Down-regulation of the Neurotrophin Receptor TrkB following Ligand Binding (2000)

This study examines the mechanisms by which the tyrosine kinase receptor TrkB is down-regulated following binding of brain-derived neurotrophic factor (BDNF). In primary cultures of cerebellar granule neurons, BDNF-induced reduction of TrkB receptors was largely prevented by the addition of specific proteasome inhibitors. HN10 cells, a neuronal cell line that can be readily transfected, also showed a marked down-regulation of cell surface TrkB following BDNF exposure. In addition, we observed that prolonged exposure to nerve growth factor of TrkA-transfected cells did not lead to the down-regulation seen with BDNF and TrkB. TrkA and TrkB chimeric molecules were therefore expressed in HN10 cells and tested for ligand-induced regulation. These experiments led to the conclusion that the motives responsible for down-regulation are contained in the cytoplasmic domain of TrkB, and a short sequence in the juxtamembrane domain of TrkB was identified that confers nerve growth factor-induced down-regulation when inserted into TrkA. This study examines the mechanisms by which the tyrosine kinase receptor TrkB is down-regulated following binding of brain-derived neurotrophic factor (BDNF). In primary cultures of cerebellar granule neurons, BDNF-induced reduction of TrkB receptors was largely prevented by the addition of specific proteasome inhibitors. HN10 cells, a neuronal cell line that can be readily transfected, also showed a marked down-regulation of cell surface TrkB following BDNF exposure. In addition, we observed that prolonged exposure to nerve growth factor of TrkA-transfected cells did not lead to the down-regulation seen with BDNF and TrkB. TrkA and TrkB chimeric molecules were therefore expressed in HN10 cells and tested for ligand-induced regulation. These experiments led to the conclusion that the motives responsible for down-regulation are contained in the cytoplasmic domain of TrkB, and a short sequence in the juxtamembrane domain of TrkB was identified that confers nerve growth factor-induced down-regulation when inserted into TrkA. nerve-growth factor brain-derived neurotrophic factor neurotrophin-3 neurotrophin-4/5 polyacrylamide gel electrophoresis wheat germ agglutinin N-acetyl-Leu-Leu-norleucinal N-acetyl-Leu-Leu-methional transmembrane domain juxtamembrane domain tyrosine kinase domain carboxyl-terminal domain PLC-γ, phospholipase C-γ The members of the nerve-growth factor (NGF)1 family, also designated neurotrophins, are factors that play a critical role in the development of the vertebrate nervous system (for reviews, see Refs.1.Snider W.D. Cell. 1994; 77: 627-638Abstract Full Text PDF PubMed Scopus (1314) Google Scholar, 2.Davies A.M. Philos. Trans. R. Soc. Lond-B Sci. 1996; 351: 389-394Crossref PubMed Scopus (121) Google Scholar, 3.Barde Y.A. Hefti F. Neurotrophic Factors. Springer-Verlag, Berlin1999: 1-31Google Scholar). In mammals, they comprise NGF, BDNF (brain-derived neurotrophic factor), NT3 (neurotrophin 3), and NT4/5 (neurotrophin 4/5). Their trophic actions are mainly mediated by tyrosine kinase receptors belonging to the Trk family. Whereas NGF is the ligand for the TrkA receptor, BDNF and NT4/5 are the ligands for TrkB, and NT3 is a promiscuous ligand with some selectivity for TrkC. Binding of a neurotrophin to the appropriate Trk receptor leads to dimerization and transphosphorylation of tyrosine residues in the intracellular domain of the Trk receptor and subsequent activation of cytoplasmic signaling pathways, such as the Ras/Raf/MAP kinase pathway (for review, see Ref.4.Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (545) Google Scholar). Neurotrophins and their receptors are also involved in the function of the mature central nervous system, where some of them, in particular BDNF (5.Hofer M. Pagliusi S.R. Hohn A. Leibrock J. Barde Y.-A. EMBO J. 1990; 9: 2459-2464Crossref PubMed Scopus (850) Google Scholar) and TrkB (6.Klein R. Parada L.F. Coulier F. Barbacid M. EMBO J. 1989; 8: 3701-3710Crossref PubMed Scopus (484) Google Scholar), are widely expressed. For example, neurotrophins play a role in the modulation of synaptic transmission, including neurotransmitter release, postsynaptic responses to neurotransmitters, and facilitation of long term potentiation (for reviews, see Refs. 7.Berninger B. Poo M.M. Curr. Opin. Neurobiol. 1996; 6: 324-330Crossref PubMed Scopus (160) Google Scholarand 8.Thoenen H. Science. 1995; 270: 593-598Crossref PubMed Scopus (1721) Google Scholar). These functions are reminiscent of the actions of neurotransmitters, and it is conceivable that the duration of the signal elicited by the neurotrophins, like that of neurotransmitters, may be regulated. Yet comparatively little is known on the regulation of neurotrophin receptors following ligand binding, although a few previous studies have indicated unexpected differences in the duration of signaling elicited by BDNF and NGF. Whereas both stimulate GTP binding to Ras in chick sensory neurons (9.Ng N.F.L. Shooter E.M. J. Biol. Chem. 1993; 268: 25329-25333Abstract Full Text PDF PubMed Google Scholar, 10.Carter B.D. Zirrgiebel U. Barde Y.-A. J. Biol. Chem. 1995; 270: 21751-21757Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), prolonged exposure to BDNF leads to a complete loss of Ras activation, in contrast to NGF (10.Carter B.D. Zirrgiebel U. Barde Y.-A. J. Biol. Chem. 1995; 270: 21751-21757Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In cerebellar granule cells, desensitization of Ras signaling by BDNF is accompanied by a disappearance of TrkB-binding sites (10.Carter B.D. Zirrgiebel U. Barde Y.-A. J. Biol. Chem. 1995; 270: 21751-21757Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Down-regulation of TrkB signaling upon BDNF exposure has also been reported in cultures of cortical (11.Knüsel B. Gao H. Okazaki T. Yoshida T. Mori N. Hefti F. Kaplan D.R. Neuroscience. 1997; 78: 851-862Crossref PubMed Scopus (108) Google Scholar, 12.Widmer H.R. Ohsawa F. Knüsel B. Hefti F. Brain Res. 1993; 614: 325-334Crossref PubMed Scopus (29) Google Scholar) and hippocampal neurons (12.Widmer H.R. Ohsawa F. Knüsel B. Hefti F. Brain Res. 1993; 614: 325-334Crossref PubMed Scopus (29) Google Scholar,13.Frank L. Ventimiglia R. Anderson K. Lindsay R.M. Rudge J.S. Eur. J. Neurosci. 1996; 8: 1220-1230Crossref PubMed Scopus (168) Google Scholar), reflected in either an impairment of phosphatidylinositol hydrolysis (12.Widmer H.R. Ohsawa F. Knüsel B. Hefti F. Brain Res. 1993; 614: 325-334Crossref PubMed Scopus (29) Google Scholar) or decreased phosphorylation of PLC-γ1 (11.Knüsel B. Gao H. Okazaki T. Yoshida T. Mori N. Hefti F. Kaplan D.R. Neuroscience. 1997; 78: 851-862Crossref PubMed Scopus (108) Google Scholar). Furthermore, treatment of both striatal and hippocampal cultures for 24 h with BDNF decreased c-Fos induction upon acute BDNF stimulation (13.Frank L. Ventimiglia R. Anderson K. Lindsay R.M. Rudge J.S. Eur. J. Neurosci. 1996; 8: 1220-1230Crossref PubMed Scopus (168) Google Scholar). Marked decrease of binding to radiolabeled BDNF (10.Carter B.D. Zirrgiebel U. Barde Y.-A. J. Biol. Chem. 1995; 270: 21751-21757Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,13.Frank L. Ventimiglia R. Anderson K. Lindsay R.M. Rudge J.S. Eur. J. Neurosci. 1996; 8: 1220-1230Crossref PubMed Scopus (168) Google Scholar) and/or of Trk tyrosine phosphorylation (11.Knüsel B. Gao H. Okazaki T. Yoshida T. Mori N. Hefti F. Kaplan D.R. Neuroscience. 