The traditional view of growth factor receptors and hormone receptors in general is
that a specific ligand directly recognizes a highly selective binding site on its
cognate receptor and, thereby, activates receptor-dependent signaling and biological
responses. In the case of the EGF receptor, several structurally related proteins
(EGF, transforming growth factor alpha, amphiregulin, betacellulin, epiregulin, heparin-binding
EGF) are recognized as direct agonists. Each of these growth factors binds to the
ectodomain of the EGF receptor and provokes its activation through a mechanism that
involves dimerization, activation of the receptor tyrosine kinase cytosolic domain,
and autophosphorylation of the receptor. This process initiates signaling pathways
that lead to mitogenesis.
Recently it has become apparent that the EGF receptor is also part of signaling networks
activated by stimuli that do not directly interact with this receptor. These stimuli
include agonists that specifically bind to other membrane receptors, membrane depolarization
agents, and environmental stressors. The data not only show EGF-independent tyrosine
phosphorylation of the EGF receptor, but also provide experimental evidence that the
EGF receptor participates in the signaling events and cellular responses initiated
by these various stimuli. Collectively, the results imply that in the absence of direct
agonists the EGF receptor is employed in a wide array of biological signaling processes.
The purpose of this article is to review and evaluate these nonclassical uses of the
EGF receptor.
G Protein–coupled Receptor Agonists
Agonists for a diverse group of G protein–coupled receptor (GPCR)1 agonists (purogenic,
muscarnic acetylcholine, angiotensin, lysophosphatidic acid [LPA], thrombin, endothelin,
adrenergic, and bombesin) have been demonstrated to bring about increased levels of
phosphotyrosine on the EGF receptor (reviewed in Hackel et al. 1999; Luttrell et al.
1999a). Since GPCRs can induce cell proliferation in certain circumstances (reviewed
in Gutkind et al., 1998), these observations suggest that GPCR-dependent mitogenic
activity involves receptor networking that couples GPCRs to a growth factor receptor
tyrosine kinase. The important issue is whether the EGF receptor is a necessary component
for GPCR mitogenic signaling. Numerous reports have now demonstrated that either over-expression
of a dominant-negative EGF receptor or the presence of a specific pharmacologic inhibitor
of EGF receptor tyrosine kinase activity significantly uncouples GPCR-driven mitogenic
responses (Daub et al. 1996, Daub et al. 1997; Tsai et al. 1997; Zwick et al. 1997;
Cunnick et al. 1998; Eguchi et al. 1998; Gohla et al. 1998, Gohla et al. 1999; Iwasaki
et al. 1998; Keely et al. 1998; Li et al. 1998; Murasawa et al. 1998; Soltoff 1998;
Vainganker and Martins-Green, 1998; Adomeit et al. 1999; Della Rocca et al. 1999;
Eguchi et al. 1999; Moriguchi et al. 1999). In these reports, a variety of biological
endpoints (activation of MAP kinase, tyrosine phosphorylation of known substrates,
gene expression, stress fiber formation, DNA synthesis) have been measured in both
transfected and untransfected cell lines as well as primary cells. In a different
approach, Cunnick et al. 1998 demonstrated that B82L cells (an EGF receptor negative
cell line) did not produce a mitogenic response to LPA unless exogenous EGF receptors
were expressed. When kinase-negative EGF receptors were expressed in these cells,
LPA did not produce a mitogenic response. Hence, the capacity of GPCRs to transduce
a mitogenic response requires an EGF receptor and its tyrosine kinase activity. The
consistency of these reports reinforces the overall conclusion and its biological
significance. Clearly, EGF receptor transactivation may be only one of several independent
pathways emanating from GPCRs and inputs from other pathways may also be essential
for mitogenic signaling. Bradykinin stimulation of protein kinase C seems to be such
a required pathway functioning in parallel to EGF receptor–dependent signaling (Adomeit
et al. 1999).
In nearly all examples cited, heterologous modulation of the EGF receptor occurs too
quickly to invoke a mechanism involving the induced synthesis of EGF-like ligands
and in some cases this possibility has been directly tested and ruled out. Less well
explored, however, is the possibility that heterologous stimuli might stimulate the
proteolytic release of cell surface EGF-like growth factor precursors to soluble and
diffusible receptor agonists. Since GPCR activation produces a small amount of EGF
receptor tyrosine phosphorylation compared with a saturating dose of EGF, only a low
level of growth factor would have to be produced. However, in view of other data cited
below and in the absence of direct data, this mechanism seems unlikely at present.
