Activation of tyrosine kinase receptors is associated with human tumors. Tumorigenic versions of several RTKs, such as Ret, Kit and Met carry activating mutations at highly conserved residues of the tyrosine kinase domain. We have investigated the effect of some of these mutations on the NTRK1/NGF receptor, for which no naturally occurring activating point mutations have been so far detected. We introduced the following mutations in NTRK1 tyrosine kinase domain: (i) D668N equivalent to Met D1246N associated to HPRC; (ii) D668V modelled on Kit D816V found in mastocytosis; (iii) M688T corresponding to Ret M918T associated to the cancer syndrome MEN2B. The Met-like mutation rendered the NTRK1 receptor more responsive to ligand, as observed for the corresponding mutation in Met. On the contrary the Kit-like D668V resulted as neutral mutation. Surprisingly, the MEN2B-like M688T completely abrogated NTRK1 receptor activity, resulting as a loss of function mutation. Our results show that the mutations tested, although involving conserved amino acids in highly homologous regions, exert distinct effects in different receptors, and suggest a very peculiar auto-inhibitory mechanism for NTRK1.
Receptor tyrosine kinases (RTKs) are critical components of signalling pathways and control cellular proliferation and differentiation; their activity in resting untransformed cells is tightly controlled. Relief or perturbation of one or several of the auto-control mechanisms of RTKs can result in their oncogenic activation or pathogenic inactivation. Consistently, gain or loss of function mutations of several RTKs have been found associated with malignancies or developmental abnormalities.
NTRK1 (also known as TrkA) is one of the receptors for the Nerve Growth Factor (NGF). Binding to NGF results in phosphorylation of five different tyrosine residues in the intracellular domain of NTRK1 (Y490, Y670, Y674, Y675 and Y785). Y490 and Y785 provide docking sites for Shc, FRS2 and PLCγ, whereas Y670, Y674 and Y675 are essential for kinase activity (Kaplan and Miller, 2000).
Deregulation of NTRK1 activity is associated with several human diseases. Missense mutations causing inactivation of the receptor are associated with the genetic disorder Congenital Insensitivity to Pain with Anhidrosis (CIPA) (Indo, 2001). Conversely, constitutive activation of NTRK1, caused by autocrine and paracrine loops, has been shown in breast, pancreatic and prostate cancer (Descamps et al., 1998; Miknyoczki et al., 1999; Weeraratna et al., 2001). Ligand-independent NTRK1 activation, due to an intragenic deletion, has been reported in a case of acute myeloid leukemia (Reuther et al., 2000). Finally, in a consistent fraction of human papillary thryoid carcinoma NTRK1 is involved in chromosomal rearrangements producing chimeric oncogenes with constitutive tyrosine kinase activity (Pierotti et al., 1996). Despite the evidence that NTRK1 can be activated as an oncogene, so far no NTRK1 missense mutations leading to ligand-independent activation have been reported.
The tyrosine kinase (TK) domain is highly conserved among different receptors (Hanks et al., 1988). Mutations occurring at conserved residues have been found in the tumorigenic version of several receptors, such as Kit, Met and Ret. In Kit, the conserved residue D816, located in the DFGXXRD816 motif in the activation loop, is mutated into V in human mastocytosis, causing constitutive activation of the receptor (Nagata et al., 1995; Furitsu et al., 1993). The equivalent Met D1246 is mutated into N in human papillary renal carcinomas (HPRC) and renders the receptor more responsive to HGF (Schmidt et al., 1997; Maritano et al., 2000). In Ret M918, located in the P+1 loop, is mutated into T in the Multiple Endocrine Neoplasia (MEN) 2B tumor syndrome and causes shift of substrata (van Heyningen, 1994; Songyang et al., 1995). The same M to T substitution at the homologous residue induces activation of the Met receptor in HPRC (Schmidt et al., 1997). Moreover, mutations modelled on Kit and Ret cause activation of the Ron receptor (Santoro et al., 1998). Thus, the above reported residues appear to represent hot spots for activating mutations in RTKs.
