TrkA Amino Acids Controlling Specificity for Nerve Growth Factor
2000; Elsevier BV; Volume: 275; Issue: 11 Linguagem: Inglês
10.1074/jbc.275.11.7870
ISSN1083-351X
AutoresLori O'Connell, Jo-Anne Hongo, Leonard G. Presta, Pantelis Tsoulfas,
Tópico(s)Signaling Pathways in Disease
ResumoNeurotrophins are important for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the Trk family. The extracellular portion of the Trk receptors has been previously proposed to consist of a cysteine-rich motif, a leucine-rich motif, a second cysteine-rich motif followed by two immunoglobulin-like domains. Earlier studies have shown that a major neurotrophin-binding site in the Trk receptors resides in the second immunoglobulin-like domain. Although the individual amino acids in TrkA involved in binding to nerve growth factor (NGF) and those in TrkC involved in binding to neurotrophin-3 have been mapped in this domain, the Trk amino acids that provide specificity remained unclear. In this study, a minimum set of residues in the human TrkC second immunoglobulin-like domain, which does not bind nerve growth factor (NGF), were substituted with those from human TrkA. The resulting Trk variant recruited binding of NGF equivalent to TrkA, maintained neurotrophin-3 binding equivalent to TrkC, and also bound brain-derived neurotrophin, although with lower affinity compared with TrkB. This implies that the amino acids in the second immunoglobulin-like domain that determine Trk specificity are distinct for each Trk. Neurotrophins are important for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the Trk family. The extracellular portion of the Trk receptors has been previously proposed to consist of a cysteine-rich motif, a leucine-rich motif, a second cysteine-rich motif followed by two immunoglobulin-like domains. Earlier studies have shown that a major neurotrophin-binding site in the Trk receptors resides in the second immunoglobulin-like domain. Although the individual amino acids in TrkA involved in binding to nerve growth factor (NGF) and those in TrkC involved in binding to neurotrophin-3 have been mapped in this domain, the Trk amino acids that provide specificity remained unclear. In this study, a minimum set of residues in the human TrkC second immunoglobulin-like domain, which does not bind nerve growth factor (NGF), were substituted with those from human TrkA. The resulting Trk variant recruited binding of NGF equivalent to TrkA, maintained neurotrophin-3 binding equivalent to TrkC, and also bound brain-derived neurotrophin, although with lower affinity compared with TrkB. This implies that the amino acids in the second immunoglobulin-like domain that determine Trk specificity are distinct for each Trk. nerve growth factor neurotrophin-3 brain-derived neurotrophin monoclonal antibody enhanced green fluorescent protein phosphate-buffered saline bovine serum albumin leucine-rich motif The neurotrophins form a highly homologous family of growth factors responsible for differentiation, survival, and function of neurons sensitive to their presence (reviewed in Refs. 1.Chao M. Casaccia-Bonnefil P. Carter B. Chittka A. Kong H. Yoon S.O. Brain Res. Rev. 1998; 26: 295-301Crossref PubMed Scopus (149) Google Scholar, 2.Frade J.M. Barde Y.A. BioEssays. 1998; 20: 137-145Crossref PubMed Scopus (208) Google Scholar, 3.Lewin G.R. Barde Y.A. Annu. Rev. Neurosci. 1996; 19: 289-317Crossref PubMed Scopus (1772) Google Scholar, 4.Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (544) Google Scholar, 5.Barbacid M. Ann. N. Y. Acad. Sci. 1995; 766: 442-458Crossref PubMed Scopus (233) Google Scholar). These molecules may also play a role outside the nervous system (6.Tam S.-Y. Tsai M. Yamaguchi M. Yano K. Butterfield J.H. Galli S.J. Blood. 