The mechanism by which GPCRs actually bring about tyrosine phosphorylation of the
EGF receptor is centered on the mediation of the non-receptor tyrosine kinase c-Src,
which is reported to be coupled to nearly all GPCRs that lead to EGF receptor phosphorylation
(reviewed in Thomas and Brugge 1997). Overexpression of either a dominant-negative
Src construct or Csk, a regulatory kinase that inhibits Src function, decreases EGF
receptor tyrosine phosphorylation provoked by activation of LPA or α2 adrenergic receptors
(Luttrell et al. 1997). The mediator role of Src may, in fact, be direct in that Src
is able to associate with and phosphorylate the EGF receptor in vivo and in vitro
(Thomas and Brugge 1997). This mechanism would predict the existence of Src–EGF receptor
complexes provoked by activation of GPCR. The evidence for this is shown by the demonstrations
that angiotensin II (Eguchi et al. 1998) or LPA (Luttrell et al. 1997) rapidly increase
the amount of Src coprecipitated with EGF receptors.
The EGF receptor autophosphorylates at five known tyrosine residues after the addition
of EGF. Src-induced EGF receptor phosphorylation has been mapped to most of these
sites as well as novel sites. Of particular interest is phosphorylation of Tyr 845
in the EGF receptor, which has been mapped for both in vivo and in vitro Src phosphorylation
and which is not a known autophosphorylation site (Sato et al. 1995; Biscardi et al.
1999). This residue is highly conserved in tyrosine kinases and in many kinases has
a regulatory role. In the EGF receptor, Tyr 845 is predicted to be in the activation
loop of the tyrosine kinase domain and, therefore, could function as an activation
trigger to increase activity of the kinase domain. There are several indications that
the kinase catalytic activity of the EGF receptor is necessary to mediate GPCR-dependent
mitogenic responses. First, chemical inhibitors reasonably specific for the EGF receptor
tyrosine kinase and competitive with ATP, which suggests an active site mechanism,
block GPCR induction of mitogenic responses. Second, a point mutant, kinase-negative
EGF receptor does not support GPCR mitogenic signaling (Cunnick et al. 1998). Therefore,
it seems unlikely that the receptor is simply phosphorylated by Src and acting as
an inert docking site for SH2-containing signal transducers. An outstanding issue
is whether GPCRs and/or Src induce dimerization of EGF receptors, a hallmark of EGF-dependent
receptor activation. There is but one report (Tsai et al. 1997) which describes carbachol-induced
EGF receptor dimerization.
Mutagenesis of Tyr 845 in the EGF receptor does not attenuate EGF-dependent receptor
autophosphorylation or signaling, but does prevent DNA synthesis induction by the
GPCR agonist LPA (Gotoh et al. 1992; Tice et al. 1999). However, reports of this mutant
include an inconsistency. In the latter report this mutant also attenuated EGF-induced
DNA synthesis, while in the former report it did not. Recently, cells genetically
deficient for multiple Src family kinase have been described (Klinghoffer et al. 1999).
It will be informative to test the Src requirement for GPCR coupling to the EGF receptor
in those cells. That Src may act upstream of the EGF receptor is complicated by the
fact that EGF often produces Src activation. Hence, depending on the agonist Src may
be upstream and/or downstream of the EGF receptor.
The means by which GPCRs activate Src is not understood. However, there is sufficient
data to support two alternative potential mechanisms. Many GPCRs that lead to EGF
receptor phosphorylation are coupled to G proteins that activate phospholipase C activity
provoking Ca2+ mobilization. Ca2+ mobilization can lead to the activation of the cytoplasmic
tyrosine kinase Pyk2. There is evidence that in certain cell types after GPCR activation,
Pyk2 can associate with Src and that this association may lead to Src activation,
through a mechanism that is unclear (Dikic et al. 1996; Eguchi et al. 1999; Sayeski
et al. 1999). This putative pathway proceeds as follows: agonist → GPCR → heterotrimeric
G protein → PLC → IP3 → Ca2+ → Pyk2 → Src → EGF receptor. However, this pathway cannot
explain all GPCR activation of Src, as not all agonists that activate PLC activity
bring about Src activation. While dominant-negative forms of Pyk2 have been shown
to block GPCR activation of MAP kinases, these assays have not included tyrosine phosphorylation
of the EGF receptor. Also, the expression of Pyk2 is not ubiquitous and the above
scenario may not be applicable in all cells.
