(Received for publication, February 22, 1995; and in revised form, May 24, 1995)
From the
We have reported that overexpression of Neu leads to heregulin-stimulated neurite outgrowth and the tyrosine-phosphorylation of Neu and other cellular proteins in PC12 cells. Considering that Neu/ErbB2 alone is not able to functionally couple to heregulin, we looked for the possible involvement of ErbB3 in these neurite outgrowth and tyrosine phosphorylation responses. We found that heregulin stimulates the tyrosine phosphorylation of endogenous ErbB3 protein in PC12 cells and that this phosphorylation, like that of Neu, is greatly enhanced in cells that overexpress Neu. Furthermore, overexpression of ErbB3 in PC12 cells led to heregulin-stimulated neurite extension. In addition to becoming tyrosine-phosphorylated, Neu/ErbB2 and ErbB3 associate with each other, and each associates with the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase in a heregulin-dependent manner. Thus, Neu/ErbB2 and ErbB3 appear to cooperate to mediate the heregulin signal in PC12 cells. Like heregulin, epidermal growth factor (EGF) also stimulates the tyrosine phosphorylation of both Neu and ErbB3. However, there are clear differences between the EGF- and heregulin-stimulated phosphorylations of ErbB3. In the heregulin response, two tyrosine-phosphorylated forms of ErbB3 are detected. Of these, only the more quickly migrating form (on SDS-polyacrylamide gel electrophoresis) is found to be associated with Neu, whereas the other, more slowly migrating form is uniquely capable of forming stable complexes with p85. In the EGF response, at least two tyrosine-phosphorylated forms of ErbB3 are detected, but these phosphoproteins have distinctly lower apparent molecular weights compared with the heregulin-stimulated ErbB3 phosphoproteins and do not complex with p85. Thus the formation of a stable ErbB3-p85 complex in PC12 cells is a unique outcome of heregulin signaling that correlates with the differences in cell morphology induced by the activated EGF receptor and the Neu tyrosine kinase.
The subclass 1 receptor tyrosine kinases include the epidermal
growth factor (EGF) ()receptor, the Neu/ErbB2 tyrosine
kinase, and the ErbB3 and ErbB4 proteins. This receptor family has
received a great deal of attention because of the suspected involvement
of different members of the family in the development of human cancers.
This has especially been true for Neu/ErbB2. The rat Neu tyrosine
kinase was first identified in rat neuroblastomas induced by the
chemical mutagenesis of rat embryos(1) . More recently, the
human homolog of the rat Neu protein, commonly designated ErbB2 (or
Her2), has been implicated in the development of human breast and
cervical cancers(2) .
It is interesting that a variety of studies also have implicated the involvement of receptor tyrosine kinases in developmental processes; the most well known examples being in the development of the compound eye in Drosophila(3) and in vulval development in Caenorhabditis elegans(4) . In the case of Neu/ErbB2, it has been suspected that this tyrosine kinase may play a role in neuronal development, because a putative activator/growth factor for Neu/ErbB2, called heregulin (5) or the Neu differentiation factor(6) , is identical to glial growth factor (7) and acetylcholine receptor-inducing activity(8) . Along these lines, we recently have shown that the expression of a transforming version of the Neu tyrosine kinase (where the valine residue at position 664 within the transmembranal domain has been changed to a glutamic acid) is capable of stimulating neurite extension in rat pheochromocytoma (PC12) cells (9) . Moreover, we have found that the addition of heregulin to PC12 cells that overexpress the normal Neu tyrosine kinase also elicits neurite extension. These results then highlight two important points. The first is that activation of the Neu tyrosine kinase, either by a point mutation or by the addition of its putative activating ligand, elicits a cellular morphology that is similar to that induced by the nerve growth factor receptor (i.e. the Trk tyrosine kinase) but distinct from the cellular effects elicited by the more similar EGF receptor. Thus, PC12 cells offer an excellent model system for distinguishing the specific features of the signaling pathways initiated by Neu/ErbB2 versus the EGF receptor.
The second major implication from the original studies of Neu effects in PC12 cells is the fact that the addition of heregulin leads to the apparent activation of the Neu/ErbB2 tyrosine kinase in these cells. This finding indicates that PC12 cells must contain the necessary components for the stimulatory regulation of Neu. This is an important point because we (10, 11) and others (12, 13) have shown that Neu/ErbB2 alone is not able to functionally couple to heregulin. Rather, it appears that the actual receptors for heregulin are the ErbB3 and ErbB4 proteins, suggesting that heregulin-stimulated heterodimer formation between Neu/ErbB2 and either ErbB3 or ErbB4 is necessary to stimulate Neu/ErbB2 tyrosine kinase activity. In the case of ErbB3, the need for coupling to Neu/ErbB2 is most clear, because ErbB3 by itself has little or no intrinsic tyrosine kinase activity(14) .
