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• W2993129623 abstract "Tropomyosin-receptor kinases (TRKs) are essential for the development of the nervous system. The molecular mechanism of TRKA activation by its ligand nerve growth factor (NGF) is still unsolved. Recent results indicate that at endogenous levels most of TRKA is in a monomer–dimer equilibrium and that the binding of NGF induces an increase of the dimeric and oligomeric forms of this receptor. An unsolved issue is the role of the TRKA transmembrane domain (TMD) in the dimerization of TRKA and the structural details of the TMD in the active dimer receptor. Here, we found that the TRKA–TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor, and validated this interface through site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo cross-linking, we found that the extracellular juxtamembrane region is reordered after ligand binding. Replacement of some residues in the juxtamembrane region with cysteine resulted in ligand-independent active dimers and revealed the preferred dimer interface. Moreover, insertion of leucine residues into the TMD helix induced a ligand-independent TRKA activation, suggesting that a rotation of the TMD dimers underlies NGF-induced TRKA activation. Altogether, our findings indicate that the transmembrane and juxtamembrane regions of TRKA play key roles in its dimerization and activation by NGF. Tropomyosin-receptor kinases (TRKs) are essential for the development of the nervous system. The molecular mechanism of TRKA activation by its ligand nerve growth factor (NGF) is still unsolved. Recent results indicate that at endogenous levels most of TRKA is in a monomer–dimer equilibrium and that the binding of NGF induces an increase of the dimeric and oligomeric forms of this receptor. An unsolved issue is the role of the TRKA transmembrane domain (TMD) in the dimerization of TRKA and the structural details of the TMD in the active dimer receptor. Here, we found that the TRKA–TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor, and validated this interface through site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo cross-linking, we found that the extracellular juxtamembrane region is reordered after ligand binding. Replacement of some residues in the juxtamembrane region with cysteine resulted in ligand-independent active dimers and revealed the preferred dimer interface. Moreover, insertion of leucine residues into the TMD helix induced a ligand-independent TRKA activation, suggesting that a rotation of the TMD dimers underlies NGF-induced TRKA activation. Altogether, our findings indicate that the transmembrane and juxtamembrane regions of TRKA play key roles in its dimerization and activation by NGF. Nerve growth factor (NGF) 4The abbreviations used are: NGFnerve growth factorTRKtropomyosin-receptor kinaseTMtransmembraneTMDTM domainJTMjuxtamembraneeJTMextracellular JTMRTKreceptor-tyrosine kinaseICDintracellular domainDPCdodecylphosphocholineDPRdetergent-to-protein molar ratioHSQCheteronuclear single quantum coherenceeJTMextracellular juxtamembrane regionPEIpolyethylenimineANOVAanalysis of variance. is a member of the mammalian neurotrophin protein family implicated in the maintenance and survival of the peripheral and central nervous systems (1Chao M.V. Neurotrophins and their receptors: a convergence point for many signalling pathways.Nat. Rev. Neurosci. 2003; 4 (12671646): 299-30910.1038/nrn1078Crossref PubMed Scopus (1732) Google Scholar, 2Bothwell M. NGF, BDNF, NT3, and NT4.Handb. Exp. Pharmacol. 2014; 220 (24668467): 3-1510.1007/978-3-642-45106-5_1Crossref PubMed Scopus (151) Google Scholar, 3Ceni C. Unsain N. Zeinieh M.P. Barker P.A. Neurotrophins in the regulation of cellular survival and death.Handb. Exp. Pharmacol. 2014; 220 (24668474): 193-22110.1007/978-3-642-45106-5_8Crossref PubMed Scopus (56) Google Scholar). NGF is a dimer that interacts with two distinct receptors: TRKA, a cognate member of the Trk receptor tyrosine kinase family, and the p75 neurotrophin receptor, which belongs to the tumor necrosis factor receptor superfamily of death receptors (4Friedman W.J. Greene L.A. Neurotrophin signaling via Trks and p75.Exp. Cell Res. 1999; 253 (10579918): 131-14210.1006/excr.1999.4705Crossref PubMed Scopus (312) Google Scholar, 5Bothwell M. Recent advances in understanding neurotrophin signaling.F1000Research. 