We were interested in finding out whether this ‘hot spot’ concept applies also to the NTRK1 receptor, with the ultimate goal of identifying mutations capable of releasing the NTRK1 oncogenic potential. We inserted single amino acid substitutions in the NTRK1 TK domain, reproducing the following oncogenic mutations: the Met D1246N, associated with HPRC; the Kit D816V, associated with human mastocytosis; and the Ret M918T, associated with the MEN2B syndome. Met D1246 and Kit D816 are equivalent to D668 in NTRK1; Ret M918 corresponds to M688 in NTRK1 (Figure 1). The resulting NTRK1 mutated constructs, named NTRK1/D668N, NTRK1/D668V and NTRK1/M688T, respectively, were transiently transfected into COS1 cells. Two days after transfection cells were treated or not with NGF for 10 min, and then subjected to protein extraction. After immunoprecipitation with MGR12 antibodies, specific for NTRK1 extracellular portion (Tagliabue et al., 1999), Western blot analysis was performed. Hybridization with anti-TRK antibodies showed that the mutated proteins were properly expressed and processed, producing the expected 110 and 140 kDa receptor isoforms (Figure 2a). Hybridization with anti-phosphotyrosine antibodies detected a basal phosphorylation of the unstimulated NTRK1 D668N receptor, further increased by NGF treatment. The D668V receptor was phosphorylated only upon NGF stimulation, similarly to the wild type. The most intriguing result was obtained with the NTRK1 M688T mutant, equivalent to Ret MEN2B mutation: no phosphorylation was observed upon NGF treatment, suggesting that the mutation abrogates the kinase activity of the receptor. The inactivating effect of the M688T mutation was confirmed also by transient expression of several NTRK1 M688T clones derived from independent mutagenesis (data not shown) and by immunokinase assay (data not shown). Moreover, the MEN2B-like mutation was introduced into the corresponding residue of the thyroid TRK-T3 oncogene which has constitutive tyrosine kinase activity (Greco et al., 1995). Transient expression of the T3/M688T construct into COS1 cells showed that the mutation abrogates the constitutive phosphorylation of the oncogene (Figure 2b).
To further characterize the effect of D668N mutation on the kinase activity of NTRK1, we constructed a deletion mutant carrying only the intracellular portion of NTRK1 receptor (aa 480–790) and we introduced either the D668N or D668V mutations, in order to exclude any contribution of the extracellular portion. The cyto-TRK constructs were transiently transfected into 293T cells. As control we used the TRK-T3 oncogene. Cell extracts were immunoprecipitated with anti-TRK antibodies and subjected to immunokinase assay. As shown in Figure 2c, upper panel, cyto-TRK WT protein (39 kDa) showed an autokinase activity above the background. The D668N cyto-TRK mutant resulted in increased kinase activity compared to the wild type, although to a much lower level than the activity of TRK-T3. The D668V mutation did not have any significant effect on cyto-TRK activity compared to wild type. This result is in agreement with what we have previously shown on the full size receptor and indicates that the D668N mutation increases the kinase activity of NTRK1, independently from its extracellular portion.
To test for transforming activity wild type and mutant NTRK1 constructs were transfected into NIH3T3 cells (Figure 3); foci selection was performed both in the absence and in the presence of NGF (50 ng/ml). The transfection efficiency, deduced from the frequency of G418-resistant colonies, was similar for all the constructs (data not shown). As previously shown (Cordon-Cardo et al., 1991), the wild type receptor produced foci only in the presence of NGF. No foci were detected in cells transfected with the M688T mutant NTRK1 (Figure 3a,b), either in the absence or in the presence of NGF. However, biochemical analysis of a pool of G418-resistant colonies from the same transfection showed that the mutated receptor was expressed at a level comparable to the wild type (Figure 3c). Moreover, the transforming activity of TRK-T3 oncogene resulted abrogated by the M688T mutation (data not shown). These data indicate that the M688T mutation abrogates the NTRK1 receptor activity, in agreement with the lack of tyrosine phosphorylation observed in the transient expression experiments.
With respect to D668 mutations, the D668V mutant did not transform above background in the absence of NGF. Upon ligand stimulation, the D668V receptor induced foci with efficiency similar to wild type. The D668N mutant induced a small number of foci in the absence of NGF, confirming the basal activation deduced from Western blot analysis. Treatment with NGF increased the NTRK1 D668N transforming activity, which was significantly higher than wild type and D668V. These data suggest that the D668N mutation renders the NTRK1 receptor more responsive to NGF.
To prove this concept we performed a focus forming assay in the presence of low doses of NGF, namely 1, 2, and 5 ng/ml based on previous observation showing detectable transforming activity of NTRK1 in the presence of 1 ng/ml of NGF (Miranda et al., 2002). As shown in Figure 3d, at all the NGF doses the NTRK1 D668N mutant induced foci with efficiency higher than wild type and D668V (1.5–3-fold depending on the NGF dose). These data support the notion that the D668N mutation induces a partial activation of the NTRK1 receptor which enhances NGF responsiveness, more evident at low NGF doses.
Our data demonstrate that mutations releasing the oncogenic potential of Kit, Met and Ret show distinct effects on the NTRK1 receptor.