1997; 90: 1807-1820Crossref PubMed Google Scholar, 7.Torcia M. Bracci-Laudiero L. Lucibello M. Nencioni L. Labardi D. Rubartelli A. Cozzolino F. Aloe L. Garaci E. Cell. 1996; 85: 345-356Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The mammalian members of this family include nerve growth factor (NGF),1 brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT4/5) (reviewed in Ref. 8.Ibáñez C.F. Trends Biotechnol. 1995; 13: 217-227Abstract Full Text PDF PubMed Scopus (125) Google Scholar). Neurotrophins bind to two classes of receptors, tyrosine kinases encoded by the Trk gene family and p75NTR (1.Chao M. Casaccia-Bonnefil P. Carter B. Chittka A. Kong H. Yoon S.O. Brain Res. Rev. 1998; 26: 295-301Crossref PubMed Scopus (149) Google Scholar, 2.Frade J.M. Barde Y.A. BioEssays. 1998; 20: 137-145Crossref PubMed Scopus (208) Google Scholar, 3.Lewin G.R. Barde Y.A. Annu. Rev. Neurosci. 1996; 19: 289-317Crossref PubMed Scopus (1772) Google Scholar, 4.Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (544) Google Scholar, 9.Dechant G. Barde Y.A. Curr. Opin. Neurobiol. 1997; 7: 413-418Crossref PubMed Scopus (203) Google Scholar). Neurotrophin binding induces autophosphorylation of the Trk receptors that triggers the subsequent steps in the signal transduction cascade (reviewed in Ref. 4.Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (544) Google Scholar). Each Trk receptor can discriminate between the different neurotrophins as follows: TrkC interacts with NT-3 (10.Lamballe F. Klein R. Barbacid M. Cell. 1991; 66: 967-979Abstract Full Text PDF PubMed Scopus (913) Google Scholar), TrkB interacts primarily with BDNF and NT-4/5 (11.Berkemeier L.R. Winslow J.W. Kaplan D.R. Nikolics K. Goeddel D.V. Rosenthal A. Neuron. 1991; 7: 857-866Abstract Full Text PDF PubMed Scopus (706) Google Scholar, 12.Ip N.Y. Ibáñez C.F. Nye S.H. McClain J. Jones P.F. Gies D.R. Belluscio L. Le Beau M.M. Espinosa III, R. Squinto S.P. Persson H. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3060-3064Crossref PubMed Scopus (546) Google Scholar), and TrkA interacts primarily with NGF (13.Kaplan D.R. Hempstead B.L. Martin-Zanca D. Chao M.V. Parada L.F. Science. 1991; 252: 554-558Crossref PubMed Scopus (1135) Google Scholar), although TrkB and TrkA can bind NT-3 (12.Ip N.Y. Ibáñez C.F. Nye S.H. McClain J. Jones P.F. Gies D.R. Belluscio L. Le Beau M.M. Espinosa III, R. Squinto S.P. Persson H. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3060-3064Crossref PubMed Scopus (546) Google Scholar, 14.Belliveau D.J. Krivko I. Kohn J. Lachance C. Pozniak C. Rusakov D. Kaplan D. Miller F.D. J. Cell Biol. 1997; 136: 375-388Crossref PubMed Scopus (154) Google Scholar).The domain organization of the extracellular portion of the Trk receptors has been proposed based on sequence information (15.Schneider R. Schweiger M. Oncogene. 1991; 6: 1807-1811PubMed Google Scholar). According to this proposal, the extracellular portion of the Trk receptors is comprised of a cysteine cluster, a leucine-rich motif, a second cysteine cluster followed by two immunoglobulin-like domains. Previous studies have shown that the second immunoglobulin-like domain of the Trk receptors is involved in binding to their respective neurotrophins. Deletion of the second immunoglobulin-like domain in TrkA (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar, 17.MacDonald J.I.S Meakin S.O. Mol. Cell. Neurosci. 1996; 7: 371-380Crossref PubMed Scopus (39) Google Scholar) and TrkC (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar) abrogates neurotrophin binding. Exchanging the second immunoglobulin-like domain from TrkA or TrkB into TrkC resulted in high affinity NGF and BDNF binding, respectively (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar), and exchanging the two immunoglobulin-like domains from TrkA into TrkB also transferred NGF binding (18.Pérez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar). In cells transfected with receptors comprising only the two immunoglobulin-like domains of TrkA (17.MacDonald J.I.S Meakin S.O. Mol. Cell. Neurosci. 