In the present studies, we set out to expand upon these two implications. Specifically, we wanted to determine if the ErbB3 protein participated with Neu/ErbB2 in eliciting neurite extension in PC12 cells, and we set out to identify differences between the signaling cascades initiated by heregulin and those stimulated by EGF. We found that overexpression of either Neu/ErbB2 or ErbB3 renders PC12 cells biochemically and morphologically responsive to heregulin. Neu/ErbB2 and ErbB3 associate with each other, become tyrosine-phosphorylated, and associate with the p85 regulatory subunit of phosphatidylinositol 3-kinase after heregulin treatment. The events triggered by heregulin are distinguished from those initiated by EGF by the formation of a tyrosine-phosphorylated ErbB3 species with a retarded electrophoretic mobility and unique ability to complex with p85.
Figure 1:
Heregulin-stimulated
tyrosine phosphorylation of Neu/ErbB2 and ErbB3 and specificity of
anti-ErbB3 antibody (Ab). A and B, parental
PC12 or PC12/NeuN (line I77.2, (9) ) cells (2 10
each) were incubated for 5 min with (+) or without(-)
10 nM heregulin (HRG) in serum-free medium, and cell
lysates were prepared as described under ``Materials and
Methods.'' The lysates were divided equally for
immunoprecipitation with antibodies against Neu (
Neu) or
phosphotyrosine (
PY). A, the precipitates were
analyzed by immunoblotting with antibody against Neu/ErbB2. B,
a separate experiment in which the precipitates were analyzed by
immunoblotting with the 2F12 monoclonal antibody against ErbB3. C, a lysate of PC12/NeuN cells was divided equally for
immunoprecipitation with anti-Neu (
Neu) or anti-ErbB3 (
ErbB3) antibodies. The precipitates were first analyzed
by immunoblotting anti-ErbB3, and then the blot was stripped and
reprobed with anti-Neu.
Lanes 3 and 4 in Fig.1A also show that Neu is tyrosine-phosphorylated in a heregulin-stimulated manner in parental PC12 cells. As expected, the tyrosine phosphorylation of Neu is stronger in Neu transfectants (Fig.1A, lanes 7 and 8), and in fact a doublet is detectable for Neu transfectants treated with heregulin.
Given that we previously had shown that the ErbB3 protein, by binding heregulin, enabled Neu to become responsive to heregulin(10) , we examined whether the addition of heregulin to PC12 cells stimulated the formation of a complex between Neu/ErbB2 and ErbB3. We approached this in a separate experiment using a monoclonal antibody that is highly specific for ErbB3 and does not cross-react with the rat Neu protein (monoclonal 2F12, (15) ; see also Fig.1C) as the first probe (Fig.1B). In the parental PC12 cells, we had a difficult time detecting ErbB3 above background and have been unable to draw any conclusions regarding a heregulin-stimulated ErbB3-Neu complex. However, in cells that overexpress Neu, we can clearly detect ErbB3 in the anti-Neu precipitated pellets, and this appears to be a heregulin-stimulated event (see Fig.1B, lanes 5 and 6). The fact that substantially more of this heregulin-stimulated ErbB3-Neu complex is found in the PC12/NeuN cells compared with the parental PC12 cells suggests that the affinity of Neu for ErbB3 is relatively weak, such that sufficient complex formation can only be detected by immunoprecipitation under conditions where the amount of one or the other of these proteins is relatively high (also see below).
The ErbB3 protein can be clearly detected in anti-phosphotyrosine immunoprecipitates (Fig.1B, lanes 4 and 8). Again, the presence of ErbB3 in these pellets is entirely dependent upon the treatment of cells with heregulin, and the amount of ErbB3 is significantly increased in the PC12/NeuN cells compared with parental PC12 cells. Note that the broad ErbB3 band detected in anti-phosphotyrosine precipitates (Fig.1B, lane 8) appears to contain a component with lesser electrophoretic mobility than that found in the anti-Neu precipitates (this will be considered further below). It should also be noted that although the ErbB4 protein can be detected by Western blotting anti-phosphotyrosine precipitates with a specific anti-ErbB4 antibody, the apparent tyrosine phosphorylation of ErbB4 is not stimulated by heregulin in PC12 cells (data not shown).
Figure 2: Heregulin-stimulated neurite outgrowth in PC12/ErbB3 (C59.3) cells. Cells were incubated in 2 nM heregulin (HRG) in 0.5% fetal bovine serum for 3 days. Neurites were scored as described by Gamett and Cerione(9) .