The NTRK1 D668 residue is located in the activation loop (A-loop) downstream of the DFG motif, considered important to coordinate MgATP in the insulin receptor (Hanks et al., 1988). The NTRK1 D668N mutation produced effects similar to those of the equivalent Met D1246N mutation. The NTRK1 D668N protein showed phosphorylation in the absence of ligand stimulation; treatment with NGF further increased the phosphorylation level, indicating that the mutation releases only in part the receptor auto-inhibition. This effect is independent from NTRK1 extracellular portion, since the D668N mutation induces partial activation of the isolated NTRK1 intracellular domain. Transforming activity assays in NIH3T3 cells produced results in agreement with the biochemical data. The non-stimulated NTRK1 D668N receptor showed a low, but significant, transforming activity. In the presence of NGF the transforming activity of NTRK1 D668N was higher than wild type. The enhanced NGF responsiveness was further demonstrated by the higher transforming activity of NTRK1 D668N with respect to the wild type receptor, even in the presence of low doses of NGF. Comparable results have been obtained for the Met receptor carrying the equivalent mutation D1246N (Maritano et al., 2000), thus demonstrating a similar effect of same amino acidic substitution in the context of two different receptors.
The NTRK1 D668V mutation is equivalent to the Kit D816V activating mutation associated with human mastocytosis (Furitsu et al., 1993; Nagata et al., 1995). The same mutation in Met (D1246V) reduced the requirement for HGF with respect to wild type, similarly to D1246N mutation (Maritano et al., 2000). When reproduced in the Ron receptor (D1232V) it released its oncogenic potential (Santoro et al., 1998). However, when introduced into NTRK1 the D668V mutation was neutral, since the mutated receptor showed biochemical and biological features similar to wild type. In fact, the NTRK1 D668V receptor did not show any constitutive activation, was phosphorylated and induced foci formation only upon NGF treatment, with no differences with respect to wild type at all doses used for the analysis. Conflicting results have been observed also for another mutation affecting the D668 residue. We have recently shown that the D668Y mutation, detected in CIPA patients, causes partial inactivation of the NTRK1 receptor (Miranda et al., 2002), while the equivalent mutation (D814Y) activates murine Kit and is associated with mastocytosis (Tsujimura et al., 1994). The analogy of effects of the mutations observed between NTRK1 and Met and the divergence with Kit would suggest that the NTRK1 receptor adopts a very peculiar auto-inhibitory mechanism in which other residues, perhaps unique rather than conserved, might play a crucial role.
The 3-D structure of the NTRK1 receptor is not available. However, due to the high degree of homology with the insulin receptor, it is possible to use the IRK structure to model NTRK1 with good approximation (Hubbard, 1997; Cunningham and Greene, 1998). Thermodynamical studies on the insulin receptor showed that the unphosphorylated form of the enzyme is more stable than the phosphorylated one (Till et al., 2001). Mutations in the A-loop perturb interactions between residues, thus affecting the stability of the enzyme and changing the equilibrium between the unphosphorylated and phosphorylated forms. The effect of the D668N mutation in NTRK1 possibly destabilizes the inactive form. In the molecular model built on the base of IRK, D668 can make a salt bridge with R593, which in turn interacts with D590, forming a network of charged residues. The substitution D to N destroys this network, and could make the inactive conformation less stable and shift the equilibrium towards the active form. The mutation D668V introduces a hydrophobic residue in this area; this might make the inactive conformation even less stable, but at the same time also destabilize the active form, in which the hydrophobic residue is exposed to the solvent. Consequently, both the active and inactive forms could be similarly destabilized so that the equilibrium of non-phosphorylated versus phosphorylated form remains unaltered.
The M688T mutation, equivalent to the Ret MEN2B, produced results very different from those expected. The Ret M918T mutation makes the surrounding region more similar to non-receptor PTKs and has been shown to cause a shift of substrata (Songyang et al., 1995). Several studies have shown that such mutation, naturally occurring or experimentally introduced, is able to activate not only Ret but also other RTKs such as Met and Ron (Schmidt et al., 1997; Santoro et al., 1998). Although all these evidences would foresee an activating effect, the M688T mutation rendered the NTRK1 receptor unable to respond to NGF. In fact, neither phosphorylation nor NIH3T3 transformation were observed upon ligand stimulation.
M688 is located in the hydrophobic central patch of the C-terminal lobe of the kinase: this area, although relatively far from the active site, is important for the overall stabilization of the enzyme conformation. The substitution of the hydrophobic M with a partially hydrophilic T probably destabilizes the area, with a drastic consequence for the enzyme conformation and activity.
In summary, our data indicate that missense mutations releasing the oncogenic potential of different RTKs show distinct effects on the NTRK1 receptor. In particular, D668N partially activates the NTRK1 tyrosine kinase activity, as expected by analogy with the Met receptor; D668V shows a neutral effect, while M688T has the effect of a loss of function mutation. Thus, despite the high degree of conservation of certain amino acid residues, the NTRK1 receptor seems to diverge from other RTKs in terms of tridimensional structure, and to have a distinct auto-inhibitory mechanism.
