Crucial Role of Neuron-enriched Endosomal Protein of 21 kDa in Sorting between Degradation and Recycling of Internalized G-protein-coupled Receptors

Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway. Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway. G-protein-coupled receptors (GPCR) 1The abbreviations used are: GPCR, G-protein-coupled receptor; EE, early endosome; RE, recycling endosome; Tf, transferrin; SNARE, soluble NSF attachment protein receptors; NT, neurotensin; GFP, green fluorescent protein; PBS, phosphate-buffered saline; AS, antisense. activated by their agonists are sequestrated into intracellular compartments as ligand-receptor complexes. Complexes are then routed to early endosomes in which vesicular acidification dissociates the ligand from the receptor. At the level of early endosomes (EEs), receptors and/or ligands are sorted either to the degradation or to the recycling pathways (1von Zastrow M. Life Sci. 2003; 74: 217-224Crossref PubMed Scopus (153) Google Scholar, 2Trejo J. J. Pharmacol. Exp. Ther. 2003; 307: 437-442Crossref PubMed Scopus (100) Google Scholar). Molecular events leading to the choice of the sorting pathway are still unknown despite their importance in the regulation of cellular responses to various stimuli. The degradation pathway always uses the late endosome network to deliver receptors into lysosomes. By contrast, receptor recycling to the plasma membrane can be achieved either directly from early endosomes or through tubular vesicular recycling endosomes (REs) (3Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (553) Google Scholar). These two recycling pathways can be distinguished by their recycling rate, by their sensitivity to different drugs, and by the involvement of various small Rab GTPases. Each Rab GTPase interferes with a particular trafficking step (4Sonnichsen B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. J. Cell Biol. 2000; 149: 901-913Crossref PubMed Scopus (808) Google Scholar) and can be used as a specific endosomal marker. Recently, neuron-enriched endosomal protein of 21 kDa (NEEP21) has been shown to be involved in receptor recycling. NEEP21 is strongly expressed during neuronal maturation (5Sutcliffe J.G. Milner R.J. Shinnick T.M. Bloom F.E. Cell. 1983; 33: 671-682Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar) and is able to form a complex with the SNARE protein syntaxin 13 (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar), which is bound to EE and RE membranes. Syntaxin 13 plays an essential role in the fusion of EE vesicles (7McBride H.M. Rybin V. Murphy C. Giner A. Teasdale R. Zerial M. Cell. 1999; 98: 377-386Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar) as well as in transferrin (Tf) receptor recycling (8Prekeris R. Klumperman J. Chen Y.A. Scheller R.H. J. Cell Biol. 1998; 143: 957-971Crossref PubMed Scopus (234) Google Scholar), because antibodies blocking syntaxin 13 inhibited both events. Syntaxin 13, the Rab5 effector protein EEA1, and Rabaptin-5 are part of the multiprotein complex that is necessary for membrane fusion between various vesicles (7McBride H.M. Rybin V. Murphy C. Giner A. Teasdale R. Zerial M. Cell. 1999; 98: 377-386Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). NEEP21, as well as syntaxin 13, is localized in the membrane receptor recycling structures and particularly in the Rab4-containing vesicles (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar). Down-regulation of NEEP21 expression leads to an important delay of recycling for both the Tf and AMPA receptors (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar). Although NEEP21 has been shown to be crucial in the recycling of transferrin and glutamate receptors nothing is known about its role on the GPCR internalization/recycling processes. In the present study, we used the two receptors of the neuropeptide neurotensin (NT) belonging to the GPCR family, namely the NTSR1 and NTSR2. These two receptors can be distinguished by their different affinities for NT. Moreover, although both receptors are internalized upon NT activation, only the NTSR2 is able to recycle back to the plasma membrane (9Botto J.M. Chabry J. Sarret P. Vincent J.P. Mazella J. Biochem. Biophys. Res. Commun. 