1997; 78: 851-862Crossref PubMed Scopus (108) Google Scholar, 13.Frank L. Ventimiglia R. Anderson K. Lindsay R.M. Rudge J.S. Eur. J. Neurosci. 1996; 8: 1220-1230Crossref PubMed Scopus (168) Google Scholar) was also observed upon BDNF treatment for 2 or more days. As the mechanisms underlying the loss of response to BDNF with time are largely unknown, we investigated the regulation of the Trk receptors by their ligands, using both cultured cerebellar granule cells and the neuronal cell line HN10 (14.Lee H.J. Hammond D.N. Large T.H. Roback J.D. Sim J.A Brown D.A. Otten U.H. Wainer B.H. J. Neurosci. 1990; 10: 1779-1787Crossref PubMed Google Scholar). These cells, unlike A293 cells transfected with TrkB (10.Carter B.D. Zirrgiebel U. Barde Y.-A. J. Biol. Chem. 1995; 270: 21751-21757Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), do show a marked down-regulation of TrkB following prolonged stimulation with BDNF, and as they can be readily transfected with cDNA constructs, we could investigate the TrkB domains accounting for its down-regulation by comparison to TrkA. Recombinant human NGF, BDNF, NT3, and NT4/5 produced in Chinese hamster ovary cells or Escherichia coliwere obtained from Genentech Inc. or Regeneron/Amgen Partners. BDNF produced using the vaccinia virus system (15.Götz R. Kolbeck R. Lottspeich F. Barde Y.-A. Eur. J. Biochem. 1992; 204: 745-749Crossref PubMed Scopus (41) Google Scholar) was also used, with identical results. HN10 cells (14.Lee H.J. Hammond D.N. Large T.H. Roback J.D. Sim J.A Brown D.A. Otten U.H. Wainer B.H. J. Neurosci. 1990; 10: 1779-1787Crossref PubMed Google Scholar) were kept in culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, in an atmosphere of 5% CO2. Cerebellar granule cells were prepared from P6 rats as described (16.Zafra F. Hengerer B. Leibrock J. Thoenen H. Lindholm D. EMBO J. 1990; 9: 3545-3550Crossref PubMed Scopus (773) Google Scholar). Briefly, cerebella were dissected and incubated for 20 min at 37 °C in phosphate-buffered saline containing 10 mm glucose, 1 mg/ml albumin, 10 mg/ml DNase, and 0.5 mg/ml papain. The cells were dissociated by trituration through a plastic pipette, pelleted at 900 × g for 5 min, and resuspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The cells were plated at a density of 20 × 106 on 10-cm polyornithine-coated dishes, and the medium was changed after 12 h to a serum-free complete medium (17.Zirrgiebel U. Ohga Y. Carter B. Berninger B. Inagaki N. Thoenen H. Lindholm D. J. Neurochem. 1995; 65: 2241-2250Crossref PubMed Scopus (126) Google Scholar) with or without 20 ng/ml BDNF. Rat TrkA, TrkB, and TrkA/TrkB chimeras cDNAs, without signal peptide sequences, were cloned, using NheI and NotI into pRC CMV AC7 (pAC7), a vector that provides the signal sequence peptide of basal membrane protein 40 (18.Mayer U. Poschl E. Nischt R. Specks U. Pan T.C. Chu M.L. Timpl R. Eur. J. Biochem. 1994; 225: 573-580Crossref PubMed Scopus (41) Google Scholar). Chimeric molecules between TrkA and TrkB were constructed by polymerase chain reaction, using an overlap extension procedure. All constructs were checked by sequencing. For the construction of the chimeras, the following oligodeoxynucleotides were used: TrkA/B, GGAGAAGAAGGACGAAACACCTCTCTCGGTCTATGCTGTGGTG (fusion at TM domain) and CACCACAGCATAGACCGAGAGAGGTGTTTCGTCCTTCTTCTCC (fusion at TM domain); TrkB/A, GACCAAACCAATCGGGAGCATTTTGGGGTCTCTGTGGCTGTG (fusion at TM domain) and CACAGCCACAGAGACCCCAAAATGCTCCCGATTGGTTTGGTC (fusion at TM domain); TrkATM/B, CTCCTCCTAGTGCTCAACAAGTTGGCGAGACATTCCAA (fusion between TM and JM); TrkA/JMB, CTCCTCCTAGTGCTCAACAAGTTGGCGAGACATTCCAA (fusion between TM and JM) and AGCTCCCACTTGAGAATGATGTTGTGTCTCTTGATGTGCT (fusion between TM