Vaccinia virus and the EGF receptor: A portal for infectivity?

We previously demonstrated that occupancy of the epidermal growth factor (EGF) receptor reduced the ability of vaccinia virus to infect L cells [Eppstein et al: Nature 318:663, 1985]. This result suggested that vaccinia virus was utilizing the EGF receptor as one pathway to infect cells. We have studied this system further, and now find that antibodies to the EGF receptor also reduce the ability of vaccinia virus to infect cells productively. Inclusion of both EGF and antibodies to the EGF receptor did not cause inhibition over that obtained by EGF alone, providing another line of evidence that the antiviral effects on vaccinia virus were at the level of the EGF receptor. The antiviral effects of EGF or synthetic peptides corresponding to the third disulfide loop of TGF-alpha or the vaccinia virus growth factor were specific to vaccinia virus and did not inhibit replication of herpes simplex virus type 2 or vesicular stomatitis virus. The inhibitory effects on replication of vaccinia virus were obtained when EGF (but not insulin or growth hormone) was present prior to, but not after, productive viral adsorption. These results provided further evidence that the antivaccinia viral effects of EGF were at the level of initial receptor occupancy. As interferon (IFN) treatment has been shown to interfere with the action of some growth factors, including EGF, we examined the effects of IFN treatment of cells on the antivaccinia viral activity of EGF. Our results show that the antivaccinia effect of IFN-beta either interfered with or partially coalesced with the inhibitory effects of EGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

The C11R Gene, Which Encodes the Vaccinia Virus Growth Factor, Is Partially Responsible for MVA-Induced NF-κB and ERK2 Activation

MVA is an attenuated strain of vaccinia virus (VACV) that is a popular vaccine vector. MVA infection activates NF-κB. For 293T cells, it is known that MVA early gene expression activates extracellular signal-regulated kinase 2 (ERK2), resulting in NF-κB activation. However, other viral and cellular mechanisms responsible for this event are ill defined. The data presented here show that the epidermal growth factor receptor (EGFR) is at least one apical trigger in this pathway: ERK2 and NF-κB activation was diminished when MVA infections occurred in cells devoid of the EGFR (CHO K1 cells) or in the presence of a drug that inhibits EGFR activation (AG1478) in 293T cells. The expression of dominant negative Ras or Raf proteins still permitted NF-κB activation, suggesting that a nonclassical EGFR-based signal transduction pathway triggered ERK2-NF-κB activation. C11R is an early gene present in MVA and other orthopoxviruses. It encodes the soluble, secreted vaccinia virus growth factor (VGF), a protein that binds to and stimulates the EGFR. Here it was observed that NF-κB was activated in 293T cells transfected with a plasmid encoding the C11R gene. Silencing by small interfering RNA (siRNA) or deletion of the C11R gene (MVAΔC11R) reduced both MVA-induced ERK2 and NF-κB activation in 293T cells or the keratinocyte line Hacat, suggesting that this mechanism of MVA-induced NF-κB activation may be common for several cell types.     

A functional insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the epidermal growth factor receptor.

When wild-type mouse embryo cells are stably transfected with a plasmid constitutively overexpressing the epidermal growth factor (EGF) receptor (EGFR), the resulting cells can grow in serum-free medium supplemented solely with EGF. Supplementation with EGF also induces in these cells the transformed phenotype (growth in soft agar). However, when the same EGFR expression plasmid is introduced and overexpressed in cells derived from littermate embryos in which the insulin-like growth factor I (IGF-I) receptor genes have been disrupted by homologous recombination, the resulting cells are unable to grow or to be transformed by the addition of EGF. Reintroduction into these cells (null for the IGF-I receptor) of a wild-type (but not of a mutant) IGF-I receptor restores EGF-mediated growth and transformation. Our results indicate that at least in mouse embryo fibroblasts, the EGFR requires the presence of a functional IGF-I receptor for its mitogenic and transforming activities.     

