(Received for publication, April 26, 1995; and in revised form, November 8, 1995)
From the
The potential for the activation of one Trk receptor by ligand binding to another Trk receptor was explored by determining if transphosphorylation on tyrosine residues can occur between receptors. For most of these experiments, functional chimeric receptors were used that contained the extracellular domain of the human type 2 tumor necrosis factor receptor and the transmembrane and cytoplasmic domains of rat TrkA, TrkB, or TrkC and that, when activated by the tumor necrosis factor, mediated the nerve growth factor-like biological activities in PC12 cells. Cotransfection experiments in COS-7 cells and fibroblasts showed that despite the presence of different extracellular regions, intermolecular transphosphorylation of homologous cytoplasmic domains occurred between TrkA or TrkB and their cognate chimeras. Heterologous transphosphorylation between TrkB and TrkC kinase domains was also observed when one partner was a chimeric receptor; however, TrkA did not transphosphorylate the TrkB or TrkC kinase domains of chimeric receptors or act as a transphosphorylation substrate for these two receptors. The failure of TrkA to take part in transphosphorylation reactions with TrkB and TrkC was confirmed using the natural receptors. Trk receptor transphosphorylation occurs in the two non-neuronal cell types, but TrkA is excluded from these reactions.
The nerve growth factor (NGF) ()is a prototype for a
structurally related family of neurotrophic factors, the neurotrophins.
Three other members of this gene family have been identified,
brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and
neurotrophin-4/5 (reviewed in (1, 2, 3) ).
The neurotrophins share
50% amino acid homology, but they exhibit
characteristic patterns of activity. For instance, NGF, BDNF, and NT-3
can support the survival of embryonic sensory dorsal root ganglial
neurons, but only NGF can effectively support sympathetic neurons (4, 5) from undergoing programed cell death.
Conversely, embryonic placode-derived sensory neurons are sustained by
both BDNF and NT-3, but not by NGF(6, 7, 8) .
This selectivity is believed to depend on the presence of specific
cell-surface receptors with tyrosine kinase activity, known as Trk
receptors. A family of three related receptors, TrkA, TrkB, and TrkC,
has been characterized. In neurons, NGF, BDNF, and NT-3 are the
preferred ligands for TrkA, TrkB, and TrkC, respectively, while all the
neurotrophins bind to a common receptor, designated p75
(where NGFR is NGF receptor) (reviewed in (1, 2, 3) ).
The ligand-induced dimerization of TrkA tyrosine kinase receptors leads to activation of the tyrosine kinase(9) . Receptor phosphorylation occurs by a dimerization mechanism whereby the two receptor molecules representing the dimer phosphorylate each other(10) . It is known that the presence of homologous intra- or extracellular domains is sufficient to mediate transphosphorylation between some heterologous receptors(11, 12, 13) , while intermolecular transphosphorylation can also occur between two different but related receptors(14, 15) . Furthermore, the transphosphorylation involving an inactive tyrosine kinase receptor can lead to partial restoration of the functional internalization of this receptor (12) or to amplification of the response by an active receptor(12, 16) .
Dose-response survival experiments with the various neurotrophins suggest that some neurons express more than one type of Trk. Furthermore, it has been demonstrated that subsets of dorsal root ganglial neurons coexpress TrkB with either TrkA or TrkC, suggesting that the neurotrophin activity can overlap in the same cell population (17) . The colocalization of TrkA, TrkB, or TrkC receptor mRNA has also recently been reported in primary sensory neurons(18) . It was of interest to determine if the homologous kinase domains of the various Trk receptors represent compatible substrates for heterologous transphosphorylation, permitting one neurotrophin to activate more than one Trk receptor. For this purpose chimeric, TNFR2-Trk receptors, which differ in mobility from the corresponding Trk receptors, have been used. Accordingly, active or inactive Trk receptors were coexpressed in COS-7 cells and fibroblasts with inactive or active chimeric TNFR2-Trk receptors, respectively, and the effects of one or more of the neurotrophins on receptor phosphorylation were determined. Intermolecular transphosphorylation occurred between homologous Trk receptors in these cells, while a restricted TrkB/TrkC heterologous transphosphorylation was observed.
