Mariano Barbacid

trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3

We report the isolation and molecular characterization of trkC, a new member of the trk family of tyrosine protein kinase genes. trkC is preferentially expressed in the brain. In situ hybridization studies revealed trkC transcripts in the hippocampus, cerebral cortex, and the granular cell layer of the cerebellum. The product of the trkC gene has been identified as a glycoprotein of 145,000 daltons, gp145trkC, which is equally related to the previously characterized gp140trk and gp145trkB tyrosine kinases. gp145trkC is a functional receptor for neurotrophin-3 (NT-3). However, gp145trkC does not bind the highly related neurotrophic factors NGF or BDNF. In proliferating cells, the interaction between gp145trkC and NT-3 elicits a more efficient biological response than when NT-3 binds to its other receptors gp140trk and gp145trkB. These results indicate that gp145trkC may play an important role in mediating the neurotrophic effects of NT-3.

The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.

Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death

We have generated mice carrying a germline mutation in the tyrosine kinase catalytic domain of the trkB gene. This mutation eliminates expression of gp145trkB, a protein-tyrosine kinase that serves as the signaling receptor for two members of the nerve growth factor family of neurotrophins, brain-derived neurotrophic factor and neurotrophin-4. Mice homozygous for this mutation, trkBTK(-/-), develop to birth. However, these animals do not display feeding activity, and most die by P1. Neuroanatomical examination of trkBTK (-/-) mice revealed neuronal deficiencies in the central (facial motor nucleus and spinal cord) and peripheral (trigeminal and dorsal root ganglia) nervous systems. These findings illustrate the role of the gp145trkB protein-tyrosine kinase receptor in the ontogeny of the mammalian nervous system.

trkB, a novel tyrosine protein kinase receptor expressed during mouse neural development.

We have isolated a novel member of the tyrosine protein kinase family of cell surface receptors. This gene, designated trkB, is highly related to the human trk proto‐oncogene. At the amino acid level, their respective products share a 57% homology in their extracellular regions including 9 of the 11 cysteines present in the trk proto‐oncogene. This homology increases to 88% within their respective tyrosine kinase catalytic domains. Both trk and trkB are equally distantly related to the other members of this gene family of receptors. A biologically active cDNA clone of trkB can direct the synthesis of gp145trkB, a glycoprotein of 145 kd of which only 93 kd correspond to its polypeptide backbone. In adult mice, trkB is preferentially expressed in brain tissue, although significant levels of trkB RNA have also been observed in lung, muscle and ovaries. In addition, trkB transcripts can be detected in mid and late gestation embryos. The trkB locus exhibits a complex pattern of transcription. At least seven RNA species ranging in size from approximately 9 kb to 2 kb have been identified in brain. However, only a subset of these transcripts appears to be expressed in the other tissues. In situ hybridization analysis of 14 and 18 day old mouse embryos indicates that trkB transcripts are localized in the central (CNS) and peripheral (PNS) nervous systems, including brain, spinal cord, spinal and cranial ganglia, paravertebral trunk of the sympathetic nervous system and various innervation pathways. These results suggest that trkB may code for a novel cell surface receptor involved in neurogenesis.

Similarities and differences in the way neurotrophins interact with the Trk receptors in neuronal and nonneuronal cells.

We have exploited a battery of approaches to address several controversies that have accompanied the expansion of the nerve growth factor (NGF) family of neurotrophic factors and the identification of the Trk tyrosine kinases as receptors for these factors. For example, we find that a recently cloned mammalian neurotrophin, known as either neurotrophin-4 or neurotrophin-5 and assigned widely differing receptor specificities, represents the functional counterpart of Xenopus neurotrophin-4 and is a preferred ligand for TrkB. However, its interactions with TrkB can be distinguished from those of brain-derived neurotrophic factor (BDNF) with TrkB. We also find that all of the Trks display similar dose responses to their preferred ligands in neuronal as compared with nonneuronal cells (i.e., NGF for TrkA, BDNF and NT-4/5 for TrkB, and NT-3 for TrkC), providing evidence against a role for accessory molecules expressed in neurons in generating receptors that would allow for responses to lower concentrations of the neurotrophins. However, we find that a neuronal environment does restrict the Trks in their ability to respond to their nonpreferred neurotrophin ligands.

The trk tyrosine protein kinase mediates the mitogenic properties of nerve growth factor and neurotrophin-3

The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.

The trkB tyrosine protein kinase is a receptor for neurotrophin-4.