2016; 5 (27540475)188510.12688/f1000research.8434.1Crossref Google Scholar, 6Vilar M. Structural characterization of the p75 neurotrophin receptor: a stranger in the TNFR superfamily.Vitam. Horm. 2017; 104 (28215307): 57-8710.1016/bs.vh.2016.10.007Crossref PubMed Scopus (19) Google Scholar). TRKA signaling is essential for sensory and sympathetic neuron survival during development (7Smeyne R.J. Klein R. Schnapp A. Long L.K. Bryant S. Lewin A. Lira S.A. Barbacid M. Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene.Nature. 1994; 368 (8145823): 246-24910.1038/368246a0Crossref PubMed Scopus (834) Google Scholar). Genetic mutations in the gene that encodes TRKA, NTRK1, cause congenital insensitivity to pain with anhidrosis (8Indo Y. Tsuruta M. Hayashida Y. Karim M.A. Ohta K. Kawano T. Mitsubuchi H. Tonoki H. Awaya Y. Matsuda I. Mutations in the TRKA–NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis.Nat. Genet. 1996; 13 (8696348): 485-48810.1038/ng0896-485Crossref PubMed Scopus (512) Google Scholar), and somatic mutations and chromosomal rearrangements generate aberrant protein fusions with constitutive kinase activation causing several types of cancer (9Martin-Zanca D. Hughes S.H. Barbacid M. A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences.Nature. 1986; 319 (2869410): 743-74810.1038/319743a0Crossref PubMed Scopus (531) Google Scholar, 10Cocco E. Scaltriti M. Drilon A. NTRK fusion-positive cancers and TRK inhibitor therapy.Nat. Rev. Clin. Oncol. 2018; 15 (30333516): 731-74710.1038/s41571-018-0113-0Crossref PubMed Scopus (694) Google Scholar). nerve growth factor tropomyosin-receptor kinase transmembrane TM domain juxtamembrane extracellular JTM receptor-tyrosine kinase intracellular domain dodecylphosphocholine detergent-to-protein molar ratio heteronuclear single quantum coherence extracellular juxtamembrane region polyethylenimine analysis of variance. Despite all these important roles, the molecular mechanisms of TRKA activation have been poorly studied compared with those of other receptor-tyrosine kinase (RTK) family members (11Endres N.F. Barros T. Cantor A.J. Kuriyan J. Emerging concepts in the regulation of the EGF receptor and other receptor tyrosine kinases.Trends Biochem. Sci. 2014; 39 (25242369): 437-44610.1016/j.tibs.2014.08.001Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141 (20602996): 1117-113410.1016/j.cell.2010.06.011Abstract Full Text Full Text PDF PubMed Scopus (3105) Google Scholar). The first three extracellular domains of TRKA consist of a leucine-rich region (Trk-d1) that is flanked by two cysteine-rich domains (Trk-d2 and Trk-d3). The fourth and fifth domains (Trk-d4 and Trk-d5) are Ig-like domains, and they are followed by a 30-residue-long linker that connects the extracellular portion of the receptor to the single transmembrane domain and a juxtamembrane intracellular region that is connected to the kinase domain. TRKA is activated by NGF a member of the neurotrophin family (3Ceni C. Unsain N. Zeinieh M.P. Barker P.A. Neurotrophins in the regulation of cellular survival and death.Handb. Exp. Pharmacol. 2014; 220 (24668474): 193-22110.1007/978-3-642-45106-5_8Crossref PubMed Scopus (56) Google Scholar). The NGF-binding domain is located in the Trk-d5(Ig2) domain (13Wiesmann C. Ultsch M.H. Bass S.H. de Vos A.M. Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TRKA receptor.Nature. 1999; 401 (10490030): 184-18810.1038/43705Crossref PubMed Scopus (313) Google Scholar), although other domains also participate in the activation by neurotrophins through an unknown mechanism (14Arevalo J.C. Conde B. Hempstead B.I. Chao M.V. Martín-Zanca D. Pérez P. A novel mutation within the extracellular domain of TRKA causes constitutive receptor activation.Oncogene. 