1996; 7: 371-380Crossref PubMed Scopus (39) Google Scholar) or TrkC (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar) or only the second immunoglobulin-like domain of TrkC (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar), these truncated receptors bound neurotrophin and exhibited autophosphorylation. In addition, a fragment of TrkA comprising the two immunoglobulin-like domains has been shown to bind NGF and inhibit neurite outgrowth (19.Holden P.H. Asopa V. Robertson A.G.S. Clarke A.R. Tyler S. Bennett G.S. Brain S.D. Wilcock G.K. Allen S.J. Smith S.K.F. Dawbarn D. Nat. Biotechnol. 1997; 15: 668-672Crossref PubMed Scopus (48) Google Scholar, 20.Tannahill L. Klein R. Schachner M. Eur. J. Neurosci. 1995; 7: 1424-1428Crossref PubMed Scopus (18) Google Scholar).A second neurotrophin-binding site on Trk receptors, the leucine-rich motif (LRM) domain, has also been implicated in binding neurotrophins (21.Windisch J.M. Marksteiner R. Lang M.E. Auer B. Schneider R. Biochemistry. 1995; 34: 11256-11263Crossref PubMed Scopus (61) Google Scholar, 22.Windisch J.M. Marksteiner R. Schneider R. J. Biol. Chem. 1995; 270: 28133-28138Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23.Haniu M. Montestruque Bures E.J. Talvenheimo J. Toso R. Lewis-Sandy S. Welcher A.A. Rohde M.F. J. Biol. Chem. 1997; 272: 25296-25303Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 24.Windisch J.M. Auer B. Marksteiner R. Lang M.E. Schneider R. FEBS Lett. 1995; 374: 125-129Crossref PubMed Scopus (32) Google Scholar, 25.Ninkina N. Grashchuck M. Buchman V.E. Davies A.M. J. Biol. Chem. 1997; 272: 13019-13025Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Peptides corresponding to segments of the LRM domain can bind to neurotrophins and inhibit neurotrophin binding to Trk receptors, although only with a K i value in the micromolar range (24.Windisch J.M. Auer B. Marksteiner R. Lang M.E. Schneider R. FEBS Lett. 1995; 374: 125-129Crossref PubMed Scopus (32) Google Scholar). In contrast to the studies showing that transferring the second immunoglobulin-like domain among Trk receptors also transfers neurotrophin binding specificity (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar, 18.Pérez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar), no BDNF binding was observed when the TrkB LRM domain was substituted into TrkC (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar) nor was NGF binding recruited when the TrkA LRM domain was substituted into TrkC (26.Urfer R. Tsoulfas P. O'Connell L. Hongo J.-A. Zhao W. Presta L.G. J. Biol. Chem. 1998; 273: 5829-5840Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) or TrkB (18.Pérez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar). Although the role of the LRM domain in binding neurotrophins remains unclear, the function of this domain was suggested in a study in which cells were transfected with TrkA receptors in which the LRM domain was deleted. These cells bound NGF, showed autophosphorylation of the Trk receptor, and activation of the Shc-dependent Ras pathway, but they failed to fasciculate and showed delayed aborization (17.MacDonald J.I.S Meakin S.O. Mol. Cell. Neurosci. 1996; 7: 371-380Crossref PubMed Scopus (39) Google Scholar).The amino acids in the second immunoglobulin-like domain of TrkA involved in binding NGF and those in TrkC involved in binding NT-3 have been previously mapped (26.Urfer R. Tsoulfas P. O'Connell L. Hongo J.