Soluble receptor-mediated targeting of mouse hepatitis coronavirus to the human epidermal growth factor receptor

The mouse hepatitis coronavirus (MHV) infects murine cells by binding of its spike (S) protein to murine CEACAM1a. The N-terminal part of this cellular receptor (soR) is sufficient for S binding and for subsequent induction of the conformational changes required for virus-cell membrane fusion. Here we analyzed whether these characteristics can be used to redirect MHV to human cancer cells. To this end, the soR domain was coupled to single-chain monoclonal antibody 425, which is directed against the human epidermal growth factor receptor (EGFR), resulting in a bispecific adapter protein (soR-425). The soR and soR-425 proteins, both produced with the vaccinia virus system, were able to neutralize MHV infection of murine LR7 cells. However, only soR-425 was able to target MHV to human EGFR-expressing cancer cells. Interestingly, the targeted infections induced syncytium formation. Furthermore, the soR-425-mediated infections were blocked by heptad repeat-mimicking peptides, indicating that virus entry requires the regular S protein fusion process. We conclude that the specific spike-binding property of the CEACAM1a N-terminal fragment can be exploited to direct the virus to selected cells by linking it to a moiety able to bind a receptor on those cells. This approach might be useful in the development of tumor-targeted coronaviruses.     

Molecular imaging of epidermal growth factor receptor kinase activity

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types, leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades, including cell proliferation, differentiation, and migration. To investigate the EGFR signaling events in real time and in living cells and animals, here we describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thereby providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with epidermal growth factor. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.     

Epidermal growth factor receptor is not required for human cytomegalovirus entry or signaling

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain beta1 and beta3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and beta3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between beta1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.     

Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.     

Epidermal growth factor receptor induced apoptosis: potentiation by inhibition of Ras signaling

Previous studies have shown that certain tumor cell lines which naturally express high levels of the epidermal growth factor receptor (EGFR) undergo apoptosis when exposed to epidermal growth factor. Whether this phenomenon is a direct result of receptor overexpression or some other genetic alteration renders these cells sensitive to apoptosis is yet to be established. We show that experimentally increasing the level of EGFR expression predictably leads to apoptosis in a variety of cell types which requires an active tyrosine kinase but not EGFR autophosphorylation sites. Expression of a dominant negative Ras mutant in EGFR overexpressing cells results in a significant potentiation of EGFR induced apoptosis suggesting that Ras activation is a key survival signal generated by the EGFR. We propose that potentiation of EGFR induced apoptosis by dominant negative Ras results, at least in part, by a block of Akt activation.     

Protein kinase Calpha mediates feedback inhibition of EGF receptor transactivation induced by Gq-coupled receptor agonists

While a great deal of attention has been focused on G-protein-coupled receptor (GPCR)-induced epidermal growth factor receptor (EGFR) transactivation, it has been known for many years that the tyrosine kinase activity of the EGFR is inhibited in cells treated with tumor-promoting phorbol esters, a process termed EGFR transmodulation. Because many GPCR agonists that elicit EGFR transactivation also stimulate the G_q/phospholipase C (PLC)/protein kinase C (PKC) pathway, we hypothesized that PKC-mediated inhibition of EGFR transactivation operates physiologically as a feedback loop that regulates the intensity and/or duration of GPCR-elicited EGFR transactivation. In support of this hypothesis, we found that treatment of intestinal epithelial IEC-18 cells with the PKC inhibitors GF 109203X or Ro 31-8220 or chronic exposure of these cells to phorbol-12,13-dibutyrate (PDB) to downregulate PKCs, markedly enhanced the increase in EGFR tyrosine phosphorylation induced by angiotensin II or vasopressin in these cells. Similarly, PKC inhibition enhanced EGFR transactivation in human colonic epithelial T84 cells stimulated with carbachol, as well as in bombesin-stimulated Rat-1 fibroblasts stably transfected with the bombesin receptor. Furthermore, cell treatment with inhibitors with greater specificity towards PKCα,  including Gö6976, Ro 31-7549 or Ro 32-0432, also increased GPCR-induced EGFR transactivation in IEC-18, T84 and Rat-1 cells. Transfection of siRNAs targeting PKCα  also enhanced bombesin-induced EGFR tyrosine phosphorylation in Rat-1 cells. Thus, multiple lines of evidence support the hypothesis that conventional PKC isoforms, especially PKCα, mediate feedback inhibition of GPCR-induced EGFR transactivation.     