2001; 20 (11313867): 1229-123410.1038/sj.onc.1204215Crossref PubMed Scopus (36) Google Scholar, 15Zaccaro M.C. Ivanisevic L. Perez P. Meakin S.O. Saragovi H.U. p75 Co-receptors regulate ligand-dependent and ligand-independent Trk receptor activation, in part by altering Trk docking subdomains.J. Biol. Chem. 2001; 276 (11425862): 31023-3102910.1074/jbc.M104630200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Two models for TRKA activation are postulated; a ligand-induced dimerization of TRKA monomers and a ligand-induced conformational activation of preformed inactive dimers. The first model, which is based on the crystal structure of NGF with the ligand-binding domain of TRKA (13Wiesmann C. Ultsch M.H. Bass S.H. de Vos A.M. Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TRKA receptor.Nature. 1999; 401 (10490030): 184-18810.1038/43705Crossref PubMed Scopus (313) Google Scholar, 16Wehrman T. He X. Raab B. Dukipatti A. Blau H. Garcia K.C. Structural and mechanistic insights into nerve growth factor interactions with the TRKA and p75 receptors.Neuron. 2007; 53 (17196528): 25-3810.1016/j.neuron.2006.09.034Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), assumed that the dimerization of TRKA is solely ligand-mediated and that receptor–receptor interactions are not present in the absence of its ligand. In the second model, TRKA exists as a preformed inactive dimer, suggesting receptor–receptor contacts in the absence of NGF (17Mischel P.S. Umbach J.A. Eskandari S. Smith S.G. Gundersen C.B. Zampighi G.A. Nerve growth factor signals via preexisting TRKA receptor oligomers.Biophys. J. 2002; 83 (12124278): 968-97610.1016/S0006-3495(02)75222-3Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Shen J. Maruyama I.N. Nerve growth factor receptor TRKA exists as a preformed, yet inactive, dimer in living cells.FEBS Lett. 2011; 585 (21187090): 295-29910.1016/j.febslet.2010.12.031Crossref PubMed Scopus (34) Google Scholar). The most recent data using single-particle tracking (19Marchetti L. Callegari A. Luin S. Signore G. Viegi A. Beltram F. Cattaneo A. Ligand signature in the membrane dynamics of single TRKA receptor molecules.J. Cell Sci. 2013; 126 (23886941): 4445-445610.1242/jcs.129916Crossref PubMed Scopus (35) Google Scholar) and FRET studies (20Ahmed F. Hristova K. Dimerization of the Trk receptors in the plasma membrane: effects of their cognate ligands.Biochem. J. 2018; 475 (30366959): 3669-368510.1042/BCJ20180637Crossref PubMed Scopus (17) Google Scholar) suggest that TRKA is, at endogenous levels, predominantly monomer (80%), and NGF binding induces an increase and a stabilization of the TRKA dimers and the formation of oligomers, together with a conformational change leading to kinase activation. This mechanism of activation has been called the “transition model” (20Ahmed F. Hristova K. Dimerization of the Trk receptors in the plasma membrane: effects of their cognate ligands.Biochem. J. 2018; 475 (30366959): 3669-368510.1042/BCJ20180637Crossref PubMed Scopus (17) Google Scholar) and postulates a dynamic transition from a monomer to an inactive dimer to a ligand-bound active dimer, suggesting that Trk receptors are activated through a combination of the two mentioned models. Whatever the model, it is clear that dimerization of TRKA is required for its activation. Deletion constructs suggested that dimerization of TRKA is mediated by the transmembrane (TM) and by the intracellular domains (ICDs) (20Ahmed F. Hristova K. Dimerization of the Trk receptors in the plasma membrane: effects of their cognate ligands.Biochem. J. 2018; 475 (30366959): 3669-368510.1042/BCJ20180637Crossref PubMed Scopus (17) Google Scholar). In the case of the ICDs, this is supported by the crystal structure of the kinase domain of TRKA that showed the presence of dimers in the crystallographic unit (21Artim S.C. Mendrola J.M. Lemmon M.A. Assessing the range of kinase autoinhibition mechanisms in the insulin receptor family.Biochem. J. 2012; 448 (22992069): 213-22010.1042/BJ20121365Crossref PubMed Scopus (64) Google Scholar, 22Bertrand T. Kothe M. Liu J. Dupuy A. Rak A. Berne P.F. Davis S. Gladysheva T. Valtre C. Crenne J.Y. Mathieu M. The crystal structures of TRKA and TrkB suggest key regions for achieving selective inhibition.J. Mol. Biol. 2012; 423 (22902478): 439-45310.1016/j.jmb.2012.08.002Crossref PubMed Scopus (79) Google Scholar). However, the structural determinants of the TMD dimerization are not known, and in this regard, it is important to understand the conformation of the TRKA–TMD dimer and identify the active dimer interface that may