NIH-3T3 fibroblasts expressing TrkB receptors (a gift of Hua Zhou, Stanford University) were transiently transfected using LipofectAMINE (Life Technologies, Inc.) strategy, with 6 mg of an expression vector coding for TA, TB, or TC receptors in three different experiments. After BDNF treatment, the receptors were immunoprecipitated from the cell lysate using the pan-Trk antiserum. The phosphotyrosine content was determined by Western blotting as reported for the COS-7 cell protocols.
Figure 1:
Schematic representation, protein
expression, and tyrosine phosphorylation of the TB set of chimeric
molecules. A, graphic representation of the TB set of
chimeras. The extracellular domains of the human TNFR2 receptors are
shown as black boxes; rat TrkB kinase domains are represented
by white boxes. The extracellular domain of TNFR2 and the
transmembrane and cytoplasmic domains of TrkB represent the prototypic
molecule designated as TB (lanes 1 and 2). The
transmembrane domain of TNFR2 is represented as an extension of the
extracellular region (black box) into the cell membrane
(TB-TM) (lanes 3 and 4). The lysine-mutated construct
is indicated with a K (TB-K) (lanes 5 and 6). The carboxyl-terminal deletion of the last 15 amino acid
residues is represented by the missing vertical bar under the
kinase domain (TB-) (lanes 7 and 8). B,
protein expression of the TB set of chimeras. Chimeric molecules were
transiently expressed in COS-7 cells; lysed cells were
immunoprecipitated using anti-TNFR2 and resolved by SDS-polyacrylamide
gel electrophoresis on 8% acrylamide gels. The blot was
electrophoretically transferred to nitrocellulose, probed with
anti-TNFR2, and developed by the ECL detection system. C,
autophosphorylation of the TB set of chimeras. Transfected COS-7 cells
were treated for 5 min with (lanes 1, 3, 5,
and 7) or without (lanes 2, 4, 6,
and 8) 50 ng/ml human TNF, and the phosphotyrosine contents
were analyzed by immunoblotting with
anti-pTyr.
Figure 2: Schematic representation, protein expression, and tyrosine phosphorylation of the TC set of chimeras. Graphic representation, protein expression, and autophosphorylation activity were determined as described for the TB set of chimeras in the legend of Fig. 1.
Receptors were evaluated for
function by measuring the TNF-dependent phosphorylation of the tyrosine
kinase domains. The TNF incubation time was optimized to result in
maximal autophosphorylation of the chimeras (data not shown). The TB
and TC chimeras showed a TNF-dependent increase in phosphotyrosine
content of both bands of the doublet, with probably a greater relative
increase for the upper band (Fig. 1C and Fig. 2C, lane 1). In the absence of ligand, a
background of phosphorylation was detected in both TB and TC chimeras (Fig. 1C and Fig. 2C, lane 2)
that was comparable to the phosphorylation observed by expressing
wild-type TrkB and TrkC in COS-7 cells (see Fig. 4, A-C, lanes 4 and 6). These results
indicate that the TNFR2 extracellular ligand-binding site is able to
transduce the enzymatic activation of the TrkB and TrkC kinase domains,
as it was with TrkA(19) . TNF was unable to induce
autophosphorylation of the kinase-deficient molecules TB-K and TC-K,
indicating that the phosphorylation activity of TB and TC is a function
of the TNF-mediated activation of their tyrosine kinase domains. As
reported for the chimeric molecule between the extracellular domain of
the epidermal growth factor and the transmembrane and cytoplasmic
domains of TrkA (EGF-TrkA)(28) , the C-terminal deletions
(TB- and TC-
) prevented receptor autophosphorylation,
confirming that this region is important for tyrosine kinase activity.
Surprisingly, the TB-TM and TC-TM chimeras were also inactive,
indicating that the transmembrane domain of TNFR2 cannot functionally
replace this domain in the Trk receptors.
