trkC encodes multiple neurotrophin-3 receptors with ... - NCBI - NIH

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May 3, 1993 - Key words: neurotrophin-3/trkC/tyrosine protein kinase receptors ... of the trk proto-oncogene,gpl40'r (Martin-Zanca et al.,. 1989), and the highly ...
The EMBO Journal vol.12 no.8 pp.3083-3094, 1993

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

Fabienne Lamballe2, Peter Tapley2 and Mariano Barbacid1 Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA 'Corresponding author 2The first two authors contributed equally to this study Communicated by J.Schlessinger

The trkC gene product gpl45"*C 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 gp145"C (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 Ki 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 Ki phosphorylates phospholipase C-yl 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. Key words: neurotrophin-3/trkC/tyrosine protein kinase receptors

Introduction The complexity of the vertebrate nervous system requires multiple neurotrophic factors to modulate its precise developmental programs and to provide the necessary plasticity to the adult brain. The nerve growth factor (NGF) family of neurotrophins are known to play an important role in mediating the survival of neurons and perhaps in promoting proliferation and/or differentiation of neuronal and glial precursors (reviewed in Levi-Montalcini, 1987; Barde 1990). In addition to NGF, this family of neurotrophins includes brain-derived neurotrophic factor (BDNF) (Barde et al., 1982; Leibrock et al., 1989), neurotrophin-3 (NT-3), also known as NGF-2, and

hippocampus-derived neurotrophic factor (HDNF) (Ernfors et al., 1990; Hohn et al., 1990; Jones and Reichardt, 1990; Kaisho et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990) and neurotrophin-4 (NT-4), also known as neurotrophin-5 (NT-5) (Berkemeier et al., 1991; Hallbook etal., 1991; Ip etal., 1992). Recent studies have demonstrated that these neurotrophins mediate their trophic activities through the Trk family of tyrosine kinase receptors (reviewed in Meakin and Shooter, 1992; Barbacid, 1993). These receptors include the product of the trk proto-oncogene, gpl40'r (Martin-Zanca et al., 1989), and the highly related gpl45'rkB and gpl45'rc tyrosine kinases encoded by the trkB (Klein et al., 1989; Middlemas et al., 1991) and trkC (Lamballe et al., 1991) genes, respectively. Whereas NGF is the cognate ligand for gp140'tk (Hempstead et al., 1991; Kaplan et al., 1991a,b; Klein et al., 199 1a), gpl45trkB serves as the signal transducing receptor for both BDNF (Klein et al., 1991b; Soppet et al., 1991; Squinto et al., 1991) and NT-4 (Berkemeier et al., 1991; Ip et al., 1992; Klein et al., 1992). The third member of this tyrosine kinase receptor family, gpl45"*c, is the primary receptor for NT-3 (Lamballe et al., 1991). In addition, NT-3 binds and activates gpl40't' and gpl45t*B when ectopically expressed in NIH3T3 cells. However, these receptors do not appear to mediate the trophic properties of NT-3 in a more physiological environment such as PC 12 cells or sensory neurons (Ip et al., 1993). Recent studies have indicated that the trkC locus is widely expressed in the embryonic and adult central and peripheral nervous systems (Lamballe et al., 1991, 1993; Ernfors et al., 1992; Merlio et al., 1992). In addition, trkC is expressed in certain ganglions of the autonomous enteric nervous system (Lamballe et al., 1993). During embryonic development, trkC expression correlates, both temporally and spatially, with the outgrowth of axons towards their peripheral targets. In the adult mouse, trkC mRNA is heterogeneously distributed throughout the brain, with highest levels of expression in limbic and diencephalic structures. In addition, trkC is expressed in certain nonneuronal tissues such as the mesenchymal cells of arterial walls, the multilocular adipose tissue and the acini of the submaxillary gland (Lamballe et al., 1993). These observations suggest that the trkC gene plays an important role in the development and/or maintenance of the three major components (central, peripheral and enteric) of the mammalian nervous system as well as in some highly specialized non-neuronal tissues. In this study, we report that the mammalian trkC locus encodes multiple tyrosine kinase receptor isoforms with distinct biochemical and biological properties. These TrkC kinase isoforms differ from the previously characterized gpl45t'*c receptor in the presence of additional amino acid residues located within their respective catalytic kinase regions. These findings represent the first report of tyrosine 3083