represent the functional state of the full-length receptor. In addition biochemical data supporting a conformational activation of TRKA are lacking. In the present work, we investigated the structural basis of TRKA–TMD dimerization in the activation of TRKA by NGF, using complementary structural and biochemical approaches. It has been shown that the isolated TMDs of all human RTKs form dimers in bacterial membranes (23Bocharov E.V. Bragin P.E. Pavlov K.V. Bocharova O.V. Mineev K.S. Polyansky A.A. Volynsky P.E. Efremov R.G. Arseniev A.S. The conformation of the epidermal growth factor receptor transmembrane domain dimer dynamically adapts to the local membrane environment.Biochemistry. 2017; 56 (28291355): 1697-170510.1021/acs.biochem.6b01085Crossref PubMed Scopus (28) Google Scholar). In addition functional studies indicate that TMDs play an important role as a modulator of RTK homodimerization and kinase activation (reviewed in Refs. 23Bocharov E.V. Bragin P.E. Pavlov K.V. Bocharova O.V. Mineev K.S. Polyansky A.A. Volynsky P.E. Efremov R.G. Arseniev A.S. The conformation of the epidermal growth factor receptor transmembrane domain dimer dynamically adapts to the local membrane environment.Biochemistry. 2017; 56 (28291355): 1697-170510.1021/acs.biochem.6b01085Crossref PubMed Scopus (28) Google Scholar, 24Li E. Hristova K. Role of receptor tyrosine kinase transmembrane domains in cell signaling and human pathologies.Biochemistry. 2006; 45 (16700535): 6241-625110.1021/bi060609yCrossref PubMed Scopus (180) Google Scholar, 25Li E. Hristova K. Receptor tyrosine kinase transmembrane domains: function, dimer structure and dimerization energetics.Cell Adhesion Migration. 2010; 4 (20168077): 249-25410.4161/cam.4.2.10725Crossref PubMed Scopus (73) Google Scholar). Switching between two dimerization modes of the transmembrane helix has recently been described as part of the activation mechanism of the epidermal growth factor, vascular endothelial growth factor, and fibroblast growth factor receptors (26Jura N. Endres N.F. Engel K. Deindl S. Das R. Lamers M.H. Wemmer D.E. Zhang X. Kuriyan J. Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment.Cell. 2009; 137 (19563760): 1293-130710.1016/j.cell.2009.04.025Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 27King C. Hristova K. Direct measurements of VEGF·VEGFR2 binding affinities reveal the coupling between ligand binding and receptor dimerization.J. Biol. Chem. 2019; 294 (31023826): 9064-907510.1074/jbc.RA119.007737Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 28Sarabipour S. Ballmer-Hofer K. Hristova K. VEGFR-2 conformational switch in response to ligand binding.eLife. 2016; 5 (27052508)e1387610.7554/eLife.13876Crossref PubMed Scopus (80) Google Scholar, 29Sarabipour S. Hristova K. Mechanism of FGF receptor dimerization and activation.Nat. Commun. 2016; 7 (26725515)1026210.1038/ncomms10262Crossref PubMed Scopus (155) Google Scholar). However, to date the role of TRKA–TMD dimerization in TRKA receptor activation has not been studied in detail. To obtain a structural insight into TRKA–TMD dimerization, we solved the structure of human TRKA–TMD dimers in detergent micelles using NMR (Fig. 1). For this study, the human TRKA–TMD was produced in a cell-free system (see “Experimental procedures”) as previously described (30Nadezhdin K.D. García-Carpio I. Goncharuk S.A. Mineev K.S. Arseniev A.S. Vilar M. Structural Basis of p75 Transmembrane Domain Dimerization.J. Biol. Chem. 2016; 291 (27056327): 12346-1235710.1074/jbc.M116.723585Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). When the peptide is solubilized in dodecylphosphocholine (DPC) micelles at a detergent-to-protein molar ratio (DPR) of 50:1, the TRKA–TMD is in equilibrium between monomeric, dimeric, and other oligomeric states. The ratio of these states varies as the DPR value is altered (Fig. S1A). We then titrated TRKA–TMD in DPC micelles to measure the standard free energy of dimerization (ΔG0) using standard methods (31Mineev K.S. Lesovoy D.M. Usmanova D.R. Goncharuk S.A. Shulepko M.A. Lyukmanova E.N. Kirpichnikov M.P. Bocharov E.V. Arseniev A.S. NMR-based approach to measure the free energy of transmembrane helix-helix interactions.Biochim. Biophys. Acta. 2014; 1838 (24036227): 164-17210.1016/j.bbamem.2013.08.021Crossref PubMed Scopus (33) Google Scholar) (see details under “Experimental procedures” and Fig. S1B). The ΔG0 value obtained (−1.9 ± 0.2 kcal/mol) suggested that the TRKA–TMD dimer is quite stable compared with the TMD dimers of other RTKs. Thus, although its dimerization energy is weaker than that of the vascular endothelial growth factor receptor 2 dimer (ΔG0 = −2.5 kcal/mol in DPC) (32Mineev K.S. Goncharuk S.A. Arseniev A.S. Toll-like receptor 3 transmembrane domain is able to perform various homotypic interactions: an NMR structural study.FEBS Lett. 2014; 588 (25217833): 3802-380710.1016/j.febslet.2014.08.031Crossref PubMed Scopus (21) Google Scholar), it is stronger than those of fibroblast growth factor receptor 3 (ΔG0 = −1.4 kcal/mol in DPC/SDS 9:1 mixture) and ErbB4 (ΔG0 = −1.4 kcal/mol in DMPC/DHPC 1:4 bicelles) (33Bocharov E.V. Mineev K.S. Goncharuk M.V. Arseniev A.S. Structural and thermodynamic insight into the process of “weak” dimerization of the ErbB4 transmembrane domain by solution NMR.Biochim. Biophys. Acta. 2012; 1818 (22579757): 2158-217010.1016/j.bbamem.2012.05.001Crossref PubMed Scopus (61) Google Scholar). The 15N HSQC spectrum of 15N-labeled TRKA–TMD (Fig. 1A) contained the expected number of cross-peaks, and the good quality of the spectra allowed solving of the structure of the dimer in DPC micelles (Figs. 1B, Figs. S2–S4, and Table S1). The α-helical region of the TRKA-TM dimer starts at Gly417, ends at Asn440, and is ∼38 Å in length (Fig. S2). The crossing angle of the TRKA-TM helices is 40°, and the minimal distance between two monomers is 8.8 Å (Fig. 1B and Table S1). The hydrophobicity plot and contact surface area of the dimer is shown in Fig. S4. The dimerization interface lies along the sequence motif 424LXXFAXXF431 (Fig. 1B and Fig. S4) that is conserved in the TRKA–TMD of several species and also in TrkC but not in TrkB (Fig. 1C). Although the TRKA–TMD sequence contains a putative dimerization motif of the form SXXXG (shown in blue in Fig. 1B), this motif resides in an opposite helix interface. These analyses of TRKA–TMD dimerization suggested the existence of the dimerization motif 424LXXFAXXF431 (Fig. 1B). The biological relevance of this motif can be questioned, because the presence of large extracellular and intracellular globular domains or the lipid environment of the plasma membrane may favor or hinder a specific interaction interface (34Cymer F. Veerappan A. Schneider D. Transmembrane helix-helix interactions are modulated by the sequence context and by lipid bilayer properties.Biochim. Biophys. Acta. 2012; 1818 (21827736): 963-97310.1016/j.bbamem.2011.07.035Crossref PubMed Scopus (91) Google Scholar). We therefore used different functional assays to verify the found dimer interface in the context of the full-length receptor. The state of full-length TRKA was followed by assay of three different aspects of its activity: dimerization of the receptor, phosphorylation of intracellular tyrosine residues, and neurite differentiation of PC12nnr5 cells. To investigate the dimerization of TRKA, we individually mutated most of the N-terminal residues of the rat TRKA–TMD to cysteine (Fig. 2A), expressed these constructs in HeLa cells, and then measured the amount of cross-linked species. To facilitate cross-linking via these cysteine residues in the transmembrane domains, we used oxidation with molecular iodine (I2) as previously described (35Hughson A.G. Lee G.F. Hazelbauer G.L. Analysis of protein structure in intact cells: crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor.Protein Sci. 1997; 6 (9041632): 315-322Crossref PubMed Scopus (39) Google Scholar). Such oxidation allows the formation of a disulfide bond between two close cysteine residues inside the lipid bilayer. Plasma membrane fractions from cells expressing different single-cysteine mutants were incubated in the absence or presence of NGF, together with molecular I2 and were then analyzed by nonreducing SDS-PAGE and Western immunoblotting. Upon transfection in HeLa cells, the mutants G417C and V418C formed covalent dimers in the absence of NGF (Fig. 2, B and C). NGF stimulation increased the amount of G417C and V418C dimers, and low quantities