Recently a second mechanism has received experimental support (Luttrell et al. 1999b).
In this scheme, developed with β2 adrenergic receptors, receptor desensitization is
coupled to Src activation. After its activation cycle, the β-adrenergic receptor is
phosphorylated by specific serine/threonine protein kinases termed βark kinases. These
receptor phosphorylation sites then bring about association of the adaptin-type molecule
β-arrestin, which recruits the receptor into coated pits. Evidence is presented that
Src is recruited to the adrenergic receptor–β-arrestin complex by interacting with
β-arrestin and is activated by this interaction. Such a pathway would proceed as follows:
agonist → GPCR → heterotrimeric G protein → βark phosphorylation of GPCR → arrestin:
GPCR complex → GPCR-arrestin-Src complex → EGF receptor.
Cytokine Receptors
This large family of receptors is coupled to JAK family non-receptor tyrosine kinases.
Recently it has been reported that activation of the growth hormone or prolactin receptors
leads to Jak2-dependent tyrosine phosphorylation of the EGF receptor (Yamaguchi et
al., 1997). In cells expressing the EGF receptor, growth hormone was able to promote
GRB-2 association with the EGF receptor, MAP kinase activation, and c-fos induction.
These growth hormone–dependent events were also produced with a kinase-negative EGF
receptor indicating that only the adaptor-docking function of the EGF receptor was
essential and not receptor kinase activity. This is in contrast to the previously
described EGF receptor kinase activity requirement for GPCR-induced mitogenesis. The
mechanism by which growth hormone stimulates tyrosine phosphorylation of EGF receptor
was shown to include JAK-2 activation and the formation of growth hormone receptor–EGF
receptor heterodimers. Src participation was ruled out. In vitro experiments indicated
that JAK-2 may directly phosphorylate the EGF receptor at sites that include Tyr 1086,
a known GRB-2–association site.
Adhesion Receptors
While there is considerable experimental evidence that activation of integrin receptors
and cell adhesion in general can modulate EGF responses and postreceptor signaling
events, the possibility that integrins influence EGF receptor function, per se, is
suggested by two reports. In one study beads coated with ligands that induce integrin
aggregation and activation were added to cells and observed to induce the clustering
of EGF receptors around the beads (Miyamoto et al. 1996). This suggested the coaggregation
of integrin and EGF receptors. Other growth factor receptors (platelet-derived growth
factor, fibroblast growth factor) were also coclustered with the beads. While the
EGF receptors that clustered around these beads were not tyrosine phosphorylated,
the growth factor–dependent activation of the EGF receptor was enhanced by the beads.
This could be explained either by exclusion of phosphatases from the clusters or by
the fact that preclustering increased cross-phosphorylation of EGF receptors after
ligand addition. Beads with ligands that induced only integrin receptor aggregation
and not activation did not produce EGF receptor clustering around the beads.
Subsequently, a second report (Jones et al. 1997) presented similar observations from
a system in which tenascin C, a collagen-binding glycoprotein that also binds to and
activates integrin receptors, was added to smooth muscle cells. However, in this cell
system cross-linking of integrin receptors, without activation, was sufficient to
induce EGF receptor clustering.
In a more recent study using fibroblasts and endothelial cells, cell plating on a
fibronectin matrix was shown to produce rapid and transient tyrosine phosphorylation
of the EGF receptor (Moro et al. 1998). Similar results were obtained when the cells
were plated on a matrix coated with integrin receptor antibodies that cluster but
do not activate integrin receptors. Cell adhesion mediated by poly-l-lysine did not
activate EGF receptors in this system. Mechanistically, it was shown that the β1 integrin
receptor subunit coprecipitated EGF receptors in a manner that depended on cell adhesion,
i.e., coprecipitation did not occur in cells kept in suspension before lysis. Also,
the capacity of fibronectin to increase EGF receptor tyrosine phosphorylation was
abrogated by the EGF receptor selective inhibitor AG1478 and did not occur in cells
expressing a kinase-negative receptor mutant. These results imply that receptor phosphorylation
is a consequence of fibronectin-enhanced activity of the receptor tyrosine kinase.
Also, this study used both the AG1478 inhibitor and a dominant-negative EGF construct
to show that fibronectin activation of MAP kinase requires EGF receptor function.