and JM); TrkA/TKB, GCTCCCTCTTCAGAACGATGTCCTGGCGCTTGATATG (fusion between JM and TK) and AGAACATCCACACACTCCTTCAGGCCTTGGCACAGGC (fusion between TK and CT); TrkA/CTB, ATGTGCACGCGCGGCTGCAGAACTTGGCGAAGGCG (fusion between TK and CT); TrkB/JMA, TGGTGATGCTGCTTCTGCTCAAATGTGGACAGAGGAGCAAAT (fusion between TM and JM) and GCTCCCTCTTCAGAACGATGTCCTGGCGCTTGATATG (fusion between JM and TK). All constructs were expressed in HN10 cells and tested for autophosphorylation by anti-phosphotyrosine blots. The functionality of all constructs was confirmed by assessing their neurite outgrowth promoting activity in nnr5-PC12 cells (19.Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar, 20.Green S.H. Rydel R.E. Connolly J.L. Greene L.A. J. Cell Biol. 1986; 102: 830-843Crossref PubMed Scopus (231) Google Scholar). Constructs that did not behave similarly to wild-type Trk receptors were excluded from further analysis. TrkATM/B molecules with point mutations to delete the binding sites for Shc (TrkATM/B.delShc), for PLC-γ (TrkA/TMB.delPLC), or for both Shc and PLC-γ (TrkATM/B.delShc-PLC) were also constructed by polymerase chain reaction site-directed mutagenesis. For each construct, deletion of the binding site was achieved by replacement of the tyrosine responsible for binding by a phenylalanine residue. When required, simultaneous introduction of silent mutations was also performed in order to allow for identification of positive clones by restriction analysis. Sequencing of the constructs confirmed the expected sequences. For the construction of these molecules, the following oligodeoxynucleotides were used: TrkATM/B.delShc, TGGCTGTTGGTGATACCGAAGAACTGGGGGTTTTCAATGACAG (replacement of tyrosine at position 499 by phenylalanine; simultaneously a uniqueScaI site was deleted) and CTGTCATTGAAAACCCCCAGTTCTTCGGTATCACCAACAGCCA; TrkATM/B.delPLC, GCTCTAGACTAGCCTAGGATATCCAGGAAGACGGGCGACGCCTTCGCC (replacement of tyrosine at position 794 by phenylalanine; introduction of aEcoRV site). HN10 cells were transfected using the calcium phosphate precipitation method (21.Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar) and incubated overnight at 37 °C in 3% CO2. After a medium change, they were placed in a 5% CO2 atmosphere. Five to six hours later, the transfected cells were split equally, kept in culture for at least 12 h, and then treated or not (control) with 100 ng/ml neurotrophin for 24 h. NGF and BDNF were iodinated using lactoperoxidase and 125I (Amersham Pharmacia Biotech IMS30) as described previously (22.Dechant G. Biffo S. Okazawa H. Kolbeck R. Pottgiesser J. Barde Y.-A. Development. 1993; 119: 545-558Crossref PubMed Google Scholar). Typically, 75–90% of the total radioactivity was incorporated into the protein, and radiolabeled material was used within 10 days. Transfected HN10 cells cultured for 24 h in the absence or presence of neurotrophin were washed intensively with a buffer containing 25 mmHepes, pH 7.5, 125 mm NaCl, 5 mm KCl, 5 mm glucose, 3 mm MgCl2 (KRH) and twice with an acid-wash buffer (phosphate-buffered saline containing 1 mg/ml albumin, pH 3.0) that has been shown to remove previously bound neurotrophin (data not shown). The cells were then incubated for 2 h on ice with the KRH buffer containing 1 mg/ml albumin and 0.25 nm125I-BDNF or 125I-NGF. The membrane-impermeable cross-linker bis-(sulfosuccinimidyl) sulfate (Pierce) was then added to a final concentration of 0.5 mm, and incubation was continued for 30 min. Unreacted reagent was quenched with Tris buffer (30 mm, pH 8); the plates were rinsed with Tris-buffered saline, and the cells were lysed in a buffer consisting of 50 mm Hepes, pH 7.5, 150 mm NaCl, 10% glycerol, and 1% Triton X-100. The plates were scraped and cell debris removed by a 10-min spin at 14,000 × g. Trk receptors in the lysates were immunoprecipitated with anti-Trk, a polyclonal antiserum raised to a peptide corresponding to the carboxyl terminus of the intracellular domain of TrkA that also immunoprecipitates TrkB and TrkC (23.Schröpel A. von Schack D. Dechant G. Barde Y.A. Mol. Cell. Neurosci. 