of dimers in the V420C, A421C, and V422C mutants were observed (Fig. 2, B and C). Overexpression of TRKA induces a ligand-independent activation. To see whether the constitutive dimerization of some of these mutants induces the activation of TRKA, we transfected the cysteine mutants in PC12nnr5 cells and studied the differentiation in NGF-independent manner. As we can see in Fig. 2D, only the mutant V418C is able to induce the differentiation in the absence of NGF, suggesting that V418C is part of the active dimer interface. To further study the significance of the found interfaces, we mutated the small residues Ala, Gly, and Ser within this region to the bulky Ile residue, and we then assayed TRKA activation upon NGF stimulation (Fig. 2, E and F). The rationale behind this approach was that the mutation of a small residue to a bulky one on the relevant interface would prevent the formation of the active dimeric state by inducing steric clashes and would therefore reduce TRKA activation. To perform this assay, we transfected HeLa cells that do not express endogenous TRKA with these mutants, stimulated these cells with nonsaturating concentrations (10 ng/ml) of NGF, and then assayed TRKA activation by analysis of TRKA autophosphorylation using Western blotting. Upon transfection, two TRKA electrophoretic bands are present in the TRKA immunoblots of HeLa cells: a lower band (∼110 kDa) of intracellular immature TRKA that has not completed Golgi-mediated processing of high-mannose N-glycans (36Schecterson L.C. Hudson M.P. Ko M. Philippidou P. Akmentin W. Wiley J. Rosenblum E. Chao M.V. Halegoua S. Bothwell M. Trk activation in the secretory pathway promotes Golgi fragmentation.Mol. Cell Neurosci. 2010; 43 (20123019): 403-41310.1016/j.mcn.2010.01.007Crossref PubMed Scopus (17) Google Scholar) and an upper band (∼140 kDa) with mature sugars that is expressed in the plasma membrane. Exposure to NGF substantially increased the phosphorylation of the upper TRKA band as assessed by blotting with a phospho-specific antibody against the phosphotyrosine residues of the activation loop, P-Tyr674 and P-Tyr675. This autophosphorylation was quantified to follow TRKA activation. Constitutive (t = 0, no NGF added) and ligand-dependent phosphorylation of plasma membrane-localized TRKA after 5 and 15 min were measured. Because overexpression of TRKA induces ligand-independent autophosphorylation, we first transfected the HeLa cells with increasing concentrations of TRKA to determine a TRKA level that could still be detected but that displayed no autophosphorylation in the upper band in the absence of NGF (Fig. S5). It is noteworthy that all mutants are expressed at the plasma membrane as evidenced by immunofluorescence localization in the absence of Triton X-100 using an antibody against an epitope in the TRKA N terminus (Fig. S6) and by flow cytometry (Fig. 2F). Of the seven single-point mutants tested, only the A428I substitution demonstrated a pronounced inhibitory effect on receptor autophosphorylation (Fig. 2E). Ala428 is the only small-chain residue that is found deep and in the closest position in the dimerization interface of the TRKA TMD structure determined using NMR, which supports the relevance of the obtained NMR structure. The inhibitory effect of A428 substitution on receptor activity was further enhanced when all three Ala residues that are at least somehow involved in the TMD dimerization in the NMR-based structure: Ala421, Ala425, and Ala428, were simultaneously substituted (TRKA-3A/3I). Lastly, we studied the effect of the same mutations on the NGF-induced differentiation of transfected PC12nnr5 cells (Fig. 2H). Again, the A428I mutant displayed substantial inhibition of this TRKA activity. Unexpectedly the mutation G423I did have an impact on cell differentiation (Fig. 2F). The residues Gly417, Val420, and Ala421 share the same helix interface as the LXXFAXXL motif found in the NMR structure (green and yellow, respectively, in Fig. 2G). However, the residues Val418 and Val422 are in a different interface. Interestingly the mutation V418C induces a ligand-independent differentiation of PC12nnr5 cells, indicating that this residue belongs to the active dimer interface (Fig. 2G). The combined