Similar reagents also were used to demonstrate that EGF receptor function was necessary
to protect cells from apoptosis when plated on fibronectin in the absence of growth
factors. Further evidence with selective chemical inhibitors indicate the role of
the EGF receptor in mediating this resistance to apoptosis was, in fact, not due to
MAP kinase activation but rather seem dependent on phosphatidylinositol 3-kinase activity.
It should be pointed out that integrin-dependent tyrosine phosphorylation of PDGF
receptors has been reported (Sunberg and Rubin, 1996; Schneller et al. 1997) and the
means by which integrins communicate with receptors tyrosine kinases may lead to the
activation of multiple growth factor receptors, depending on the cells employed.
Finally, the collagen-binding proteoglycan decorin has been reported to produce in
A-431 cells tyrosine phosphorylation of the EGF receptor and to mobilize Ca2+ and
activate MAP kinase in a manner dependent on the tyrosine kinase activity of the EGF
receptor (Moscatello et al. 1998; Patel et al. 1998). Decorin is not known to interact
with integrin or other cell adhesion receptors, but a recent report suggest that it
may, in fact, interact directly with the EGF receptor as a low-affinity agonist (Iozzo
et al. 1999).
Membrane Depolarization
In many cells and, in particular, cells of the nervous system, electrical activity
initiates intracellular signaling pathways and the generation of cellular responses,
such as secretion. In PC-12 cells the application of KCl leads to altered electrical
potential across the plasma membrane and activation of the Ras/MAP kinase pathway.
The initiating event seems to be an influx of extracellular Ca2+ elicited by the activation
of voltage-sensitive Ca2+ channels. The artificial influx of Ca2+ by ionophore treatment
can mimic these responses. In these cells, increased levels of intracellular Ca2+
result in enhanced levels of EGF, but not insulin or nerve growth factor, receptor
tyrosine phosphorylation (Rosen and Greenberg 1996). Transient expression of dominant-negative
EGF receptors or application of the selective EGF kinase inhibitor AG1478 prevents
the capacity of KCl or ionomycin to increase EGF receptor tyrosine phosphorylation
and activate MAP kinases in PC12 cells (Zwick et al. 1997). Since the EGF receptor
is widely expressed in cells of the nervous system, these results may suggest it has
role in either the nonmitogenic signaling events present in these specialized cells
or in preventing programmed cell death.
The means by which intracellular Ca2+ levels may provoke EGF receptor tyrosine phosphorylation
are thought to revolve about the Pyk2 and Src families of tyrosine kinases, which
are both known to be activated by membrane depolarization (Thomas and Brugge 1997).
This pathway (Ca2+ → Pyk → Src → EGF receptor) is analogous to that described previously
for GCPRs. Interestingly, calmodulin-dependent protein kinase has been implicated
in EGF receptor tyrosine phosphorylation after membrane depolarization by KCl, but
not by GPCRs (Zwick et al. 1999).
Stress Response
The application of certain exogenous stimuli, both physical and chemical, initiates
signal transduction pathways in cells that are part of stress responses. Typically,
such stimuli activate members of the MAP kinase family and provoke the expressions
of genes. The following stress stimuli have been shown to increase the level of tyrosine
phosphate on the EGF receptor; arsenite (Chen et al. 1998), sulfhydryl reagents (Knebel
et al. 1996), UV radiation (Zheng et al. 1993; Warmuth et al., 1993 and later references
below), gamma irradiation (Schmidt-Ullrich et al. 1996, Schmidt-Ullrich et al. 1997;
Goldkorn et al. 1997), hyperosmotic conditions (King et al. 1989; Rosette and Karin
1996), oxidants (Knebel et al. 1996; Rao 1996; Höker et al., 1998; Peus et al. 1998),
and heat shock (Lin et al. 1997). The tyrosine phosphorylation of other receptor tyrosine
kinases is also affected and the influence on the EGF receptor can not be characterized
as specific. However, in a number of instances the capacity of these stressors to
activate MAP kinases and provoke the expression of certain genes is blocked by selective
chemical inhibitors of EGF receptor tyrosine kinase activity and/or by the expression
of dominant-negative EGF receptors. These results imply that EGF receptor involvement
is a necessary element for the initiation of signaling in response to such stimuli.