Mechanisms for oncogenic activation of the epidermal growth factor receptor

The Epidermal growth factor receptor (EGFR) is a membrane spanning glycoprotein, which frequently has been implicated in various cancer types. The mechanisms by which EGFR becomes oncogenic are numerous and are often specific for each cancer type. In some tumors, EGFR is activated by autocrine/paracrine growth factor loops, whereas in others activating mutations promote EGFR signaling. Overexpression and/or amplification of the EGFR gene are prevalent in many cancer types leading to aberrant EGFR signaling. In addition, failure to attenuate receptor signaling by receptor downregulation can also lead to cellular transformation. Heterodimerization of EGFR with ErbB2 inhibits downregulation of EGFR and thereby prolongs growth factor signaling. This also indicates that cross-talk between EGFR and heterologous receptor systems serves as another mechanism for oncogenic activation of EGFR. Because of its role in tumor promotion, the EGFR has been intensely studied as a therapeutic target. There are currently two major mechanisms by which the EGFR is targeted: antibodies binding to the extracellular domain of EGFR and small-molecule tyrosine-kinase inhibitors. However, tumorigenesis is a multi-step process involving several mutations, which might explain why EGFR therapeutics has only been partially successful. This highlights the importance of pinpointing the mechanisms by which EGFR becomes oncogenic in a particular cancer. In this review, each of the above mentioned mechanisms will be discussed, as a detailed molecular and genetic understanding of how EGFR contributes to the malignant phenotype might offer new promise for the design, development and clinical evaluation of future tumor-specific anticancer approaches.     

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.     

Phosphorylation of the platelet-derived growth factor receptor-beta and epidermal growth factor receptor by G protein-coupled receptor kinase-2. Mechanisms for selectivity of desensitization

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-beta (PDGFRbeta) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRbeta, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRbeta in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRbeta but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRbeta and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRbeta mutant, GRK2-mediated PDGFRbeta phosphorylation required receptor tyrosine kinase activity, as does PDGFRbeta ubiquitination. Agonist-induced ubiquitination of the PDGFRbeta, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRbeta down-regulation. Like the vast majority of GRK2 substrates, the PDGFRbeta, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRbeta, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.     

Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28 , 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time a functional interaction between NCAM and EGFR in mammalian cells and investigates the molecular mechanisms underlying this interaction. First, NCAM and EGFR are shown to play opposite roles in neurite outgrowth regulation in cerebellar granular neurons. The data presented indicate that negative regulation of EGFR is one of the mechanisms underlying the neuritogenic effect of NCAM. Second, it is demonstrated that expression of the NCAM-180 isoform induces EGFR down-regulation in transfected cells and promotes EGFR down-regulation induced by EGF stimulation. It is demonstrated that the mechanism underlying this NCAM-180-induced EGFR down-regulation involves increased EGFR ubiquitination and lysosomal EGFR degradation. Furthermore, NCAM-180-mediated EGFR down-regulation requires NCAM homophilic binding and interactions of the cytoplasmic domain of NCAM-180 with intracellular interaction partners, but does not require NCAM-mediated fibroblast growth factor receptor activation.     

Purification and characterization of vaccinia virus growth factor

A growth factor detected in the medium of vaccinia-virus-infected cells was purified to homogeneity. Sequence analysis shows it to be a processed form of a polypeptide encoded in the vaccinia virus genome which is related to epidermal growth factor (EGF) and alpha-transforming growth factor (alpha TGF). The amino terminus of the processed vaccinia virus growth factor (VVGF) begins at residue 20 of the primary translation product, indicating a signal sequence has been removed. The amino acid composition of purified VVGF predicts the carboxyl terminus is at, or near, residue 96, suggesting a transmembrane sequence has also been removed from secreted VVGF, which is, therefore, approximately 77 amino acids. VVGF, unlike EGF or alpha TGF, is glycosylated. VVGF binds to the EGF receptor and stimulates its autophosphorylation, suggesting that vaccinia virus has acquired sequences encoding a growth factor that may allow it to subvert EGF receptor-dependent functions.     