1998; 243: 585-590Crossref PubMed Scopus (56) Google Scholar, 10Martin S. Vincent J.P. Mazella J. J. Cell Sci. 2002; 115: 165-173PubMed Google Scholar), with the NTSR1 being targeted to lysosomes (11Vandenbulcke F. Nouel D. Vincent J.P. Mazella J. Beaudet A. J. Cell Sci. 2000; 113: 2963-2975Crossref PubMed Google Scholar) (for review see Ref. 12Vincent J.P. Mazella J. Kitabgi P. Trends Pharmacol. Sci. 1999; 20: 302-309Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). NTSR1 and NTSR2 constitute good models to measure the effects of variation of NEEP21 expression on the different intracellular behaviors of receptors following ligand-induced sequestration. We showed here that overexpression of NEEP21 had no influence on the internalization rates of both receptors but differentially regulated their recycling properties. Indeed, NEEP21 overexpression not only slightly accelerated NTSR2 recycling but also transformed the non-recycling NTSR1 into a recycling receptor. We also demonstrated that down-regulation of NEEP21 strongly attenuated NTSR2 recycling. Finally, we showed that endogenous NEEP21-dependent NTSR2 recycling and NEEP21-forced NTSR1 recycling take different intracellular pathways. Materials—Neurotensin was purchased from Peninsula Laboratories. [125I-Tyr3]NT was prepared and purified as described previously (13Sadoul J.L. Mazella J. Amar S. Kitabgi P. Vincent J.P. Biochem. Biophys. Res. Commum. 1984; 120: 812-819Crossref PubMed Scopus (114) Google Scholar). The pcDNA3 expression vector was from Invitrogen. Dulbecco's modified Eagle's medium was from Invitrogen, gentamycin, 1–10-phenanthroline, brefeldin A, Mowiol, and paraformaldehyde were from Sigma France, and fetal calf serum was from Roche Diagnostics. Lysotracker DS Red was from Molecular Probes. The polyclonal antibody directed against NEEP21 and monoclonal anti-EE antibody have been characterized previously (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar, 14Grussenmeyer T. Scheidtmann K.H. Hutchinson M.A. Eckhart W. Walter G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7952-7954Crossref PubMed Scopus (189) Google Scholar). Anti-NTR1, mouse monoclonal anti-actin, and the rabbit polyclonal anti-green fluorescent protein (GFP) antibodies were from Santa Cruz Biotechnology, Inc. NTR Expression—Transient transfections were performed with 1–5 μg of recombinant pcDNA3-NEEP21-EE, pcDNA3-GFP-antisense-NEEP21 (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar) or pcDNA3-NTR1, and pcDNA3-NTR2 expression vector by the DEAE-dextran precipitation method (15Cullen B.R. Methods Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (662) Google Scholar). Binding, internalization and recycling assays were performed on cells plated in 12-mm cell culture dishes ∼60 h after transfection as described previously (16Chabry J. Botto J.-M. Nouel D. Beaudet A. Vincent J.-P. Mazella J. J. Biol. Chem. 1995; 270: 2439-2442Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Membranes from non-transfected COS-7 cells were totally devoid of specific [125I-Tyr3]NT binding. Binding Studies—Binding experiments were carried out on freshly prepared cell membrane homogenates as described previously (15Cullen B.R. Methods Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (662) Google Scholar). Cell membranes (10–50 μg of protein) were incubated in 250 μl of 50 mm Tris-HCl, pH 7.5, containing 0.1% bovine serum albumin (binding buffer) with 0.4 nm [125I-Tyr3]NT (2000 Ci/mmol) and various concentrations of unlabeled NT. After 20 min at 25 °C, incubation media were filtered through cellulose acetate filters (Sartorius). Filters were rinsed twice with 3 ml of ice-cold binding buffer and counted in a Packard γ-counter (counting efficiency, 80%). Nonspecific binding was determined in the presence of 1 μm unlabeled NT and represented less than 5% of the total binding. Internalization and Recycling Studies on Whole Transfected Cells, Internalization Experiments—Internalization experiments were performed on cells plated in 12-mm well culture dishes. Briefly, cells were