Perhaps the most investigated stress stimulus, as it relates to the EGF receptor,
is UV radiation. UVA, UVB, and UVC have all been shown to very rapidly, within seconds,
promote enhanced tyrosine phosphorylation of the EGF receptor. Also, UV induces the
phosphotyrosine-dependent association of signaling molecules, such as GRB-2, with
the receptor and induces the tyrosine phosphorylation of EGF receptor substrates,
such as Shc and PLC-γ1 (Huang et al. 1996; Knebel et al. 1996; Rosetto and Karin,
1996). That functional receptors are produced by UV exposure is also indicated by
studies demonstrating the formation of receptor dimers, receptor aggregation, and
receptor internalization (Rosette and Karin 1996). Hence, by many parameters, UV seems
to provoke receptor activation that mimics addition of a direct ligand. It is clear,
however, that UV does not provoke receptor activation by a means that involves autocrine
production of EGF-family agonist. Less clear, though unlikely, is the possibility
that stressors promote the proteolytic release of an EGF family molecule from cell
surface precursors. However, as UV activates v-erbB, an oncogenic isoform of the chicken
EGF-receptor that lacks a ligand-binding domain, the possible role of ligand involvement
would seem unlikely (Knebel et al. 1996).
That it is the kinase activity of the EGF receptor that promotes receptor autophosphorylation
is indicated by the fact that selective kinase inhibitors, such as AG1478 (Knebel
et al. 1996) and tyrphostin 23 (Miller et al. 1994) block UV-induced EGF receptor
phosphorylation. Similarly, expression of a dominant-negative EGF receptor prevents
UV-induced receptor phosphorylation presumably by the formation of dimers incapable
of cross-phosphorylation (Huang et al. 1996). Finally, in cells expressing a kinase-negative
EGF receptor, UV exposure did not increase receptor tyrosine phosphorylation (Coffer
et al. 1995). Hence, the evidence suggests that UV stress activates the kinase domain
and that EGF receptor phosphorylation is not primarily a consequence of phosphorylation
by other kinases. There is one reported exception in cells expressing kinase-negative
EGF receptors and wild-type ErbB-2 (Knebel et al. 1996). UV does activate ErbB-2 and
ErbB-2, which heterodimerizes with EGF receptors, is able to cross-phosphorylate the
EGF receptor. While UV does seem to mimic growth factor activation of the receptor,
no phosphotyrosine maps have been published for UV-activated EGF receptors. Therefore,
it is not clear that UV and growth factors result in identical receptor activation.
UV treatment of cells activates a plethora of signaling pathways. Experiments using
chemical inhibitors of the EGF receptor or expression of dominant-negative receptor
mutants show that the EGF receptor mediates, at least in part, several UV-induced
signaling events, including activation of erk1 and 2, production of prostaglandins
and leukotrienes, and the expression of several genes including c-fos and erg-1 (Miller
et al. 1994; Huang et al. 1996; Sachsenmaier et al., 1996).
Less clear is the mechanism by which UV actually activates the EGF receptor. The prevailing
evidence would suggest that the activation is indirect through inactivation of phosphotyrosine
phosphatase activity. UV is known to produce reactive oxygen species in cells, perhaps
through the generation of H2O2. Interestingly, EGF also promotes H2O2 formation in
cells (Bae et al. 1997). Several studies have shown that preincubation with antioxidants,
such as N-acetylcysteine, prevent UV but not EGF-induced receptor autophosphorylation
(Huang et al. 1996; Knebel et al. 1996; Miller et al. 1994; Peus et al. 1998). Reducing
agents also protect against UV-induced receptor activation. As the catalytic sites
of phosphotyrosine phosphatase have a highly conserved sulfhydryl group as an essential
element, this is the likely target for oxidation-induced by UV. There is experimental
evidence at the biochemical level which shows that a phosphatase-containing membrane
preparation can dephosphorylate a second membrane preparation that contains activated
EGF receptor (Knebel et al. 1996). The rate of receptor dephosphorylation in this
system is markedly decreased by UV treatment of the phosphatase preparation before
its addition to EGF receptors. This result implies that basal EGF receptor kinase
activity is quite significant, which is true of purified receptor preparations.
It is interesting that various stimuli provoke EGF receptor tyrosine phosphorylation
by two distinct means. While physical and chemical stressors inactivate downstream
phosphotyrosine phosphatases, heterologous receptors and membrane depolarization bring
about a similar result by activating upstream tyrosine kinases. It is possible, however,
that this division into separate mechanisms is not absolute and some level of contribution
by kinases and phosphatases exists with each stimulus, as recently described for carbachol
regulation of the Kv1.2 potassium channel (Tsai et al. 1999).