-A. Zhao W. Presta L.G. J. Biol. Chem. 1998; 273: 5829-5840Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), although the amino acids that controlled specificity were not elucidated. In order to determine the residues that control specificity of TrkA for NGF, a TrkC-based variant was generated by replacing TrkC residues with the minimum set of TrkA residues necessary to recruit NGF binding equivalent to native TrkA. Unexpectedly, this TrkC-based variant maintained NT-3 binding equivalent to native TrkC and also bound BDNF, although less well than native TrkB. This implies that the amino acids in the Trk receptors that determine specificity for their respective neurotrophins occupy distinct, separate positions in the second immunoglobulin-like domain sequence.RESULTSA previously described TrkA/TrkC chimera, S5A (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar), was used as the initial template to generate the variants in this study; S5A has TrkC domain 5 (second immunoglobulin-like domain) exchanged with that from TrkA (i.e. domains 1–4 from TrkC and domain 5 from TrkA). S5A bound NGF approximately 6-fold reduced compared with full-length TrkA (EC50 S5A/EC50 TrkA = 6.22 ± 1.7, n = 8). This contrasts to equivalent binding of S5A and TrkA found in a previous study (16.Urfer R. Tsoulfas P. O'Connell L. Shelton D.L. Parada L.F. Presta L.G. EMBO J. 1995; 14: 2795-2805Crossref PubMed Scopus (127) Google Scholar); however, the studies differ in the assay used to measure NGF binding.Initially, groups of residues in S5A domain 5 that differed from human TrkC were exchanged for their TrkC counterparts. For each group exchanged, the new variant was evaluated for NGF binding. Exchanging amino acids at positions 305, 306, 308, 312, 313, 328, 330, 332, 337, 364, and 365 (residue numbers in the text refer to human TrkC as noted in Fig. 1) did not affect NGF binding (data not shown). In addition, when all of these positions were simultaneously changed to the TrkC sequence (variant C13), NGF binding remained equivalent to wild type TrkA (Table I and Fig.2 A). Variant C13 functioned as the template for a “TrkA-to-TrkC” scan, i.e. all residues in C13 domain 5 that differed between TrkA and TrkC were individually converted from TrkA to TrkC.Table IBinding of C13-based variantsEC50 C13bEC50 TrkA 207.5 ± 56.4 ng/ml (∼2.1 nm), n = 20. EC50 C13 217.3 ± 89.0 ng/ml, n = 20.VariantaC13 was used as template for all variants (see Fig.1). TrkA numbering is in parentheses. Residue numbers in this study can be converted to residue numbers in Ref. 26 by subtracting 31 from TrkC residue numbers and 33 from TrkA residue numbers.EC50variantTrkAcEC50 variant/EC50 TrkA alanine-scan mutants from Ref. 26.MeanS.D.nTrkA1.020.2824His311 (291) → Glu0.930.02212Val314 (294) → Glu0.250.01214Glu315 (295) → Leu0.550.1121Met316 (296) → Arg0.250.0227His317 (297) → Leu0.270.01215His318 (298) → Glu0.370.0221Pro322 (302) → Glu0.190.064>100Ser324 (304) → Val0.420.02211Asp326 (306) → Arg0.480.0225Arg334 (314) → His0.890.0423His360 (343) → Glu0.570.02279Gln367 (350) → Lys1.390.1426Val371 (354) → Tyr1.390.08224Leu379 (362) → Ile0.830.04224eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Ala381 (364) → Lys0.720.0422Phe384 (367) → Leu0.810.072NEdNE, not evaluated.Gln386 (369) → Thr1.040.1722eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Ser388(371) → Asn1.190.0822eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Ala389 (372) → Gln1.180.102NESer390 (373) → Thr0.860.09248eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Met392 (375) → Asn0.900.07448eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Ala393(376) → Gly0.840.144NEAla394 (377) → His1.730.3541Met396 (379) → Leu0.290.044NEAsp397 (380) → Lys0.580.2441eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Asn398 (381) → Glu0.820.2041eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Glu401(384) → Pro1.180.2345eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Phe402 (385) → Val0.320.114NEAsn403 (386) → Asp0.900.0845eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Pro404 (387) → Glu0.960.124NEGlu405 (388) → Val0.570.06418eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Asp406 (389) → Ser0.620.14418eThe double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A.Ile408(391) → Thr0.720.0428a C13 was used as template for all variants (see Fig.1). TrkA numbering is in parentheses. Residue numbers in this study can be converted to residue numbers in Ref. 26.Urfer R. Tsoulfas P. O'Connell L. Hongo J.