preincubated at 37 °C in an Earle's buffer (25 mm HEPES, pH 7.4, 140 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 3.6 mm MgCl2) for 30 min. Cells were then incubated with 0.1–0.3 nm [125I-Tyr3]NT for various times and washed twice with 0.5 ml of equilibration buffer (neutral wash) or with 0.5 ml of the same buffer (pH 4) containing 0.5 m NaCl for 3 min to remove non-sequestrated radioactivity (acid-NaCl wash). Cells were harvested with 1 ml of 0.1 n NaOH and counted in a γ-counter. Non-specific internalization was determined in the presence of 1 μm unlabeled NT. Recycling Experiments—Cells were first preincubated for 30 min at 37 °C in the absence of drug or with 1 μm wortmannin or 10 μg/ml brefeldin A in the Earle's buffer. Then the cells were incubated in the presence or in the absence of drug with 100 nm unlabeled NT for 15 min at 37 °C to induce internalization. The peptide remaining on the cell surface was removed by a series of ice-cold washes, three with the Earle's buffer, two with 150 mm NaCl, 5 mm acetic acid, and three with the Earle's buffer. Fresh Earle's buffer was added, and cells were incubated at 37 °C for various times in the presence or in the absence of drugs. The amount of cell-surface receptor was then measured in binding assays carried out with 0.5–2 nm [125I-Tyr3]NT for 30 min at 37 °C in the presence of the internalization blocker PheAsO. The nonspecific binding was determined in the presence of 1 μm unlabeled NT. There was no significant increase in cell surface binding sites for the NTR1 or the NTR2 after 30 min, indicating that de novo synthesis was negligible during the time course of the experiment. The total number of NTR1 binding sites at the cell surface, upon persistent NT stimulation, was measured as described for the human β1-adrenergic receptor down-regulation (17Liang W. Austin S. Hoang Q. Fishman P.H. J. Biol. Chem. 2003; 278: 39773-39781Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Briefly, cells transfected with NTR1 alone, with NTR1 plus NEEP21, or with NTR1 plus antisense (AS)-NEEP21 were plated in 24-well plates and exposed at 37 °C to 100 nm NT for increasing times up to 24 h. The plates were rapidly washed several times with the Earle's buffer and incubated with 0.5–2 nm [125I-Tyr3]NT for 30 min at 37 °C in the presence of the internalization blocker PheAsO. Radioactivity remaining that was associated with cells was counted as described above. Western Blot Experiments—Proteins from cells transfected with various constructs were denatured by boiling at 95 °C for 3 min using 2× or 6× Laemmli sample buffer, resolved using 10% acrylamide gels, and subsequently electroblotted onto nitrocellulose membranes. Membranes were blocked and incubated with primary antibody either overnight at 4 °C or for 2–4 h at room temperature. The bound antibody was visualized using a horseradish peroxidase-conjugated goat anti-rabbit or a horseradish peroxidase-conjugated goat anti-mouse followed by chemiluminescence reagents. Confocal Microscopy Experiments—Cells were transfected with plasmids coding either for NTR1 alone or for NTR1 and NEEP21 and grown on 12-mm coverslips. After for 10 min at 37 °C in Earle's buffer, cells were incubated with 100 nm NT at 37 °C for min in the presence of nm Lysotracker After a cells were with paraformaldehyde in phosphate-buffered for 20 min at room washed twice in PBS, then incubated with 50 mm in for 10 min to cells were by incubation in containing 10% serum and for 20 min at room then incubated with a of a rabbit polyclonal antibody in buffer containing 5% serum and for 1 h. Cells were rinsed three times in buffer and incubated with anti-rabbit antibody in containing 5% serum and for min. After two with and with were on with for cells were visualized a with a and an were both and nm were as by the of The of NTR1 localized into the was from cells for using the The to the of vesicles with the NEEP21 in COS-7 first the endogenous expression of NEEP21 in COS-7 Western of cell using the antibody a but expression of a protein of 21 kDa protein to NEEP21, because cell from COS-7 cells transfected with the showed no expression Then