1995; 6: 544-556Crossref PubMed Scopus (36) Google Scholar). The cross-linking and immunoprecipitation procedures were performed at 4 °C. After washing with a buffer containing 50 mm Hepes, pH 7.5, 150 mm NaCl, 10% glycerol and 0.1% Triton X-100, the immunoprecipitates were denatured by heating to 100 °C for 5 min in Laemmli buffer (63 mmTris, pH 6.8, 15% glycerol, 5% mercaptoethanol, 2.25% SDS, and bromphenol blue 0.1 mg/ml). Following electrophoresis (SDS-PAGE 6%), the gels were dried and detected by exposing overnight to a PhosphorImager plate. Densitometric analysis of the gels was performed using the PhosphorImager Software TINA. Cerebellar granule neurons cultured in the absence or presence of 20 ng/ml BDNF were washed twice with phosphate-buffered saline and lysed as described above. Lysates were centrifuged, and the supernatants were precipitated with WGA-Sepharose 6MB (Amersham Pharmacia Biotech). The precipitates were washed, run on 6% SDS-PAGE, transferred to Immobilon-P membranes (Millipore Corp.), and incubated with anti-TrkB serum that was raised to a peptide corresponding to amino acids 155–318 in the extracellular domain of TrkB (Transduction Laboratories) or with a pan-Trk antiserum (23.Schröpel A. von Schack D. Dechant G. Barde Y.A. Mol. Cell. Neurosci. 1995; 6: 544-556Crossref PubMed Scopus (36) Google Scholar). Detection was performed with a secondary antibody conjugated to horseradish peroxidase and an enhanced chemiluminescence substrate solution (Pierce). Cerebellar granule cells were cultured for 2 h in serum-free medium (17.Zirrgiebel U. Ohga Y. Carter B. Berninger B. Inagaki N. Thoenen H. Lindholm D. J. Neurochem. 1995; 65: 2241-2250Crossref PubMed Scopus (126) Google Scholar) containing the inhibitor at the indicated concentrations. BDNF was then added, and incubation proceeded at 37 °C for 3 h. ALLN, ALLM, and E64 were obtained from Sigma, and lactacystin was purchased from Calbiochem. The neurotrophin receptors TrkB and TrkC, but not TrkA, have been shown to be present in the developing and adult rodent cerebellum (17.Zirrgiebel U. Ohga Y. Carter B. Berninger B. Inagaki N. Thoenen H. Lindholm D. J. Neurochem. 1995; 65: 2241-2250Crossref PubMed Scopus (126) Google Scholar, 24.Ernfors P. Merlio J.-P. Persson H. Eur. J. Neurosci. 1992; 4: 1140-1158Crossref PubMed Scopus (449) Google Scholar). By Western blot analysis using dissociated P6 cerebellar granule neurons and either anti-Trk antiserum or TrkB-specific antibodies, TrkB was shown to be the predominant receptor type in this system (data not shown). As previously reported (10.Carter B.D. Zirrgiebel U. Barde Y.-A. J. Biol. Chem. 1995; 270: 21751-21757Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), we observed that pre-treatment of cerebellar granule cells with 20 ng/ml BDNF for 24 h resulted in a dramatic reduction of radiolabeled BDNF cross-linked to TrkB (data not shown). To determine if the loss of cell-surface binding was a consequence of decrease in expression levels of TrkB, Western blot analyses of total cell lysates were performed with TrkB-specific antibodies following WGA precipitation. Two major bands with molecular masses of ∼130 and ∼100 kDa were stained by the antibody (Fig.1 A). Since only the upper band reacted with antibodies raised against the carboxyl terminus of Trk (Fig. 1 B), it appears that the upper band corresponds to the full-length form of TrkB, whereas the lower band presumably represents a truncated, kinase-defective, form of TrkB (25.Klein R. Conway D. Parada L.F. Barbacid M. Cell. 1990; 61: 647-656Abstract Full Text PDF PubMed Scopus (621) Google Scholar), already described to be present in rodent cerebellum (17.Zirrgiebel U. Ohga Y. Carter B. Berninger B. Inagaki N. Thoenen H. Lindholm D. J. Neurochem. 