-A. Zhao W. Presta L.G. J. Biol. Chem. 1998; 273: 5829-5840Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar by subtracting 31 from TrkC residue numbers and 33 from TrkA residue numbers.b EC50 TrkA 207.5 ± 56.4 ng/ml (∼2.1 nm), n = 20. EC50 C13 217.3 ± 89.0 ng/ml, n = 20.c EC50 variant/EC50 TrkA alanine-scan mutants from Ref. 26.Urfer R. Tsoulfas P. O'Connell L. Hongo J.-A. Zhao W. Presta L.G. J. Biol. Chem. 1998; 273: 5829-5840Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar.d NE, not evaluated.e The double mutants used are as follows: T377A/L379A, Q386A/S388A, S390T/M392N, D397A/N398A, E401A/N403A, and E405A/D406A. Open table in a new tab Figure 2Binding of NGF and NT-3 to wild type and variant TrkA and TrkC. A, binding of human NGF to TrkA (open square, large dashed line), TrkC (open triangle), variant C13 (filled circle, solid line), and variant CR1 (filled square, small dashed line).B, binding of human NT-3 to TrkA (open square), TrkC (open triangle, solid line), variant C13 (filled circle, large dashed line), and variant CR1 (filled square, small dashed line).View Large Image Figure ViewerDownload Hi-res image Download (PPT)All five residues in loop AB (Val314-His318) were required to be the TrkA amino acid in order to retain NGF binding equivalent to C13; individually changing these residues to their TrkC counterpart reduced NGF binding by 2–4-fold (Table I). Other residues that affected binding when changed to their TrkC counterparts were Pro322, Ser324, Asp326, His360, Met396, Asp397, Phe402, Glu405, and Asp406 (TableI). Note that no individual changes were made in the segment connecting β-strands C and E (TrkC residues 340–360). The crystal structures of domain 5 of TrkA and TrkC (30.Ultsch M.H. Wiesmann C. Simmons L.C. Heinrich J. Yang M. Reilly D. Bass S.H. de Vos A.M. J. Mol. Biol. 1999; 290: 149-159Crossref PubMed Scopus (140) Google Scholar) show that this segment differs between TrkC and TrkA not only in the nature of the amino acids but also in its size and conformation; therefore, changing residues in this segment in a one-to-one correspondence was not possible.Based on the results in Table I, all TrkA residues that did not have an effect on binding were simultaneously changed from TrkA to TrkC sequence, except at position 371. The resulting variant CR1 bound NGF 2.5-fold better than C13 (Fig. 1 and TableII). Position 371 is part of domain 5 loop EF, and most of this loop is conserved among TrkA, TrkB, and TrkC, although position 371 varies. Since it had been previously shown that loop EF plays a major role in neurotrophin binding to TrkA and TrkC and that Val371 was a major determinant in NGF binding to TrkA (26.Urfer R. Tsoulfas P. O'Connell L. Hongo J.-A. Zhao W. Presta L.G. J. Biol. Chem. 1998; 273: 5829-5840Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), Val371 was retained in further variants.Table IIBinding of NGF and NT-3 to TrkC variantsNGFNT-3EC50 C13EC50 TrkCEC50 VariantEC50 VariantMeanS.D.nMeanS.D.nC131.00aAt least two independent protein lots were evaluated for each variant. EC50 TrkA/NGF 207.5 ± 56.4 ng/ml (∼2.1 nm), n = 20. EC50 TrkC/NT3 61.1 ± 22.5 ng/ml (∼0.63 nm), n = 20.0.75bFull titration curves for these receptors could not be obtained since NT-3 saturation was not achieved even at the highest receptor concentration used. The values are the ratio of C13/TrkC and TrkA/TrkC for NT-3 binding at receptor concentration of 1 μg/ml.0.038CR12.500.7661.600.6310CR20.270.0221.650.688CR30.680.0840.470.248CR41.340.0821.490.528CR1A1.570.7661.310.444C13A0.820.288TrkA1.030.26200.11bFull titration curves for