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Fig. 1. Nucleotide and deduced amino acid sequence of a region of the tyrosine kinase domain of the porcine TrkC KI (amino acid residues 697-726) encompassing the conserved amino acid residues (solid boxes) of domains VII and VHI as defined by Hanks et al. (1988). TrkC KI was previously designated as gpl45trkc (Lamballe et al., 1991). The nucleotide and deduced amino acid sequences of the 42 bp sequences specific for the TrkC K2 receptor isoform and the 75 nucleotides unique to the TrkC K3 receptor kinase are also indicated. The circled tyrosine residues (YY) correspond to the putative major autophosphorylation site(s) of these TrkC receptors.

protein kinase receptor isoforms with distinct catalytic domains and different substrate specificities.

Results Multiple TrkC tyrosine protein kinase receptors Molecular characterization of trkC cDNA clones from a porcine brain cDNA library (Lamballe et al., 1991) revealed a cDNA, designated pFL6, which contained 42 nucleotides not present in the previously reported trkC cDNA clone, pFL19 (Lamballe et al., 1991). These 42 bp were located between nucleotides 2164-2165 of pFL19, a region coding for the tyrosine kinase catalytic domain of gpl45trkc (Lamballe et al., 1991). This sequence is in-frame with the pFL19 open reading frame and does not contain any terminator codons. These observations suggest the existence of a TrkC receptor isoform identical to gpl45"*c except for the presence of 14 additional amino acid residues within its catalytic domain (Figure 1). These residues are located at the end of subdomain VII (Hanks et al., 1988) following the TDYYR motif characteristic of the insulin/Trk receptor subfamilies (reviewed in Barbacid et al., 1991). This motif has homology with the major autophosphorylation site of the Src kinase family (reviewed in Cooper, 1990) and is tyrosine-phosphorylated in the insulin receptor after ligand binding (Tornqvist et al., 1988). We have designated this putative TrkC receptor isoform as TrkC K2. The previously characterized gpl45trIc receptor (Lamballe et al., 1991) will now be designated as TrkC Ki (Figure 1). To determine whether the additional 42 nucleotides present in pFL6 were a cloning artifact, we performed PCR-aided amplification of an adult mouse brain cDNA using amplimers corresponding to sequences flanking these nucleotides. Parallel PCR amplifications were performed on pFL19 and pFL6 plasmid DNAs as controls. As shown in Figure 2, the amplified product from the mouse brain cDNA contained at least three distinct DNA fragments. The two smaller DNA fragments co-migrated with DNAs amplified from pFL19 (184 bp) and pFL6 (226 bp) and correspond to sequences amplified from cDNAs encoding TrkC KI and TrkC K2 receptor isoforms. The other PCR product(s) exhibited an electrophoretical mobility corresponding to a DNA fragment of 259 bp, suggesting the presence of previously uncharacterized trkC cDNA species containing additional sequences in the amplified region (Figure 2). -

3084

Fig. 2. Expression of multiple trkC transcripts in adult mouse brain. Ethidium bromide staining of a 2% Nu Sieve agarose gel containing electrophoresed PCR-amplified DNAs from (a) TrkC Kl-coding pFLl9 DNA; (b) TrkC K2-coding pFL6 DNA; (c) an adult mouse brain cDNA; (d) PCR reaction performed without template DNA; (M) size markers. The open arrowheads indicate the migration of mouse brain PCR products whose sizes correspond to those expected for PCR products amplified from transcripts encoding TrkC Kl (bottom open arrowhead) and TrkC K2 (top open arrowhead) receptors. The solid arrowhead indicates the migration of a novel mouse brain PCR product(s) containing additional cDNA sequences.