1995; 65: 2241-2250Crossref PubMed Scopus (126) Google Scholar). Treatment of the cells with 20 ng/ml BDNF resulted in a marked reduction of the 130-kDa band, while not affecting the 100-kDa band (Fig. 1 A). This marked decrease in the levels of the upper band was also seen after precipitation with Trk-specific antibodies (data not shown) and with antibodies raised against the carboxyl terminus of the receptor in the Western blotting (Fig. 1 B). This finding suggests that the loss of BDNF-binding sites following prolonged exposure of the cells to BDNF is a consequence of down-regulation of the full-length TrkB receptor. The time course of BDNF-induced TrkB down-regulation was then analyzed (Fig. 1 B). Exposure of cells to BDNF for only 1–2 h already reduced the TrkB protein levels to about 50%, and only very low levels of TrkB protein could be detected after 3 h in the presence of BDNF. The rapid onset of down-regulation suggests that BDNF acts at the protein level, rather than reducing the transcription rates of TrkB mRNA. In view of the that TrkB is by its ligands at the protein level, we then tested of or of for their to receptor a of known to protein of the proteasome were to be in BDNF-induced down-regulation of TrkB By comparison with cells (Fig. 1 and BDNF-induced down-regulation of full-length TrkB was reduced in the presence of the proteasome at a concentration of 10 (Fig. Since can also we also tested the which has the activity as ALLN, but a lower proteasome activity C. L. K. R. L. D. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar). identical on TrkB down-regulation (Fig. a inhibitor of did not down-regulation of TrkB at a concentration of 10 (Fig. that the of is to its of this of To the of the proteasome in the down-regulation of TrkB, the specific proteasome inhibitor lactacystin G. S. Science. 1995; 268: PubMed Scopus Google Scholar) was tested as has not been to the activity of including and papain. to ALLN, lactacystin also prevented down-regulation of TrkB (Fig. The of these on TrkB down-regulation was they were used or in (Fig. 3 and BDNF-induced down-regulation of TrkB therefore to the proteasome As protein by the proteasome pathway is by the of a chain M. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google L. Cell Biol. 9: Full Text Full Text PDF PubMed Scopus Google Scholar), we for of TrkB in lysates of cerebellar granule for TrkB following BDNF could be observed in these cells used the neuronal cell line HN10 (14.Lee H.J. Hammond D.N. Large T.H. Roback J.D. Sim J.A Brown D.A. Otten U.H. Wainer B.H. J. Neurosci. 1990; 10: 1779-1787Crossref PubMed Google Scholar) to more on the of TrkB for activation of the HN10 cells were for these experiments in contrast to cerebellar granule cells, they can be Following with a wild-type TrkB radiolabeled BDNF was cross-linked to HN10 cell surface receptors (Fig. After immunoprecipitation of cell lysates with a Trk-specific a band at kDa was detected (Fig. corresponding to a cross-linked between TrkB and radiolabeled BDNF. This band was in the cells transfected with an vector (Fig. or when binding of the radiolabeled neurotrophin was for by a BDNF (Fig. from cells treated for 24 h with 100 ng/ml BDNF a dramatic decrease in receptor a loss of cell sites by BDNF treatment (Fig. Since previous experiments with primary neuronal cultures to a regulation of signaling by NGF and BDNF (10.Carter B.D. Zirrgiebel U. Barde Y.