Fabienne Lamballe

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.

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.

Combined signaling through ERK, PI3K/AKT, and RAC1/p38 is required for met-triggered cortical neuron migration.

Cell migration is a complex biological process playing a key role in physiological and pathological conditions. During central nervous system development, positioning and function of cortical neurons is tightly regulated by cell migration. Recently, signaling events involving the urokinase-type plasminogen activator receptor, which is a key regulator for the activation of hepatocyte growth factor (HGF), have been implicated in modulating cortical neuron migration. However, the intracellular pathways controlling neuronal migration triggered by the HGF receptor Met have not been elucidated. By combining pharmacological and genetic approaches, we show here that the Ras/ERK pathway and phosphatidylinositol 3-kinase (PI3K) are both required for cortical neuron migration. By dissecting the downstream signals necessary for this event, we found that Rac1/p38 and Akt are required, whereas the c-Jun N-terminal kinase (JNK) and mTOR/p70s6k pathways are dispensable. This study demonstrates that concomitant activation of the Ras/ERK, PI3K/Akt, and Rac1/p38 pathways is required to achieve full capacity of cortical neurons to migrate upon HGF stimulation.

Proapoptotic Function of the MET Tyrosine Kinase Receptor through Caspase Cleavage

ABSTRACT The MET tyrosine kinase, the receptor of hepatocyte growth factor-scatter factor (HGF/SF), is known to be essential for normal development and cell survival. We report that stress stimuli induce the caspase-mediated cleavage of MET in physiological cellular targets, such as epithelial cells, embryonic hepatocytes, and cortical neurons. Cleavage occurs at aspartic residue 1000 within the SVD site of the juxtamembrane region, independently of the crucial docking tyrosine residues Y1001 or Y1347 and Y1354. This cleavage generates an intracellular 40-kDa MET fragment containing the kinase domain. The p40 MET fragment itself causes apoptosis of MDCK epithelial cells and embryonic cortical neurons, whereas its kinase-dead version is impaired in proapoptotic activity. Finally, HGF/SF treatment does not favor MET cleavage and apoptosis, confirming the known survival role of ligand-activated MET. Our results show that stress stimuli convert the MET survival receptor into a proapoptotic factor.

Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth

Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo

J. Neurochem. (2011) 117, 892–903.

Pool-Specific Regulation of Motor Neuron Survival by Neurotrophic Support

The precise control of motor neuron (MN) death and survival following initial innervation of skeletal muscle targets is a key step in sculpting a functional motor system, but how this is regulated at the level of individual motor pools remains unclear. Hepatocyte growth factor (HGF) and its receptor Met play key developmental roles in both muscle and MNs. We generated mice (termed “Nes-Met”) in which met is inactivated from midembryonic stages onward in the CNS only. Adult animals showed motor behavioral defects suggestive of impaired innervation of pectoral muscles. Correspondingly, in neonatal spinal cords of Nes-Met mutants, we observed death of a discrete population of pea3-expressing MNs at brachial levels. Axonal tracing using pea3 reporter mice revealed a novel target muscle of pea3-expressing MNs: the pectoralis minor muscle. In Nes-Met mice, the pectoralis minor pool initially innervated its target muscle, but required HGF/Met for survival, hence for proper maintenance of muscle innervation. In contrast, HGF/Met was dispensable for the survival of neighboring Met-expressing MN pools, despite its earlier functions for their specification and axon growth. Our results demonstrate the exquisite degree to which outcomes of signaling by receptor tyrosine kinases are regulated on a cell-by-cell basis. They also provide a model for one way in which the multiplicity of neurotrophic factors may allow for regulation of MN numbers in a pool-specific manner.

MET signaling in GABAergic neuronal precursors of the medial ganglionic eminence restricts GDNF activity in cells that express GFRα1 and a new transmembrane receptor partner.

GDNF (glial cell line-derived neurotrophic factor) promotes the differentiation and migration of GABAergic neuronal precursors of the medial ganglionic eminence (MGE). These functions are dependent on the GPI-anchored receptor GFRα1, but independent of its two known transmembrane receptor partners RET and NCAM. Here we show that soluble GFRα1 is also able to promote differentiation and migration of GABAergic MGE neurons. These activities require endogenous production of GDNF. Although GDNF responsiveness is abolished in Gfra1−/− neurons, it can be restored upon addition of soluble GFRα1, a result that is only compatible with the existence of a previously unknown transmembrane signaling partner for the GDNF-GFRα1 complex in GABAergic neurons. The roles of two candidate transmembrane receptors previously implicated in GABAergic interneuron development - MET, a receptor for hepatocyte growth factor (HGF), and ErbB4, the neuregulin receptor – were examined. GDNF did not induce the activation of either receptor, nor did inhibition of MET or ErbB4 impair GDNF activity in GABAergic MGE neurons. Unexpectedly, however, inhibition of MET or HGF per se promoted neuronal differentiation and migration and enhanced the activity of GDNF on MGE neurons. These effects were dependent on endogenous GDNF and GFRα1, suggesting that MET signaling negatively regulates GDNF activity in the MGE. In agreement with this, Met mutant MGE neurons showed enhanced responses to GDNF and inhibition of MET or HGF increased Gfra1 mRNA expression in MGE cells. In vivo, expression of MET and GFRα1 overlapped in the MGE, and a loss-of-function mutation in Met increased Gfra1 expression in this region. Together, these observations demonstrate the existence of a novel transmembrane receptor partner for the GDNF–GFRα1 complex and uncover an unexpected interplay between GDNF–GFRα1 and HGF–MET signaling in the early diversification of cortical GABAergic interneuron subtypes.