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Fig. 3. Expression of different trkC RNAs in adult rat brain. Ethidium bromide staining of a 2% Nu Sieve agarose gel containing electrophoresed PCR-amplified DNAs from (a) pFLl9 (trkC Kl); (b) pFL22 (trkC K2); (c) pFL39 (trkC K3); (d-j) cDNAs prepared from rat RNAs isolated from (d) kidney, (e) total adult brain, (f) hippocampal pyramidal neurons; (g) glial cells derived from the cerebral hemispheres; (h) cerebral hemispheres; (i) midbrain and (j) cerebellum. The size of the PCR products of pFLl9 (184 bp), pFL22 (226 bp) and pFL39 (259 bp) are indicated.

To investigate the nature of these PCR products, they were subcloned into pBluescript and representative clones submitted to nucleotide sequence analysis. All clones derived from the smallest DNA fragment exhibited the expected sequences for the mouse trkC Kl cDNA which is 90% identical to the corresponding region of the porcine pFL19

trkC encodes three tyrosine protein kinase receptors

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Fig. 7. Phosphorylation of PLC'y by TrkC receptors. Quiescent NIH3T3 cells expressing TrkC Ki (G4-611 cell line), TrkC K2 (G4-811 cell line) and TrkC K3 (G31-52 cell line) receptors were either (-) mock treated or (+) stimulated for 10 min with 50 ng/ml of baculovirus-synthesized NT-3. Cells were lysed in P-TYR buffer and immunoprecipitated with (A) anti-panTrk or (B) anti-PLC-y antibodies. The resulting immunoprecipitates were analyzed by 7.5% SDS-PAGE, transferred to nitrocellulose and blotted with antiphosphotyrosine monoclonal antibody 4G10. Filters were incubated with [125I]protein A and exposed with the help of an intensifying screen to Kodak X-Omat film at -700C for 48 h. The migrations of PLC-y and the TrkC receptors are indicated by solid arrows. Coelectrophoresed molecular weight markers are those described in the legend to Figure 5.

activation, exhibiting significant levels of tyrosine phosphorylation even after 2 min of incubation with NT-3. These results suggest that the differences observed in the in vitro kinase assays

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50% of the resting G4-61 1 cells were capable of entering S phase 16 h after exposure to NT-3. These results closely resemble those previously obtained with an independent cell line, R4-31, expressing the TrkC KI

Table H. Induction of DNA synthesis in cells expressing TrkC receptor isoforms Cell line

NIH3T3

G4-611

G4-811

G31-52

3088

Receptor isoform

-

TrkC KI

TrkC K2

TrkC K3

Additions

None Serum NT-3 NT-3 BDNF NGF None Serum NT-3 NT-3 BDNF NGF None Serum NT-3 NT-3 BDNF NGF None Serum NT-3 NT-3 BDNF NGF

Bromodeoxyuridine incorporation

20% 10 ng/ml 50 ng/ml 50 ng/ml 50 ng/ml

20% 10 ng/ml 50 ng/ml 50 ng/ml 50 ng/ml 20% 10 50 50 50

ng/ml ng/ml

ng/ml ng/ml

20%

10 ng/ml 50 ng/ml 50 ng/ml 50 ng/ml

Positive/total cells

Percent positive (%)

7/546 380/411 10/463 10/448 12/486 11/391 8/519 467/511 256/516 279/501 21/441 9/454 8/258 209/219 155/255 138/276 8/256 5/234 3/219 286/293 107/260 105/242 10/195 9/178