Neurotrophin-4 is a novel member of the nerve growth factor family of neurotrophins recently isolated from Xenopus and viper DNA. We now report that the Xenopus NT-4 protein (XNT-4) can mediate some of its biological properties through gp145trkB, a murine tyrosine protein kinase previously identified as a primary receptor for the related brain-derived neurotrophic factor (BDNF). XNT-4 displaces 125I-labeled BDNF from binding to cells expressing gp145trkB receptors, induces their rapid phosphorylation on tyrosine residues, and causes the morphologic transformation of NIH 3T3 cells when coexpressed with gp145trkB. Moreover, XNT-4 induces the differentiation of PC12 cells into sympathetic-like neurons only if they ectopically express gp145trkB receptors. None of these biochemical or biological effects could be observed when XNT-4 was added to cells expressing the related receptors. Replacement of one of the extracellular cysteines (Cys-345) of gp145trkB by a serine residue prevents its activation by XNT-4 but not by BDNF. Therefore, XNT-4 and BDNF may interact with at least partially distinct domains within the gp145trkB receptor.

trkC encodes multiple neurotrophin-3 receptors with distinct biological properties and substrate specificities.

The trkC gene product gp145trkC is a high affinity signaling receptor for neurotrophin‐3 (NT‐3), a member of the NGF family of neurotrophic factors. We now report that trkC encodes at least two additional tyrosine protein kinase receptors. These receptors, designated TrkC K2 and TrkC K3, have the same amino acid sequences as gp145trkC (now designated TrkC K1) except for the presence of 14 and 25 additional amino acid residues between kinase subdomains VII and VIII, just downstream from the TDYYR motif which encompasses the putative autophosphorylation site of the Trk receptor family. Upon interaction with their cognate ligand, NT‐3, all three TrkC receptor isoforms become rapidly phosphorylated on tyrosine residues and induce DNA synthesis in quiescent cells. However, only TrkC K1 has mitogenic activity in NIH3T3 cells and induces neuronal differentiation of PC12 cells. The different biological properties of these TrkC receptor isoforms probably result from their engagement with different signaling pathways. Whereas TrkC K1 phosphorylates phospholipase C gamma 1 and phosphatidylinositol‐3 kinase, TrkC K2 and TrkC K3 do not. TrkC K2 and transcripts encoding TrkC K3 have been identified in various structures of the adult murine brain. These observations suggest that the trophic activities of NT‐3 in the mammalian nervous system might be mediated by different TrkC receptor isoforms.

High-affinity nerve growth factor receptor (Trk) immunoreactivity is localized in cholinergic neurons of the basal forebrain and striatum in the adult rat brain

Trk-immunoreactivity was observed in basal forebrain and striatal cholinergic neurons, whereas low-affinity NGF receptor immunoreactivity was observed in basal forebrain but not striatal cholinergic neurons. Since NGF exerts trophic actions on both basal forebrain and striatal cholinergic populations, the presence of Trk in these neurons lends strong support for an essential role of Trk in NGF-responsive neurons, but suggests that the low affinity receptor is not necessary for NGF actions in the striatum.

Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain.

Malignant activation of the human trk proto-oncogene, a member of the tyrosine protein kinase receptor family, has been implicated in the development of certain human cancers, including colon and thyroid papillary carcinomas. trk oncogenes have also been identified in cultured cells transfected with various DNAs. In this study, we report the characterization of three in vitro-generated trk oncogenes, trk2, trk4, and trk5 (R. Oskam, F. Coulier, M. Ernst, D. Martin-Zanca, and M. Barbacid, Proc. Natl. Acad. Sci. USA 85:2964-2968, 1988), in an effort to understand the spectrum of mutational events that can activate the human trk gene. Nucleotide sequence analysis of cDNA clones of trk2 and trk4 revealed that these oncogenes were generated by a head-to-tail arrangement of two trk tyrosine protein kinase domains connected by a purine-rich region. These oncogenes code for cytoplasmic molecules of 67,000 (p67trk2) and 69,000 (p69trk4) daltons. In contrast, the product of the trk5 oncogene, gp95trk5, is a cell surface glycoprotein of 95,000 daltons. This oncogene was generated by a 153-base-pair in-frame deletion within sequences coding for the extracellular domain of the trk receptor. This activating deletion encompasses a triplet coding for one of the nine cysteine residues that the trk receptor shares with the product of the highly related trkB tyrosine protein kinase gene. Introduction of a single point mutation (TGT----AGT) in this codon resulted in a novel trk oncogene whose product, gp140S345, differs from the nontransforming trk proto-oncogene receptor in a single amino acid residue, Ser-345 instead of Cys-345. These results illustrate that multiple molecular mechanisms, including point mutation, internal deletion, and kinase domain duplication, can result in the malignant activation of the human trk proto-oncogene.