(Received for publication, June 20, 1995; and in revised form, October 27, 1995)
From the
The EGF receptor family of tyrosine kinase growth factor
receptors is expressed in a variety of cell types and has been
implicated in the progression of certain human adenocarcinomas. The
most recent addition to this family of receptors, HER4, was expressed
in NIH 3T3 cells to determine its biological and biochemical
characteristics. Cells expressing HER4 were responsive to heregulin
2 as demonstrated by an increase in HER4 tyrosine phosphorylation
and ability to form foci on a cell monolayer. HER4 exhibited in
vitro kinase activity and was able to phosphorylate the regulatory
subunit of phosphatidylinositol 3-kinase and SHC. Peptide competition
studies identified tyrosine 1056 of HER4 as the phosphatidylinositol
3-kinase binding site and tyrosines 1188 and 1242 as two potential SHC
binding sites. Interestingly, transfection of HER4 into NIH 3T3 cells
conferred responsiveness to EGF with respect to colony formation in
soft agar. It was also found that in response to heregulin
2,
endogenous murine HER1 or transfected human HER1 became phosphorylated
when HER4 was present. This demonstrates that HER1 and HER4 can exist
in a heterodimer complex and likely activate each other by
transphosphorylation.
The human epidermal growth factor receptor (HER) ()family of type I receptor tyrosine kinases has been linked
to the progression of certain human adenocarcinomas. The mechanism by
which these receptors function with respect to tumor development is
thought to involve overproduction of the gene product resulting from an
increase in gene copy number or
expression(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
Receptor overexpression often correlates with a high level of
constitutive tyrosine kinase activity, and this activity is thought to
promote signals leading to uncontrolled cell growth (13) .
Several ligands that bind to and stimulate the kinase activity of
the HER family members have been identified and are classified as
EGF-like ligands. EGF, amphiregulin, betacellulin, and transforming
growth factor are specific for HER1(14) . Heregulin and
its rat homologue neu differentiation factor, are a subfamily of the
EGF-like ligands that have been shown to bind to and activate both HER3
and HER4 (15, 16, 17, 18, 19, 20, 21, 22) .
The molecular characterization of a HER2 ligand has not been reported;
however, a number of factors have been shown to activate HER2 tyrosine
kinase activity specifically(23, 24, 25) .
Activation of the EGF receptor family of tyrosine kinase receptors, using EGFR as the model, is thought to involve the binding of ligand and subsequent homodimerization of the receptor resulting in a conformational change and activation of the intrinsic tyrosine kinase activity(58) . Recent advances suggest that a more complex mechanism of HER activation can occur. Heregulin has been shown to bind and stimulate both HER3 and HER4(15, 26) . Thus, the signaling repertoire of any one ligand can be multifaceted depending upon the cellular receptor expression patterns, potentially allowing the same ligand to deliver distinct downstream signals. Further, ligand-induced receptor activation in the HER family is not limited to homodimerization, for it has been demonstrated that many of the HER family members can associate with and activate each other(15, 27, 28, 29, 30) . HER2 has been shown to heterodimerize with HER1, HER3, and HER4, and EGF stimulation of HER1 has been shown to activate HER3 signaling(31) . Therefore, the signaling of receptors by multifunctional ligands on homoreceptor and heteroreceptor complexes could result in a multiplicity of downstream signaling events.
Activation of growth factor receptors results in the transmission of
stimulatory signals from the outside to the inside of the cell. This
process makes use of intracellular proteins, or targets, that contain
Src homology region 2 (SH2) domains that recognize and bind to the
activated receptor(32, 33) . A number of these
proteins have been identified and shown to interact with
membrane-associated and cytoplasmic tyrosine
kinases(32, 34, 35, 36) . The
specificity of their binding is dictated by receptor sequences that
flank a specific phosphotyrosine residue(33, 37) .
Since the HER family members are similar but not identical in their
amino acid sequence, an expected diversity of associations with
different SH2-containing proteins has been demonstrated. HER1 has been
shown to associate with phospholipase C-, SHC, and
Grb2(13, 38, 39, 40, 41, 42) .
HER2 can associate with phospholipase C-
, SHC, and Ras-GTPase
activating protein(43, 44, 45) . HER3 has
recently been shown to activate PI
3-kinase(31, 41, 46) .
We have focused on characterizing the newest member of the HER family, HER4, with regard to its biological activity and activation of downstream targets. To this end, a collection of HER4-expressing 3T3 cell lines has been generated and used to assay the biological and enzymatic activities of this receptor.
Figure 1:
HER4 focal
transforming activity. HER4-transfected cells were mixed with parental
NIH 3T3 cells and cultured either in the presence or absence of ligand
at a concentration of 20 ng/ml. Cultures were fluid-changed every third
day and maintained for 2 weeks. Cell monolayers were stained with a
mixture of methylene blue/carboyl fuschia dyes. rHRG,
heregulin 2.