1.3 92.4 2.1 2.2 2.9 2.8 1.5 91.4 49.6 55.7 4.8 1.9

3.1 95.4 60.8 50.0

3.1 2.1 1.4 97.6 41.1 43.4 5.1 5

trkC encodes three tyrosine protein kinase receptors

isoform (Lamballe et al., 1991). NT-3 also had a similar mitogenic effect on NIH3T3 cells expressing the TrkC K2 (G4-8 11 cells) and TrkC K3 (G3 1-52 cells) receptors, but not on the parental, untransfected NIH3T3 cells (Table II). This mitogenic activity was specific for NT-3, since comparable amounts of the related NGF or BDNF neurotrophins did not have any effect. These results indicate that in spite of their restricted ability to phosphorylate PLC'y and PI-3 kinase, the TrkC K2 and K3 receptor isoforms can engage downstream signaling elements capable of inducing resting cells to initiate DNA synthesis upon addition of NT-3. receptor

Transformation of NIH3T3 cells by TrkC kinase receptor isoforms

We have previously reported that co-expression of NT-3 with TrkC KI leads to the efficient morphologic transformation of NIH3T3 cells (Lamballe et al., 1991). To investigate whether NT-3 could also transform NIH3T3 cells expressing TrkC K2 and TrkC K3 receptors, cells were co-transfected with pLL43, an expression plasmid encoding NT-3, and expression plasmids encoding either TrkC KI (pFL20), TrkC K2 (pFL23) or TrkC K3 (pFL39). These plasmids are all derived from pMEXneo, a mammalian expression vector containing a multiple cloning site flanked by an MSV LTR and a SV40 polyadenylation signal (Martin-Zanca et al., 1989). As illustrated in Table HI, co-transfection of TrkC Kl and NT-3-coding DNAs led to the efficient transformation of the recipient NIH3T3 cells. These transformed cells exhibit the same morphology as those transformed by the human trk oncogene. However, when cells were transfected with plasmids encoding NT-3 and either TrkC K2 or TrkC K3, we only observed very low levels of transformation in spite of the fact that each of these plasmids is equally efficient in directing the synthesis of their respective TrkC receptor isoforms (Table HI). Moreover, the foci observed in cells co-transfected with NT-3 and TrkC K2 or TrkC K3 were small in size and their cells did not exhibit the characteristic refractile morphology of cells transformed by the TrkC Kl isoform.

lesser extent, NT-3. In the present studies, we examined whether the TrkC receptor isoforms can also mediate the survival of NIH3T3 cells in the presence of NT-3. To this end, 75 000 NIH3T3 cells expressing each of the three TrkC kinase isoforms, TrkC Ki (G4-61 1 cells), TrkC K2 (G4-81 1 cells) and TrkC K3 (G31-52 cells), were seeded, quiesced by serum deprivation for 48 h and cultivated for 6 days in the absence or presence of 50 ng/ml of NT-3. As controls, we used parental NIH3T3 cells and Z82-71 cells, an NIH3T3-derived cell line transformed by constitutive coexpression of TrkC Kl and NT-3. As shown in Figure 9, the TrkC K 1-expressing cells were able to proliferate in the presence of NT-3 as efficiently as in the presence of 10% calf serum, albeit after undergoing a certain lag period that varied from experiment to experiment. NT-3 was also capable of temporarily supporting the survival of cells expressing the TrkC K2 isoforms, but had limited proliferative properties. Finally, NT-3 did not have a significant effect on either the survival or proliferation of the TrkC K3-expressing cells, which die at a rate similar to that observed for the parental NIH3T3 cells (Figure 9). These results suggest that the TrkC K2 and TrkC K3 tyrosine kinase receptors may not be efficiently coupled to mitogenic signaling pathways in these cells. a

TrkC K1, but not TrkC K2 or TrkC K3, induces differentiation of PC12 cells We next examined whether any of the TrkC receptor isoforms could induce the differentiation of PC 12 cells in the presence of NT-3. PC12 cells were transfected with each

106

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TrkC K3

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NT-3-dependent survival of NIH3T3 cells expressing TrkC receptors Glass et al. (1991) have previously shown that the TrkB tyrosine kinase receptor can mediate the survival and proliferation of NIH3T3 cells in response to BDNF and, to

106

trkC DNA

Receptor isoform

NT-3 DNA

Transforming

(pLL43)

activity (foci per 105 cells)a

pFL20

TrkC KI

None

1000