-A. J. Biol. Chem. 1995; 270: 21751-21757Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), we that TrkA and TrkB could be by their transfected HN10 cells with TrkA and cross-linked cell surface receptors with radiolabeled NGF (Fig. In contrast to cells treated with BDNF (Fig. cells their to NGF upon treatment for 24 h with 100 ng/ml NGF (Fig. appears that cell surface TrkB is down-regulated by BDNF, unlike TrkA following exposure to NGF. These experiments then that TrkB motives to down-regulation that may be in TrkA. To into accounting for the down-regulation of TrkB, we chimeric receptors between TrkA and TrkB (Fig. that were tested for regulation in HN10 tested chimeric receptors consisting of the extracellular domain of receptor to the intracellular domain of the When the intracellular domain of TrkB was by the corresponding domain of TrkA, the chimeric receptor Trk see could BDNF but was down-regulated by BDNF (Fig. 5 chimeric receptors of the extracellular domain of TrkA and the intracellular domain of TrkB bound NGF and were down-regulated following NGF treatment 3 and the intracellular domain of TrkA by that of TrkB was therefore to down-regulation upon NGF The transmembrane domain role in this receptors containing the extracellular domain of TrkA and the intracellular domain of TrkB with the transmembrane domain of either TrkA or TrkB down-regulated in response to NGF (Fig. 5 3 and 4 with 1 and 2 These show that the intracellular of TrkB a down-regulation to TrkA. Furthermore, this that the regulation for TrkA and TrkB is not to to the extracellular domain of the receptors or to the neurotrophins, such as binding or of the The cytoplasmic of TrkB down-regulation were further investigated by domains of the intracellular of TrkB into the TrkA The following TrkA chimeric receptors were constructed (Fig. TrkA/JMB, in which the juxtamembrane domain of TrkA sequence of amino acids between the transmembrane and the tyrosine kinase was by the corresponding domain of TrkA/TKB, containing the tyrosine kinase domain of TrkB amino to the juxtamembrane and the carboxyl-terminal domains of and TrkA/CTB, in which the carboxyl terminus of TrkA sequence of amino acids of the tyrosine kinase domain) was by that of TrkB. The for these molecules were transfected into HN10 cells, and the receptors were tested for down-regulation of the juxtamembrane domain of TrkA with the corresponding domain of TrkB resulted in a receptor that could be down-regulated by NGF (Fig. 1 In replacement of the tyrosine kinase domain of TrkA by that of TrkB could not the for down-regulation to TrkA (Fig. 3 Whereas in some the carboxyl-terminal domain of TrkB to a down-regulation to TrkA see 5 and the was not as indicated by the of the of experiments (Fig. B). the juxtamembrane domain of TrkB be the only accounting for the of TrkA and TrkB, it be expected that replacement of this domain in TrkB by the corresponding domain of TrkA in a TrkB that not showed ligand-induced down-regulation (Fig. 3 and This suggests that the juxtamembrane domain of TrkB is although not for receptor Since the juxtamembrane and the carboxyl-terminal domains of TrkB the binding sites for Shc and PLC-γ, we tested the activation of the signaling elicited by the phosphorylation of these could be involved in TrkB By using the TrkATM/B (Fig. a that NGF and is down-regulated by its ligand like TrkB we point mutations to the binding sites for PLC-γ, or both by 499 and by phenylalanine residues (Fig. As of binding of the Trk receptor to either PLC-γ, or to both did not with the of the receptor to domains of the Shc and of the PLC-γ signaling therefore to be responsible for TrkB The obtained with the chimeric and receptors constructed a