results of the functional assays suggest a transition from an inactive to an active dimer interface and support the importance of the NMR-derived TMD structure for TRKA activation. The residue Ala428 plays a critical role in TRKA activation by NGF. Its location in the closest dimer interface suggests a pivoting role in the transition from the ligand-free to the ligand-bound dimer interface (Fig. 2G). Because activation of TRKA is a consequence of this change in the dimer interface in the next sections, we study the nature of this conformational change induced by NGF binding. Stimulation of HeLa cells transfected with TRKA-wt with NGF induces the formation of TRKA dimers that are cross-linked with BS3 (Fig. 3). Although it has been described that TRKA dimers are formed in the absence of NGF, we were not able to detect cross-linking without the ligand, even at overexpression levels, supporting that NGF binding is not only promoting a TMD dimerization but is accompanied by changes in the conformation of the extracellular part of the protein. Because BS3 reacts only with free amines (the side chains of Lys residues or a free N terminus), we searched for possible sites in TRKA that might have caused the observed cross-linking. According to the crystal structure of the TRKA–NGF complex (15Zaccaro M.C. Ivanisevic L. Perez P. Meakin S.O. Saragovi H.U. p75 Co-receptors regulate ligand-dependent and ligand-independent Trk receptor activation, in part by altering Trk docking subdomains.J. Biol. Chem. 2001; 276 (11425862): 31023-3102910.1074/jbc.M104630200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) (Fig. 3A), there are no lysine residues in the TRKA–ECD that are located in a position where cross-linking of the side chains of Lys residues of two monomers could occur. Because BS3 does not cross-the plasma membrane and because we used the full-length TRKA receptor in our assays, we wondered whether the observed BS3 cross-linking was mediated via cross-linking of Lys410 and Lys411 in the extracellular juxtamembrane region (eJTM) of TRKA (Fig. 3A) because this region is not observed in the crystal structure (16Wehrman T. He X. Raab B. Dukipatti A. Blau H. Garcia K.C. Structural and mechanistic insights into nerve growth factor interactions with the TRKA and p75 receptors.Neuron. 2007; 53 (17196528): 25-3810.1016/j.neuron.2006.09.034Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). To verify this hypothesis, we mutated both Lys410 and Lys411 to Arg and repeated the initial experiment using HEK293 cells transfected with this TRKA-KK/RR construct (Fig. 3B). No BS3-induced TRKA cross-linking was observed in the TRKA-KK/RR-transfected cells, suggesting that NGF binding brings this region of the eJTM into close proximity. We considered that if NGF indeed induces contacts between these eJTM regions, then we should be able to mimic this activity of NGF by forcing the dimerization of eJTMs in the absence of NGF. For this purpose, we individually mutated most of the residues in the eJTM of TRKA to cysteine and subsequently analyzed the dimerization of these transfected single point mutants (Fig. 4A). After transfection of HeLa cells, disulfide dimers were spontaneously formed in all constructs but the amount of dimers differed between the various mutants (Fig. 4B). The amount of dimer is significantly higher in the positions D412C and K411C. As a functional assay, we then transfected these mutants into HeLa cells, which do not express endogenous TRKA and quantified the phosphorylation of the tyrosines from the kinase activation loop (Tyr674/675) in the absence and presence of NGF (Fig. 4C). This analysis showed the presence of active dimers (D406C, K410C, and K411C) that are activated constitutively in the absence of NGF and dimers that are" @default.
• W2993129623 created "2019-12-13" @default.
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• W2993129623 date "2020-01-01" @default.
• W2993129623 modified "2023-10-15" @default.
• W2993129623 title "Structural basis of the transmembrane domain dimerization and rotation in the activation mechanism of the TRKA receptor by nerve growth factor" @default.
• W2993129623 cites W12391028 @default.
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