Figure 4: Tyrosine transphosphorylation of cotransfected Trk receptors and kinase-deficient chimeric receptors in COS-7 cells. A, COS-7 cells were cotransfected with the kinase-deficient chimeric receptor TA-K and TrkA (lanes 2 and 3), TrkB (lanes 4 and 5), and TrkC (lanes 6 and 7). Cotransfection of TA-K and the vector pCDM8 is shown in lane 1. Transfected cells were treated for 5 min with NGF (lane 3), BDNF (lane 5), and NT-3 (lane 7) at a concentration of 50 ng/ml. The receptors were immunoprecipitated with a pan-Trk antibody that recognizes the carboxyl-terminal regions of all Trk and chimeric receptors and separated on 8% SDS-polyacrylamide gels, and the phosphotyrosine content was determined by immunoblotting using anti-pTyr. B, COS-7 cells were cotransfected with the kinase-deficient chimeric receptor TB-K and TrkA (lanes 2 and 3), TrkB (lanes 4 and 5), and TrkC (lanes 6 and 7). Cotransfection of TB-K and the vector pCDM8 is shown in lane 1. The neurotrophin-dependent tyrosine phosphorylation assays was carried out as described in the legend of Fig. 1. C, COS-7 cells were cotransfected with the kinase-deficient chimeric receptor TC-K and TrkA (lanes 2 and 3), TrkB (lanes 4 and 5), and TrkC (lanes 6 and 7). Cotransfection of TC-K and the vector pCDM8 is shown in lane 1. The neurotrophin-dependent tyrosine phosphorylation assays were carried out as described in the legend of Fig. 1C. Arrowheads indicate the TrkA, TrkB, and TrkC receptors (upper) and the inactive chimeric TA-K, TB-K, and TC-K receptors (lower). Molecular mass markers (in kilodaltons) are indicated. Stripped blots developed using pan-Trk antibody showed comparable expression of the receptor proteins.
For experiments to be described later, we also constructed an inactive TrkA carrying a single mutation in the kinase domain (designated TrkA-K) (see Fig. 5). As with the mutants described above, TrkA-K did not show any ligand-mediated autophosphorylation when expressed in COS-7 cells (data not shown). The construction of the remaining chimeric receptors, consisting of the extracellular domain of TNFR2 and the transmembrane and cytoplasmic domains of TrkA (designated TA) (see Fig. 5) or the corresponding construct with a kinase-deficient domain (designated TA-K) (see Fig. 4), was described earlier(19) .
Figure 5: Tyrosine transphosphorylation of cotransfected kinase-deficient TrkA-K and the chimeric TA, TB, and TC receptors in COS-7 cells. COS-7 cells were cotransfected with inactive TrkA (TrkA-K) and the pCDM8 vector (lane 1) or the chimeric receptors TA (lanes 2 and 3), TB (lanes 4 and 5), and TC (lanes 6 and 7). Transfected cells were treated with (lanes 3, 5, and 7) or without (lanes 2, 4, and 6) TNF (50 ng/ml) for 5 min, and the phosphotyrosine content was determined as described in the legend of Fig. 1C. Arrowheads indicate the migration of TrkA-K (upper) and the chimeric TA, TB, and TC receptors (lower). Stripped blots developed with pan-Trk antibody showed comparable protein expression of the cotransfected receptors.
Figure 3: Biological activity of the TB and TC sets of chimeras in PC12 cells. A, neurite outgrowth assay. PC12 cells transfected with both sets of chimeras were incubated with increasing concentrations of TNF (0.1, 1, 2.5, 5, 10, 25, and 40 ng/ml). After 48 h, cells presenting neurites longer than two-cell diameter were scored. B, cell survival assay. PC12 cells transfected with both sets of chimeras or vector alone (pCDM8) were cultured in serum-free medium (Control) or supplemented with TNF (50 ng/ml). Viable cells were scored using the MTT-based colorimetric assay, and survival was calculated on the basis that survival in the presence of NGF was 100%. Nontransfected PC12 cells were used as an additional control to evaluate the electroporation effects on cell viability.