we the expression of NEEP21 and following transfection into COS-7 shown in homogenates from cells transfected with vector a protein of 21 kDa by the monoclonal anti-EE Western using antibodies a protein of kDa only in homogenates from cells transfected with the expression vector we these different to measure the influence of NEEP21 suppression or overexpression on trafficking of of the of NEEP21 on NTR1 and NTR2 Binding measured the influence of of plasmids coding for with either the NTR1 or the NTR2 on the NT binding properties. experiments carried out on membrane from COS-7 cells transfected with NTR1 alone or with NTR1 and NEEP21 show binding and a slightly modified to 0.5 NEEP21 is overexpression of NEEP21 does not modify the of NTR2 but changes its for NT from to nm of NTR1 and NTR2 in the absence or in the presence of overexpression of in a of the of NEEP21 on NTR1 and NTR2 of overexpression or suppression of NEEP21 on the of NTR1 and NTR2 to NT was using the of plasmids NT receptors and NEEP21, or suppression overexpression of NEEP21 had on the internalization of both receptors A and results glutamate receptor internalization (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar). The internalization rates were with with about for the NTR1 and for the we that although overexpression of NEEP21 does not modify the recycling rate of the suppression of NEEP21 expression NTR2 recycling to the cell surface overexpression of NEEP21 changes the intracellular behavior of the which able to recycle after of NEEP21 the of the of the NTR1 to recycle NEEP21 is to study its cellular after internalization. We demonstrated previously by that after min of incubation at 37 °C with NT, the internalized NTR1 was in a (11Vandenbulcke F. Nouel D. Vincent J.P. Mazella J. Beaudet A. J. Cell Sci. 2000; 113: 2963-2975Crossref PubMed Google Scholar). Confocal experiments were carried out in the present work to the NTR1 after internalization NTR1 is expressed and the influence of NEEP21 NTR1 is with in in the absence of an of NEEP21, part of the NTR1 is in lysosomes NEEP21 is although the NTR1 is in intracellular no is with the Lysotracker indicating that in this NTR1 is not routed to lysosomes. was also by The of of NTR1 into lysosomes was In cells 5% of in vesicles were with this to following overexpression of We also the of NEEP21 overexpression or NEEP21 suppression on NTR1 and NTR2 down-regulation during incubation of cells with 100 nm NT. In persistent NT the level of NTR1 on the cell surface by between 1 and 3 followed by a after 24 h to of NEEP21 cells showed the same behavior as cells By contrast, the of surface NTR1 was inhibited upon suppression of NEEP21 experiments were carried out for an increase in the amount of cell surface receptors was in cells an of the receptor persistent to NT. this we were to the of NEEP21 on of NTR2 and NTR1 the pathways of NTR2 and NTR1 we used either wortmannin to receptor recycling from early endosomes or brefeldin A to recycling through tubular vesicular that the NTR1 recycling is by brefeldin A but not by By contrast, both the recycling of NTR2 These results that at part of NTR2 does not take the same recycling pathway as that of the NTR1 recycling. Recently, (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar) that NEEP21, also (5Sutcliffe J.G. Milner R.J. Shinnick T.M. Bloom F.E. Cell. 1983; 33: 671-682Abstract Full Text PDF PubMed Scopus (118) Google Scholar), is necessary for recycling of the Tf and AMPA receptors (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar). nothing is known about its in the recycling of internalized after by their In the present by using the two NT receptors to as a of recycling and non-recycling we a crucial role for NEEP21 in the of the intracellular behavior of both NEEP21, which with syntaxin 13, has been to to on EE involved in to RE or to the plasma membrane (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar). The endosomal NEEP21 is strongly expressed in and its level of expression with that of AMPA receptors during (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar, R. 2000; PubMed Scopus Google Scholar), a role