylation. Values for each receptor were expressed in arbitrary unit (basal intensity of phosphorylation in the absence of NT per pmol of receptor) and are means±s.e.m. of three independent experiments. *P<0.05. (C,D) The tyrosine phosphorylation of the WT GFP-tagged-mNTR2 and the GFP-Y237A-mNTR2 mutant upon NT treatment was assessed by phosphotyrosine immunoprecipitation. Cells transfected with the WT GFP-tagged-mNTR2 (C) or the GFP-Y237A-mNTR2 mutant (D) were incubated with 100 nM NT for indicated times in the absence or in the presence of 100 µM genistein, a tyrosine kinase inhibitor. Immunoprecipitation (IP) was performed on total cell extracts using mouse anti-phophotyrosine antibodies. Western blot (WB) were probed with rabbit anti-GFP antibodies. The western blot shown is representative of two independent experiments.

 


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  		Fig. 5. GFP-tagged mNTR2 and phosphotyrosine staining in transfected COS-7 cells. Transiently transfected COS-7 cells were either untreated (A-C) or treated with 100 nM NT at 37°C for 5 minutes (D-F) or 15 minutes (G-I) prior to fixation. GFP receptor fluorescence (A,D,G) and antiphosphotyrosine labeling revealed with Cy-5 conjugated donkey anti-mouse antibody (B,E,H) are visualized as described in Materials and Methods. Overlays of GFP-mNTR2 and phosphotyrosine fluorescent labeling are shown in C, F and I. The yellow color indicates colabeling. F' and I' are enlargements of boxes in F and I images. Arrowheads show clusters of colabeling. Bar, 10 µm.

 


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  		Fig. 6. GFP-tagged Y237A-mNTR2 and phosphotyrosine staining in transfected COS-7 cells. Transiently transfected COS-7 cells were either untreated (A-C) or treated with 100 nM NT at 37°C for 5 minutes (D-F) or 15 minutes (G-I) prior to fixation. GFP receptor fluorescence (A,D,G) and antiphosphotyrosine labeling revealed with Cy-5 conjugated donkey anti-mouse antibody (B,E,H) are visualized as described in Materials and Methods. Overlays of GFP-Y237A-mNTR2 and phosphotyrosine fluorescent labeling are shown in C, F and I. The yellow color indicates colabeling. F' and I' are enlargements of boxes in F and I images. Arrowheads show clusters of mutated receptors. Bar, 10 µm.

 


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  		Fig. 7. Time course of receptor recycling. After induction of receptor sequestration for 15 minutes with 100 nM NT, the peptide was removed by acid washes and the amount of cell surface receptor was determined after indicated times of incubation at 37°C as described in Materials and Methods. (A) Recycling of the rNTR1-WT (n=4), mNTR2-WT (n=6) and hNTR2- WT (n=4). (B) Recycling of the hNTR2-WT (n=4), 1/2-i3Nt-rNTR1 chimera (n=4), the C237Y-hNTR2 mutant (n=4) and effect of the recycling inhibitor monensin on the C237Y-hNTR2 (n=2).

 

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