Figure 2:
HER4 tyrosine phosphorylation is
potentiated by ligand. Cell extracts from control (0) and
heregulin 2-stimulated transfectants (HRG) were boiled
immediately in SDS sample buffer and analyzed on SDS-PAGE followed by
an antiphosphotyrosine immunoblot (A). Control and heregulin
2-stimulated HER4-expressing cells were lysed in the presence or
absence of Mg
, and extracts were precipitated with
anti-HER4 6-4-11 followed by analysis in an antiphosphotyrosine
immunoblot (B).
Figure 3:
HER4 in vitro kinase activity.
Antiphosphotyrosine immunoprecipitates of control and ligand-stimulated
cell lines were assayed in an in vitro kinase reaction. P-Labeled receptor complexes were analyzed on SDS-PAGE and
visualized by exposure to a PhosphorImager screen. The arrow points to radiolabeled receptor.
Figure 4: HER4 can associate with SHC and PI 3-kinase. A, antiphosphotyrosine immunoprecipitates from control and ligand-stimulated cell lines were labeled in an in vitro kinase assay. Labeled complexes were denatured by boiling in solubilization buffer, and 85-kDa PI 3-kinase (A) and SHC (B) were reimmunoprecipitated with specific antisera. Purified immune complexes were analyzed on SDS-PAGE, and gels were dried down and exposed on a PhosphorImager screen.
Figure 5: Identification of the PI 3-kinase and SHC binding sites on HER4. A, peptides were synthesized corresponding to potential tyrosine phosphorylation sites within the C-terminal domain of HER4. The peptide number correlates with the amino acid number of the tyrosine residue. The underlined sequences represent potential PI 3-kinase or SHC binding motifs. B, immunoprecipitated HER4 was radiolabeled in an in vitro kinase assay and denatured by boiling in solubilization buffer. Denatured HER4 was then reprecipitated using GST fusions of PI 3-kinase N- and C-terminal SH2 domains and full-length SHC. Peptides corresponding to regions of HER4 were added at a concentration of 50 µM with the GST fusions during the precipitation. Complexes were boiled in SDS sample buffer and analyzed by SDS-PAGE.
Figure 6:
EGF and heregulin 2 induce colony
growth in soft agar. HER4-transfected cells were plated either in the
presence or absence of EGF or heregulin
2 at a concentration of
100 ng/ml in 0.4% agar. Cultures were allowed to grow for 3 weeks and
photographed directly.
If
HER1 and HER4 can functionally interact and elicit a growth response,
then stimulation with the appropriate ligand might result in
cross-phosphorylation of one receptor by the other. Stimulation of
parental 3T3 cells resulted in increased tyrosine phosphorylation of
mouse EGFR in response to EGF but not heregulin (Fig. 7A).
However, in cells transfected with HER4, tyrosine phosphorylation of
the endogenous EGFR was elevated in the absence of ligand and further
increased in response to both EGF and heregulin 2. Increasing the
level of EGFR expression also increased the level of heregulin
2-induced EGFR tyrosine phosphorylation. In contrast, there was no
detectable increase in HER4 phosphorylation in response to EGF (Fig. 7B). These results suggest that HER1 and HER4 can
exist in a heterodimer complex and that transphosphorylation can occur
in one direction, in that activation of HER4 results in the
phosphorylation of HER1 but HER4 is not a substrate for HER1.
Figure 7:
Complex formation between HER1 and HER4.
NIH 3T3 cells expressing HER1, HER4, or both were stimulated with
either EGF or heregulin 2. Receptors were immunoprecipitated
following cell lysis and analyzed on SDS-PAGE followed by transfer onto
nitrocellulose. The blot was probed with the indicated antibody
followed by
I-labeled protein
A.
The activation of HER family members, as well as their
respective signaling through downstream targets is different for each
receptor. Further, the complexity of these signaling pathways becomes
even more diverse due to receptor heterodimerization, which may alter
the specificity or strength of response to a given ligand. It has been
demonstrated that HER1, HER2, and HER3 can interact with one another (15, 27, 28, 29, 30, 31) .
It has also been shown that HER4 activation can result in the
phosphorylation of HER2. In this paper, we describe some of the
properties of HER4 that are similar and different from those of other
family members. We were able to demonstrate that HER4 can also complex
with HER1, resulting in an enhanced growth signal in agar in response
to EGF and the transphosphorylation of EGFR by HER4 in response to
heregulin 2. The endogenous level of EGFR tyrosine phosphorylation
was elevated in cells co-expressing HER4, suggesting that the
heterodimer may exist at low levels in the absence of ligand.