Also similar
to PC12/NeuN cells is the pattern of 180-kDa proteins precipitated
by the anti-phosphotyrosine antibody in a heregulin-dependent manner
from PC12/ErbB3 cells (Fig.3A). Western blotting with
specific monoclonal antibodies indicates that these proteins include
both Neu and ErbB3 (Fig.3, B and C). In the
case of Neu (Fig.3B, lane 4), a doublet is
detectable in the Western blot (also see Fig.1A, lane 8), suggesting that there are multiple
heregulin-stimulated phosphorylation sites. For ErbB3, we frequently
detect a doublet in anti-phosphotyrosine immunoprecipitates of
heregulin-stimulated cells, (e.g.Fig.4); the more
quickly migrating band was barely visible, however, in the experiment
shown in Fig.3C. The predominant ErbB3 band detected
in anti-phosphotyrosine immunoprecipitates migrated more slowly than
either band detected upon Western blotting these precipitates with the
anti-Neu antibody. Taken together, these results suggest that both the
Neu and ErbB3 proteins are tyrosine-phosphorylated in a
heregulin-dependent manner in the ErbB3 transfectants and that a
phosphorylated ErbB3 protein has distinctly lesser mobility than
either of the phosphorylated forms of the Neu protein.
Figure 3:
Heregulin stimulation of tyrosine
phosphorylation of Neu and ErbB3 in parental PC12 and PC12/ErbB3 cells. A, both immunoprecipitation and immunoblotting were done with
anti-phosphotyrosine (PY) antibodies. B, the
cell lysates were immunoprecipitated with anti-phosphotyrosine
antibodies, and the precipitates were analyzed by immunoblotting with
anti-Neu antibody (
Neu). C, the cell lysates
were immunoprecipitated with anti-phosphotyrosine antibodies, and the
precipitates were analyzed by immunoblotting with the 2F12 monoclonal
antibody against ErbB3 (
ErbB3). HRG,
heregulin.
Figure 4:
Anti-ErbB3 immunoblots showing association
of different forms of ErbB3 protein with Neu and p85. A,
lysates from PC12/NeuN (A1.3) were immunoprecipitated with anti-Neu (Neu) (lanes 1 and 2), and the
supernatants after this precipitation were immunoprecipitated with
anti-phosphotyrosine (
PY) (lanes 3 and 4). B, the lysate of heregulin-stimulated cells was
divided equally for immunoprecipitation with antibodies against
phosphotyrosine or p85 (
p85). The arrow points
to the more slowly migrating form of ErbB3 discussed in the text. IP Ab, immunoprecipitation antibody; HRG,
heregulin.
Interestingly, we find that it is this ErbB3 species that can be co-precipitated with an anti-p85 antibody (Fig.4B). Specifically, lane 1 in Fig.4B shows the ErbB3 doublet obtained when Western blotting an anti-phosphotyrosine precipitate from PC12 cells overexpressing Neu, and lanes 2 and 3 show a similar experiment using a specific anti-p85 antibody to precipitate ErbB3. In direct comparisons, we consistently find that the form of ErbB3 that complexes with p85 has an identical mobility to the upper ErbB3 band detected in anti-phosphotyrosine immunoprecipitates. Again, the ability to co-precipitate ErbB3 and p85 is strictly dependent on heregulin addition to the PC12 cells. These results are consistent with a model where heregulin stimulates the formation of a Neu-ErbB3 complex and the multiple phosphorylation of ErbB3 (see ``Discussion''). One phosphorylated form of ErbB3 is able to remain complexed with Neu, whereas a second phosphorylated form, which migrates with a slower mobility on SDS-polyacrylamide gel electrophoresis, dissociates from Neu and is able to form a complex with p85.
Figure 5:
Comparison of heregulin- and
EGF-stimulated tyrosine phosphorylation of ErbB3. A, PC12 or
PC12/NeuN (I77.2) cells were stimulated for 5 min with 10 nM heregulin or 100 ng/ml EGF (lanes marked H and E, respectively; untreated controls are marked C).
Cell lysates were immunoprecipitated with anti-phosphotyrosine
antibodies (PY), and the precipitates were analyzed by
immunoblotting with anti-phosphotyrosine antibodies (top).
Then the blot was reprobed with anti-ErbB3 (
ErbB3, bottom). B, PC12 or PC12/NeuN (I77.2) cells were
treated as described in A, then cell lysates were
immunoprecipitated with anti-phosphotyrosine antibodies, and the
precipitates were analyzed by immunoblotting with anti-ErbB3
antibody.