these receptors could not be obtained since NT-3 saturation was not achieved even at the highest receptor concentration used. The values are the ratio of C13/TrkC and TrkA/TrkC for NT-3 binding at receptor concentration of 1 μg/ml.0.034a At least two independent protein lots were evaluated for each variant. EC50 TrkA/NGF 207.5 ± 56.4 ng/ml (∼2.1 nm), n = 20. EC50 TrkC/NT3 61.1 ± 22.5 ng/ml (∼0.63 nm), n = 20.b Full titration curves for these receptors could not be obtained since NT-3 saturation was not achieved even at the highest receptor concentration used. The values are the ratio of C13/TrkC and TrkA/TrkC for NT-3 binding at receptor concentration of 1 μg/ml. Open table in a new tab Two sections that differ in length and sequence between TrkA and TrkC are segment C-E and the C-terminal half (i.e. residues 408–419) of the juxtamembrane segment connecting the end of domain 5 with the transmembrane segment (Fig. 1). When segment C-E was changed from TrkA to TrkC sequence, NGF binding was reduced 9-fold (CR2, TableII) and when the C-terminal half of the juxtamembrane segment was changed to TrkC sequence, NGF binding was reduced by almost 4-fold (CR3, Table II). The role of the amino acid at position 319 was also evaluated; when changed from TrkA Trp319 to TrkC His319, NGF binding was reduced 2-fold (CR4, Table II). In the Trk domain 5, crystal structures residue 319 is buried between loop AB and the N-terminal portion of the juxtamembrane segment (Fig.3) (30.Ultsch M.H. Wiesmann C. Simmons L.C. Heinrich J. Yang M. Reilly D. Bass S.H. de Vos A.M. J. Mol. Biol. 1999; 290: 149-159Crossref PubMed Scopus (140) Google Scholar).Figure 3Stereo ribbon diagram of NGF/TrkA domain 5 crystal structure (36.Wiesmann C. Ultsch M.H. Bass S.H. de Vos A.M. Nature. 1999; 401: 184-188Crossref PubMed Scopus (309) Google Scholar). NGF is shown in gray; TrkA domain 5 in brown. Residues that were retained as TrkA in variant CR1 are shown in green or in yellow(segment C-E residues). Residues that differed between CR1 and C13 are shown in cyan. Side chain nitrogen atoms areblue, and side chain oxygen atoms are red.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The variant with optimal binding, CR1, bound NGF better than native TrkA (Table II and Fig. 2 A). This variant included the entire TrkA segment C-E, most of TrkA loop AB, and the entire TrkA C-terminal half of the juxtamembrane segment (residues 408–419). Additional residues required were as follows: three in β-strand B (Pro322, Ser324, and Asp326), one in loop EF (Val371), and three in the N-terminal half of the juxtamembrane segment (Met396, Asp397, and Phe402). Comparing the sequence of CR1 with that of TrkC, a total of 37 TrkC residues was replaced with TrkA sequence (33%) in domain 5, of which almost half (17.MacDonald J.I.S Meakin S.O. Mol. Cell. Neurosci. 1996; 7: 371-380Crossref PubMed Scopus (39) Google Scholar) were in segment C-E.Unexpectedly, variant CR1 maintained binding to NT-3 that was equivalent to native TrkC (Table II and Fig. 2 B). Variant C13, however, showed reduced NT-3 binding compared with native TrkC (Table II and Fig. 2 B). Replacing the TrkA sequence with TrkC sequence at residue 319 (CR4) or in segment C-E (CR2) did not improve NT-3 binding (Table II). In contrast, changing the juxtamembrane segment to TrkC reduced NT-3 binding (CR3).Domain 5 from C13 and CR1 were substituted for domain 5 in native TrkA (variants C13A and CR1A; Table II). Having domains 1–4 from TrkA (instead of TrkC) did not improve binding or specificity. Hence the amino acids most important for NGF and NT-3 binding and specificity seem to reside in domain 5, in agreement with previous studies (18.Pérez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar, 19.Holden P.H. Asopa V. Robertson A.G.S. Clarke A.R. Tyler S. Bennett G.S. Brain S.D. Wilcock G.K. Allen S.J. Smith S.K.F. Dawbarn D. Nat. Biotechnol. 