Increasing the level of EGFR by transfecting HER1 resulted in a higher
level of both ligand-independent and heregulin-stimulated HER1
phosphorylation, suggesting that the stoichiometry of receptor
heterodimers is dependent upon receptor expression levels. We did not
observe an EGF-dependent phosphorylation of HER4, which suggests that
HER4 is not a substrate for HER1. Nevertheless, EGF could induce colony
formation in cells expressing HER4, which suggests that a HER1-HER4
complex is being formed. It remains to be investigated whether or not
HER4 and HER3 can form a complex that would complete the possible
combinations of currently known HER family member heterodimerizations.
Biologically, we have shown that HER4 can stimulate 3T3 cells to
grow and overcome cell-to-cell contact inhibition at a low level in the
absence of exogenous ligand and that this activity can be further
induced by the addition of heregulin 2 but not EGF. Conversely,
both heregulin
2 and EGF were able to induce the growth of
colonies in soft agar in HER4-expressing cells but not the control NIH
3T3 cells. It is unclear why there is a difference in EGF
responsiveness between the focal transformation assay and the agar
assay. Perhaps the heterodimer complex is providing the necessary
downstream signals needed for growth in soft agar but not the signals
needed to overcome contact inhibition.
In analyzing HER4 tyrosine
phosphorylation in rapidly denatured extracts, HER4 is slightly
phosphorylated in the absence of ligand and can be further induced by
the addition of heregulin 2. Immunoprecipitation of HER4 in the
presence of Mg
results in the in vitro stimulation of HER4 tyrosine phosphorylation and stimulation of
HER4 in vitro kinase activity. This activation, in the absence
of ligand, is likely a result of concentrating the receptor by
immunoprecipitation and is unique to HER4, for it does not occur with
HER1, HER2, and HER3. Removal of Mg
from the lysis
buffer restores ligand responsiveness to our biochemical analysis.
While HER1 and HER4 are both ligand-responsive, HER2 is constitutively
hyperphosphorylated.
The in vitro kinase activity associated with these receptors provided a means to identify downstream targets activated by these receptors. HER4 was able to induce the phosphorylation of both SHC and 85-kDa PI 3-kinase. HER1 could activate SHC but not 85-kDa PI 3-kinase, which identifies a divergence of activities between HER1 and HER4. HER2 kinase activity and subsequent association with downstream targets could not be determined due to the lack of in vitro kinase activity associated with the receptor. HER2 is constitutively tyrosine-phosphorylated and appears to associate with downstream targets in other types of assays(43, 44, 45, 53, 54) . The lack of HER2 in vitro kinase activity remains to be investigated.
The association of SH2-containing proteins with growth factor receptors is integral in transmitting growth-stimulatory signals. The elements required for the association of SH2-containing proteins to tyrosine kinases involve a phosphorylated tyrosine residue flanked by specific sequences. Specifically, YXXM (34) and NPXY (37) are essential motifs for the binding of PI 3-kinase and SHC, respectively, to their target kinases. The C-terminal domain of HER4 contains one YXXM motif (YTMP). Peptide 1056, corresponding to this motif, was able to inhibit binding of both N- and C-terminal PI 3-kinase SH2 domains. HER1 does not contain a YXXM motif in its C terminus, which correlates with the inability to phosphorylate the 85-kDa subunit of PI 3-kinase in our assay. These data confirm the essential nature of the YXXM motif in HER4 for binding PI 3-kinase and that each of the PI 3-kinase SH2 domains alone is sufficient for association with HER4.
HER4 contains three potential SHC binding sites (NPXY) within its C-terminal coding sequence located at amino acids 1188, 1242, and 1284. Full-length SHC was able to form a complex with HER4, and this interaction was inhibited by peptides 1188 and 1242. This suggests that there are two potential SHC binding sites on HER4. Recent observations have identified a second motif in the N-terminal portion of SHC, distinct from the SH2, that is able to associate with tyrosine-phosphorylated EGFR (55) and a SHC-associated protein, p145 (56) . Identification of the domain within SHC required to associate with HER4 is under investigation.
The relationship between the HER family of receptors
and their ability to respond to similar ligands resulting in both homo-
and heterodimerized complexes lends itself to a very complex system of
cell signaling. Since multiple HER members are often expressed
concomitantly, the specific combination and relative level of
expression of different receptors will determine the response to a
given ligand. This is evident in human tumor cell lines in which
heregulin can induce either a mitogenic (21, 57) or a
terminally differentiating signal(17) , depending upon the cell
type. Thus, altering the pattern of expression of HER family members
may offer a selective growth advantage during tumor progression in the
presence of HER family ligands. It has been shown that overexpression
of several EGF-like growth factors, such as transforming growth factor
, amphiregulin, and cripto-1, represents a hallmark feature of
many solid tumors. A better understanding of the downstream signals
produced from both receptor homo- and heterodimers, as well as the
ligands involved, is critical to a better understanding of the biology
of HER family signal transduction and its role in tumor progression.