When the same anti-phosphotyrosine precipitates are reblotted with an anti-ErbB3 antibody, the overall profile obtained is very similar to that observed in the anti-phosphotyrosine blot (Fig.5A, lower panel). This similarity argues that both heregulin and EGF stimulate the tyrosine phosphorylation of ErbB3 and that the stimulatory effects of heregulin but not EGF are greatly enhanced in PC12 cells overexpressing Neu. In order to be certain that these results were not artifactual due to incomplete stripping of anti-phosphotyrosine antibody prior to reprobing, we repeated the experiment and blotted first with anti-ErbB3 (Fig.5B). Again, the electrophoretic mobilities of the ErbB3 bands are clearly different for heregulin and EGF treatment (compare lanes 5 and 6 of Fig.5B). The heregulin-stimulated cells showed the two forms of ErbB3 described above (Fig.4). The faster mobility ErbB3 band observed upon heregulin treatment co-migrated with the slower mobility EGF-stimulated ErbB3 band. These results suggest that both the EGF receptor and Neu can elicit a common phosphorylation event within the ErbB3 protein and that this phosphorylated ErbB3 species (when generated by heregulin treatment) can remain complexed with Neu. There also is a broad ErbB3 band detected in EGF-treated cells that has a greater mobility than any of the bands detected in heregulin-treated cells.
The experiment
shown in Fig.6tested for the formation of stable complexes
between the various phosphorylated forms of ErbB3 seen in heregulin-
and EGF-stimulated cells with p85. For this, lysates of parental PC12
cells or PC12/NeuN cells were first immunoprecipitated with an anti-p85
antibody, and the precipitates were blotted with the anti-ErbB3
antibody (Fig.6, upper panel). The supernatants
remaining after the anti-p85 precipitation were then immunoprecipited
with anti-phosphotyrosine antibodies, and these precipitates were also
analyzed by blotting with anti-ErbB3 (Fig.6, lower
panel). As shown in the upper panel, we only detect a
p85-ErbB3 complex in heregulin-treated cells (Fig.6, lanes
2 and 5) and not in EGF-treated cells (Fig.6, lanes 3 and 6), and this complex is strongly
stimulated in PC12 cells overexpressing Neu (compare lanes 2 and 5 of Fig.6). Again, the mobility of the ErbB3
band that co-precipitates with p85 is identical to the slower mobility
ErbB3 band detected in anti-phosphotyrosine immunoprecipitates. When
examining the lysates that remain behind following the
immunoprecipitation with anti-p85, we see both heregulin- and
EGF-stimulated phosphoprotein bands in the 180-kDa region. Thus,
whereas EGF stimulates the phosphorylation of ErbB3 (as indicated in Fig.5), this phosphorylated ErbB3 species is not able to form a
stable complex with p85. As will be discussed further below, these
results then point to a model where both the EGF-activated EGF receptor
and the heregulin-activated Neu tyrosine kinase can phosphorylate ErbB3
in a distinct manner and that only ErbB3 proteins that have been
phosphorylated by Neu can go on to form a stable complex with p85 in
PC12 cells.
Figure 6:
Comparison of heregulin- and
EGF-stimulated complex formation between ErbB3 and p85. PC12 and
PC12/NeuN (I77.2) cells were stimulated with heregulin or EGF as
described in the legend for Fig.5. Cell lysates were
immunoprecipitated with anti-p85 antibodies (p85), and
the supernatants were then immunoprecipitated with anti-phosphotyrosine
antibodies (
PY). Both the anti-p85 (top) and
anti-phosphotyrosine (bottom) precipitates were analyzed by
immunoblotting with ErbB3 antibodies (
ErbB3).
It originally was reported that heregulin, a 144-kDa transmembranal glycoprotein with an EGF-like region within its extracellular domain, was the ligand/growth factor for Neu/ErbB2(5) . However, recent studies have indicated that the true receptors for heregulin are the ErbB3 and ErbB4 proteins (10, 12) and that it is heterodimer formation between Neu and ErbB3 (or Neu and ErbB4) that enables the Neu tyrosine kinase activity to be stimulated by heregulin (11) . Here we looked for the possible involvement of ErbB3 or ErbB4 in the neurite outgrowth and tyrosine phosphorylation responses we had previously seen associated with overexpression of Neu in PC12 cells. We found that heregulin stimulates the tyrosine phosphorylation of endogenous ErbB3 protein in PC12 cells and that this phosphorylation, like that of Neu, is greatly enhanced in cells that overexpress Neu. Furthermore, we found that overexpressing the ErbB3 protein in PC12 cells led to heregulin-stimulated neurite extension, similar to the phenotypes obtained upon heregulin addition to cells overexpressing the normal Neu protein or when transforming Neu was expressed in PC12 cells. Two obviously important questions were whether the Neu and ErbB3 proteins actually formed a complex in PC12 cells and if this complex formation was stimulated by heregulin. The results of immunoprecipitation experiments using anti-Neu antibody indicated that the addition of heregulin either to PC12 cells overexpressing Neu or to PC12 cells overexpressing ErbB3 led to the co-precipitation of the Neu and ErbB3 proteins. Thus, overall, these results are consistent with a scheme where heregulin-stimulated heterodimer formation between Neu and ErbB3 results in the increased tyrosine phosphorylation of the Neu and ErbB3 proteins (see below) and accounts for the ability of PC12 cells to respond to this growth factor.