We also compared TNF-dependent cell survival mediated by the
chimeras using an assay based on the ability of NGF to sustain the
survival of PC12 cells in serum-free conditioned medium. Each set of
transfected cells was subdivided into three pools: one grown in
serum-free medium and the other two grown in serum-free medium
supplemented with either TNF or NGF. Three days later, cell survival
was determined using a MTT-based colorimetric assay. The number of
surviving cells was normalized to the cell viability observed in the
presence of NGF, which was taken to be 100%. Under these conditions,
TNF was able to sustain survival of 70-80% of the cells
transfected with either the TB or TC chimera, whereas only 37% of the
cells transfected with the vector alone were viable. In the absence of
TNF, the survival rate of TB and TC transfectants was
35-40%, which is also in the same range as the background
level (Fig. 3B). None of the mutated chimeras showed
biological activity when expressed in PC12 cells (Fig. 3, A and B). These data indicate that the ligand-mediated
phosphorylation of the tyrosine kinase domains of TrkB and TrkC
receptors, like that of TrkA (19) , is necessary and sufficient
to induce survival and a neuron-like phenotype in PC12 cells.
The kinase-deficient TA-K receptor displayed a very low level of phosphorylation when expressed alone (Fig. 4A, lane 1), but a higher level when cotransfected with TrkA (lane 2). More significantly, not only was the level of TrkA phosphorylation increased on the addition of NGF, as anticipated, but so was the level of phosphorylation of TA-K (Fig. 4A, lane 3; see also Fig. 7A, lanes 1 and 3). In the converse experiments, the effect of coexpression of the chimeric TA receptor with full-length kinase-deficient TrkA-K was examined. Expression of TrkA-K by itself did not result in autophosphorylation of the receptor (Fig. 5, lane 1). Coexpression of TrkA-K with TA resulted in significant phosphorylation of both receptors in the absence of TNF (Fig. 5, lane 2). The addition of TNF increased the phosphorylation of the chimeric TA receptor, again as anticipated, and this was accompanied by an increase in the phosphorylation of TrkA-K (Fig. 5, lane 3). Both bands of the two receptors were affected. These data suggest that transphosphorylation on tyrosine residues occurs between TrkA and the inactive chimeric receptor or between the active chimeric receptor and inactive TrkA and that an inactive receptor monomer can be transphosphorylated by an active receptor monomer irrespective of whether the extracellular domain is homologous. That the transphosphorylation results with the chimeric receptors are the same as with the normal receptors also suggests that the extracellular TNF domains may not exert any selective positive or negative influence on the heterologous interactions compared with the normal Trk extracellular domains.
Figure 7: Tyrosine transphosphorylation of cotransfected kinase-deficient TrkA-K and TrkA, TrkB, and TrkC receptors in COS-7 cells. A, COS-7 cells were cotransfected with inactive TrkA (TrkA-K) and active TrkA (lanes 1 and 2), TrkB (lanes 3 and 4), or TrkC (lanes 5 and 6). Transfected cells were treated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) NGF, BDNF, or NT-3, respectively, and the cell lysate was immunoprecipitated with the TrkA-specific antibody. TrkA and TrkA-K receptor protein expression levels (upper panel) and phosphotyrosine content (lower panel) were determined as described in the legend of Fig. 1. Aliquots of the cell lysate of each sample were used to immunoprecipitate TrkB and TrkC receptors using pan-Trk antibody, and receptor tyrosine phosphorylation was determined (data not shown). B, the selectivity of the TrkA-specific antibody was determined by transfecting COS-7 cells with TrkA-K (lanes 1 and 2), TrkB (lanes 3 and 4), and TrkC (lanes 5 and 6). Transfected cells were treated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) NGF, BDNF, or NT-3, respectively, and the cell lysate was immunoprecipitated with the TrkA-specific antibody. TrkA-K receptor protein expression levels (upper panel) and phosphotyrosine content (lower panel) were determined and indicated that neither inactive nor phosphorylated TrkB and TrkC were recognized by this antibody.
When TrkB was coexpressed with inactive TB-K, the level of phosphorylation of the latter receptor increased along with that of TrkB on addition of BDNF (Fig. 4B, lane 5), suggesting that TrkB forms heterodimers with and transphosphorylates inactive chimeric TB-K. In contrast, no increase in the phosphorylation of TC-K coexpressed with TrkC was observed on addition of NT-3 (Fig. 4C, lanes 6 and 7) even though the phosphorylation of TrkC itself was significantly increased. Whether this is because the constitutive levels of phosphorylation of TC-K when cotransfected with TrkC are already high or because heterodimers of active TrkC and inactive TC-K do not form cannot be determined from these experiments. The converse experiments investigating whether TNF-activated TB or TC can transphosphorylate inactive TrkB or TrkC, respectively, have not been done.