of NEEP21 during as shown in this study, NEEP21 is also expressed in COS-7 and although its expression level is the amount of protein present in COS-7 cells to a because variation of NEEP21 expression strongly Indeed, although overexpression down-regulation of NEEP21 the internalization process of NTR1 and its down-regulation strongly NTR2 whereas its overexpression the non-recycling NTR1 to to the plasma The of NEEP21 on GPCR trafficking is in with its role previously demonstrated on the of the Tf receptor and AMPA receptor recycling. The is that overexpression of NEEP21 Tf internalization (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar) but not the of both NTR1 and NTR2 in this the intracellular pathways by the NTR2 recycling or the NTR1 are Indeed, the cellular process that the recycling of the NTR2 uses both the phosphatidylinositol 3-kinase and the recycling previously described for the Tf receptor T. P. W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), the first recycling RE and involves a from EE to the plasma The pathway involves the through RE by an unknown These two pathways to the recycling for the NTR2 in several (9Botto J.M. Chabry J. Sarret P. Vincent J.P. Mazella J. Biochem. Biophys. Res. Commun. 1998; 243: 585-590Crossref PubMed Scopus (56) Google Scholar, 10Martin S. Vincent J.P. Mazella J. J. Cell Sci. 2002; 115: 165-173PubMed Google Scholar). The recycling process of the NTR2 that for the receptor-1 in that both phosphatidylinositol and pathways L. M. M. R. T. J. Cell Biol. 2002; 157: PubMed Scopus Google Scholar). the NTR2 is expressed to the plasma membrane (9Botto J.M. Chabry J. Sarret P. Vincent J.P. Mazella J. Biochem. Biophys. Res. Commun. 1998; 243: 585-590Crossref PubMed Scopus (56) Google Scholar), this process be of importance to its By contrast, with the NTR1 being strongly expressed at the cell surface, its recycling not be crucial to its Indeed, the absence of recycling for the internalized NTR1 has been (9Botto J.M. Chabry J. Sarret P. Vincent J.P. Mazella J. Biochem. Biophys. Res. Commun. 1998; 243: 585-590Crossref PubMed Scopus (56) Google Scholar, F. Nouel D. Vincent J.P. Mazella J. Beaudet A. J. Cell Sci. 2000; 113: 2963-2975Crossref PubMed Google Scholar). in the presence of an of NEEP21, the NTR1 able to recycle as rapidly as the this The first mechanism is that an of NEEP21 to an of of the receptor from EE to late endosome or from late endosome to because NTR1 is no localized in lysosomes in these of the cellular of internalized NTR1 NEEP21 is to be The be that NEEP21 is of to the recycling pathway and that its overexpression can these receptors to take this be of to NEEP21 can with the that the recycling of the NTR1 is only to brefeldin A that NEEP21 can this recycling the sorting of the receptor by lysosomes. to this the of NEEP21 be well with its in vesicles (6Steiner P. Floyd Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar) and be to the molecular events for the of receptors to the degradation these we an of of NEEP21 on the down-regulation of NT only NTR1 are on the cell surface after persistent with NT. In the NTR2 is slightly upon The of part of the NTR1 binding sites after 24 h of NT has been E. M. J.M. Scopus Google Scholar). overexpression of NEEP21 the down-regulation the of only the blocking of NEEP21 expression is able to the receptor of NEEP21 can modify NTR1 trafficking from a non-recycling to a recycling behavior but does not the amount of NTR1 upon to NT. be by an of NTR1 from the degradation pathway to the recycling pathway leading to an in the receptor be that the internalization rate in presence of a NT is that sorting of NTR1 to the recycling pathway by NEEP21 overexpression increase receptor surface the level in In NEEP21 plays a role in the of the because this process is inhibited by blocking its The that of NEEP21 strongly NTR1 during that de novo receptors also be routed by a NEEP21-dependent pathway. In NEEP21 is an endosomal protein that can on the recycling mechanism of a of membrane and receptors as well as The of this protein occurs at the level of the early endosomal pathway both in neuronal and We for in