Because it has been suggested that one of the primary effector/targets for phosphorylated ErbB3 is the 85-kDa regulatory subunit (p85) of the phosphatidylinositol 3-kinase(18, 19, 20) , we set out to determine if heregulin addition to PC12 cells might lead to the formation of an ErbB3-p85 complex. Immunoprecipitation experiments using a specific (precipitating) anti-p85 antibody followed by Western blotting with a specific anti-ErbB3 antibody provided evidence for a direct interaction between these proteins. This interaction was most evident in PC12 cells overexpressing Neu and was heregulin-stimulated. When comparing anti-phosphotyrosine immunoprecipitates with anti-85 immunoprecipitates, where in both cases the resuspended pellets were blotted with the anti-ErbB3 antibody, we found that the ErbB3 band that co-precipitated with p85 was identical in mobility to the slowest mobility ErbB3 band detected in anti-phosphotyrosine precipitates. However, this ErbB3 band was not detected in anti-Neu immunoprecipitates. Thus, although it appears that ErbB3 is phosphorylated at multiple tyrosine residues in a heregulin- and Neu-dependent manner, one of these phosphorylated ErbB3 species forms a stable complex with Neu but not with p85.
Overall, these results appear to be consistent with the simple scheme depicted in Fig.7. The binding of heregulin to ErbB3 on the surface of PC12 cells stimulates the formation of a Neu-ErbB3 heterodimer. We would suggest that this results in a higher affinity binding by heregulin (11) and the stimulation of the Neu tyrosine kinase activity. This in turn increases the tyrosine phosphorylation of Neu, probably at multiple sites (e.g.Fig.1), and the trans-phosphorylation of multiple tyrosine residues on ErbB3. The specific mechanism by which heregulin stimulates the phosphorylation of Neu is not clear. One possibility is that Neu is trans-phosphorylated by ErbB3; however, this is problematic because ErbB3 appears to have little or no tyrosine kinase activity(14) . It may be that within a Neu-ErbB3 heterodimer, a weak kinase like ErbB3 is still able to phosphorylate Neu because of the immediate proximity of the substrate. Once activated, the Neu tyrosine kinase should be able to trans-phosphorylate ErbB3. The timing of this trans-phosphorylation event apparently is important, because the extent of ErbB3 phosphorylation appears to influence whether or not ErbB3 remains complexed with Neu or forms a new complex with p85. In fact, these findings raise the interesting possibility that a specific, heregulin-stimulated trans-phosphorylation of ErbB3 by Neu leads to the dissociation of ErbB3 from the heregulin-Neu-ErbB3 ternary complex and promotes the specific binding of ErbB3 to a potential target, p85. Such a mechanism would be similar to hormone receptor/G protein-mediated signaling cascades where the G protein first becomes activated within a hormone-receptor-G protein ternary complex, but then the activated G protein dissociates from this complex and seeks out its target/effector molecule.
Figure 7: A model for heregulin signal transduction involving Neu/ErbB2 and ErbB3 in PC12 cells. Binding of heregulin (HRG) to ErbB3 is followed by formation of a heterodimer of Neu/ErbB2 and ErbB3, within which the Neu tyrosine kinase is activated and ErbB3 becomes phosphorylated. Phosphorylated ErbB3 and Neu/ErbB2 independently complex with p85.
It also is interesting that a potential signaling cascade involving ErbB3 and p85 within PC12 cells appears to be specifically initiated by heregulin and not by EGF. Although the addition of EGF to PC12 cells overexpressing Neu leads both to an apparent tyrosine phosphorylation of Neu (data not shown) and ErbB3 (Fig.5A), neither of these phosphorylated proteins appear to form a stable complex with p85. In the future, it will be interesting to see if the heregulin-Neu-ErbB3-p85 pathway contributes to the specificity observed in the cellular responses triggered by heregulin versus EGF.