A somewhat different picture emerges with TrkB and TrkC. NT-3 activated TrkC transphosphorylated TB-K (Fig. 4B, lane 7), although BDNF-activated TrkB does not appear to transphosphorylate TC-K (Fig. 4C, lane 5). Whether the high level of constitutive phosphorylation of TC-K cotransfected with TrkB and TrkC hides its transphosphorylation again cannot be determined. These data suggest that a heterodimer can form between the receptor monomers of TrkC and TB-K, with the inactive kinase domain of TB-K being a substrate for heterozygous transphosphorylation by TrkC.
Figure 6: Tyrosine transphosphorylation of the transfected chimeric TA, TB, and TC receptors in NIH-3T3 cells expressing TrkB. NIH-3T3 cells expressing rat TrkB were transfected with TA (lanes 1 and 2), TB (lanes 3 and 4), or TC (lanes 5 and 6). Transfected cells were treated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) BDNF (50 ng/ml) for 5 min, and the phosphotyrosine content was determined. Arrowheads indicate the migration of rat TrkB (upper) and the chimeric TA, TB, and TC receptors (lower). Stripped blots developed with pan-Trk antibody showed comparable protein expression of the cotransfected receptors.
One of the differences that sets the Trk
receptors apart from the other tyrosine kinase receptors is the short
carboxyl-terminal tail of only 15 amino acid residues. Deletion mutants
of chimeric EGF-TrkA receptors have previously indicated that this
region is essential for conferring tyrosine kinase activity and high
affinity binding to phospholipase C-(28) . Similarly, we
found that TNF failed to cause autophosphorylation of the chimeric
TB-
C and TC-
C receptors, which lack this region, and found no
TNF-dependent neurite extension and cell survival when they were
transfected in PC12 cells. These results suggest that the
carboxyl-terminal tail is also essential for the activity of TrkB and
TrkC, and it seems not to be involved in negative control of the
tyrosine kinase domain as has been reported for other tyrosine kinase
receptors(35, 36, 37) .
The transmembrane domains of the tyrosine kinase receptors can usually be exchanged in chimeric receptors without affecting their signal transduction capacity(12, 38, 39) . Also, Trk oncogenes lacking the transmembrane domain of TrkA are oncogenic, suggesting that this region is not required for the activation of the tyrosine kinase domain(3) . In contrast, exchanging the transmembrane domains of the Trk receptors with the corresponding regions of TNFR2 in TB-TM and TC-TM produced nonfunctional receptors. TNF treatment of these mutants induced neither autophosphorylation in COS-7 cells nor neurite outgrowth and survival in PC12 cells. These findings differ from the results obtained with the chimeric receptor constructed between the extracellular and transmembrane domains of type 1 TNFR (TNFR1; p55-TNFR) and the cytoplasmic domain of TrkA(19) , where the transmembrane domain from TNFR1 worked equally as well as the same domain from TrkA. It should be noted, however, that these chimeric receptors (TNFR1-TrkA) were much less efficient at autophosphorylation and signal transduction than the TNFR2-TrkA receptor. Since the cytoplasmic regions of TNFR2 lack the characteristic consensus sequences found in a tyrosine kinase domain, it is possible that its transmembrane domain might be unable to properly transduce the signals leading to receptor dimerization and autophosphorylation.
The two conclusions of this work are that Trk receptor transphosphorylation is observed in COS-7 cells and fibroblasts and that TrkA is excluded from these reactions. The nature of the mechanisms that prevent TrkA from forming heterodimers has not been addressed in this work and need to be clarified before the data can be extrapolated to neurons. Another approach to this problem will be to use the neurotrophin heterodimers that have been shown to form efficiently in cells that express more than one neurotrophin and that are stable enough to allow their purification(42, 43, 44) . These heterodimers display biological activity(42, 43) , and whether this involves the formation of Trk heterodimers will be a question of great interest.