1997; 15: 668-672Crossref PubMed Scopus (48) Google Scholar, 20.Tannahill L. Klein R. Schachner M. Eur. J. Neurosci. 1995; 7: 1424-1428Crossref PubMed Scopus (18) Google Scholar). If amino acids outside of domain 5 are important for binding and/or specificity, they must have a minimal effect.The ability of C13 and CR1 to bind NGF and NT-3 and elicit a biological response was evaluated by fusing the extracellular domains of these variants to the transmembrane/intracellular domain of TrkA. After generation of stable, transfected NIH3T3 cells expressing the chimeric proteins, the cells were evaluated for protein expression level (Fig.4). Three cell lines of each variant were then pulsed for 5 min with NGF or NT-3, followed by lysis, immunoprecipitation, and detection of tyrosine phosphorylation of the intracellular domain (Fig. 5). All three C13 cell lines expressed equivalent amounts of protein (Fig. 4) and exhibited NGF-induced phosphorylation levels similar to TrkA (Fig.5 A). NT-3-induced phosphorylation of C13 was reduced compared with NGF but was more pronounced than that of TrkA (Fig.5 A). For the three CR1 cell lines, different levels of expression were found (Fig. 4). As with C13, CR1 cells showed tyrosine phosphorylation induced by both NGF and NT-3 and the higher expressing cell line exhibited the most phosphorylation (Fig. 5 B). A cell line expressing TrkC responded to NT-3 but not NGF, as expected (Fig. 5 C).Figure 4Expression of TrkA, variant C13, and variant CR1 on NIH3T3 cells. The expression of receptors is shown after purification with wheat germ agglutinin and immunoprecipitation with the pan-Trk 45 antibody. Three independent cell lines expressing variant C13 and three expressing variant CR1 are shown, along with a cell line expressing wild type TrkA. Each of the lanes represents the same amount of total protein and the same exposure to film (15 min); hence, the intensity of the signal is a measure of the relative expression levels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5NGF - and NT-3-induced autophosphorylation of TrkA, TrkC, and TrkC-based variant receptors. NIH3T3 cell lines expressing TrkA, TrkC, variant C13, or variant CR1 were exposed for 5–10 min to 100 ng/ml human NGF, 100 ng/ml human NT-3, or no neurotrophin (−). After cell lysis, receptors were immunoprecipitated with pan-Trk antibody 45 or Trk C-14, Western-blotted, and bands stained with anti-phosphotyrosine antibody 4G10. The gels were exposed to film for 3 min. A, autophosphorylation of TrkA wild type (wt-5) and three cell lines expressing variant C13 (C13-1, C13-4, and C13-9). B,autophosphorylation of three cell lines expressing variant CR1 (CR1-5, CR1-8, and CR1-11). C,autophosphorylation of TrkC wild type (wt-18).View Large Image Figure ViewerDownload Hi-res image Download (PPT)One cell line from the C13 and CR1 variants was chosen to evaluate dose dependence. The C13–9 cell line showed dose dependence for both NGF and NT-3 with NGF eliciting a stronger response at a given neurotrophin concentration (e.g. compare 10 ng/ml neurotrophin in Fig.6 A). In contrast, BDNF at 200 ng/ml did not elicit phosphorylation of C13-9 (Fig. 6 A). Cell line CR1-5 also showed a dose dependence for NGF and NT-3 (Fig.6 B). However, in contrast to C13, the CR1-5 cell line also responded to BDNF, although a higher concentration of neurotrophin was required to elicit the same level of phosphorylation (Fig.6 C).Figure 6Dose response of NGF - and NT-3-induced autophosphorylation. NIH3T3 cell lines expressing wild type or variant Trk receptors were exposed for 5–10 min with the neurotrophin concentration noted. After cell lysis, receptors were immunoprecipitated with pan-Trk antibody 45 or Trk C-14, Western-blotted, and bands stained with anti-phosphotyrosine antibody 4G10. The gels were exposed to film for 1 min. A, NGF
Referência(s)