Cordon-Cardo C, Tapley P, Jing S, Nanduri V, O'Rorke E, Lamballe F, Kovary K, Klein R, Jones KR, Reichardt LF, Barbacid M . 1991 Cell 66: 173–183
Cunningham ME, Greene LA . 1998 EMBO J. 17: 7282–7293
Descamps S, Lebourhis X, Delehedde M, Boilly B, Hondermarck H . 1998 J. Biol. Chem. 273: 16659–16662
Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U, Sugahara H, Butterfield JH, Ashman LK, Kanayama Y . 1993 J. Clin. Invest. 92: 1736–1744
Greco A, Mariani C, Miranda C, Lupas A, Pagliardini S, Pomati M, Pierotti MA . 1995 Mol. Cell. Biol. 15: 6118–6127
Greco A, Villa R, Fusetti L, Orlandi R, Pierotti MA . 2000 J. Cell. Physiol. 182: 127–133
Hanks SK, Quinn AM, Hunter T . 1988 Science 241: 42–52
Hubbard SR . 1997 EMBO J. 16: 5572–5581
Indo Y . 2001 Hum. Mutat. 18: 462–471
Kaplan DR, Miller FD . 2000 Curr. Opin. Neurobiol. 10: 381–391
Maritano D, Accornero P, Bonifaci N, Ponzetto C . 2000 Oncogene 19: 1354–1361
Miknyoczki SJ, Lang D, Huang L, Klein-Szanto AJ, Dionne CA, Ruggeri BA . 1999 Int. J. Cancer 81: 417–427
Miranda C, Di Virgilio M, Selleri S, Zanotti G, Pagliardini S, Pierotti MA, Greco A . 2002 J. Biol. Chem. 277: 6455–6462
Nagata H, Worobec AS, Oh CK, Chowdhury BA, Tannenbaum S, Suzuki Y, Metcalfe DD . 1995 Proc. Natl. Acad. Sci. USA 92: 10560–10564
Pierotti MA, Bongarzone I, Borrello MG, Greco A, Pilotti S, Sozzi G . 1996 Genes Chrom. Cancer 16: 1–14
Reuther GW, Lambert QT, Caligiuri MA, Der CJ . 2000 Mol. Biol. Cell 20: 8655–8666
Santoro MM, Penengo L, Minetto M, Orecchia S, Cilli M, Gaudino G . 1998 Oncogene 17: 741–749
Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, Scherer SW, Zhuang Z, Lubensky I, Dean M, Allikmets R, Chidambaram A, Bergerheim URF, Feltis JT, Casadevall C, Zamarron A, Bernues M, Richard S, Lips CJ, Walther MM, Tsui LC, Geil L, Orcutt ML, Stackhouse T, Zbar B, Lipan J, Slife L, Brauch H, Decker J, Niehans G, Hughson MD, Moch H, Storkel S, Lerman MI, Lineham WM . 1997 Nat. Genet. 16: 68–73
Songyang Z, Carraway III KL, Eck MJ, Harrison SC, Feldman RA, Mohammadi M, Schlessinger J, Hubbard SR, Smith DP, Eng C, Lorenzo MJ, Ponder BAJ, Mayer BJ, Cantley LC . 1995 Nature 373: 536–539
Tagliabue E, Ghirelli C, Lombardi L, Castiglioni F, Asnaghi L, Longhi C, Borrello MG, Aiello P, Menard S . 1999 Int. J. Biol. Markers 14: 68–72
Till JH, Ablooglus AJ, Frankel M, Bishop SM, Kohanskii RA, Hubbard SR . 2001 J. Biol. Chem. 276: 10049–10055
Tsujimura T, Furitsu T, Morimoto M, Isozaki K, Nomura S, Matsuzawa Y, Kitamura Y, Kanakura Y . 1994 Blood 83: 2619–2626
van Heyningen V . 1994 Nature 367: 319–320
Weeraratna AT, Dalrymple SL, Lamb JC, Denmeade SR, Miknyoczki SJ, Dionne CA, Isaacs JT . 2001 Clin. Cancer Res. 7: 2237–2245
The authors thank Miss Cristina Mazzadi for secretarial assistance and Mrs Maria Teresa Radice for technical help. This work was supported by AIRC (Italian Association for Cancer Research) and by funds of the EC project BIO4 CT98 (0556).
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Miranda, C., Zanotti, G., Pagliardini, S. et al. Gain of function mutations of RTK conserved residues display differential effects on NTRK1 kinase activity. Oncogene 21, 8334–8339 (2002). https://doi.org/10.1038/sj.onc.1206052
- tyrosine kinase receptors
- NTRK1, oncogenic activation
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