Reactive oxygen species mediate Met receptor transactivation by G protein-coupled receptors and the epidermal growth factor receptor in human carcinoma cells

Cross-communication between the Met receptor tyrosine kinase and the epidermal growth factor receptor (EGFR) has been proposed to involve direct association of both receptors and EGFR kinase-dependent phosphorylation. Here, we demonstrate that in human hepatocellular and pancreatic carcinoma cells the Met receptor becomes tyrosine phosphorylated not only upon EGF stimulation but also in response to G protein-coupled receptor (GPCR) agonists. Whereas specific inhibition of the EGFR kinase activity blocked EGF- but not GPCR agonist-induced Met receptor transactivation, it was abrogated in the presence of a reducing agent or treatment of cells with a NADPH oxidase inhibitor. Both GPCR ligands and EGF are further shown to increase the level of reactive oxygen species within the cell. Interestingly, stimulation of the Met receptor by either GPCR agonists, EGF or its cognate ligand HGF, resulted in release of Met-associated beta-catenin and in its Met-dependent translocation into the nucleus, as analyzed by small interfering RNA-mediated knockdown of the Met receptor. Our results provide a new molecular explanation for cell surface receptor cross-talk involving the Met receptor and thereby link the wide diversity of GPCRs and the EGFR to the oncogenic potential of Met signaling in human carcinoma cells.     

E-cadherin binding modulates EGF receptor activation

We found that E-cadherin and epidermal growth factor receptor (EGFR) are associated in mammary epithelial cells and that E-cadherin engagement in these cells induces transient activation of EGFR, as previously seen in keratinocytes (37). In contrast, EGFR does not associate with and is not activated by N-cadherin. Analysis of cells expressing chimeric cadherins revealed that the extracellular domain of E-cadherin is required for interaction with and activation of EGFR. This activation results in tyrosine phosphorylation of known EGFR substrates and reduction in focal adhesions. These interactions, however, are not necessary for suppression of cell motility by E-cadherin.     

Mathematical model for the effects of epidermal growth factor receptor trafficking dynamics on fibroblast proliferation responses.

We apply a mathematical model for receptor-mediated cell uptake and processing of epidermal growth factor (EGF) to analyze and predict proliferation responses to fibroblastic cells transfected with various forms of the EGF receptor (EGFR) to EGF. The underlying conceptual hypothesis is that the mitogenic signal generated by EGF/EGFR binding on the cell surface, via stimulation of receptor tyrosine kinase activity, is attenuated when the receptors are downregulated and growth factor is depleted by endocytic internalization and subsequent intracellular degradation. Hence, the cell proliferation rate ought to depend on receptor/ligand binding and trafficking parameters as well as on intrinsic receptor signal transduction properties. The goal of our modeling efforts is to formulate this hypothesis in quantitative terms. The mathematical model consists of kinetic equations for binding, internalization, degradation, and recycling of EGF and EGFR, along with an expression relating DNA synthesis rate to EGF/EGFR complex levels. Parameter values have been previously determined from independent binding and trafficking kinetic experiments on B82 fibroblasts transfected with wild-type and mutant EGFR. We show that this model can successfully interpret literature data for EGF-dependent growth of NR6 fibroblasts transfected with wild-type EGFR. Moreover, it successfully predicts the literature observation that NR6 cells transfected with a delta 973 truncation mutant EGFR, which is kinase-active but internalization-deficient, require an order of magnitude lower EGF concentration than cells with wild-type EGFR for half-maximal proliferation rate. This result demonstrates that it may be feasible to genetically engineer mammalian cell lines with reduced growth factor requirements by a rational, nonempirical approach. We explore by further model computations the possibility of exploiting other varieties of EGFR mutants to alter growth properties of fibroblastic cells, based on relationships between changes in the primary structure of the EGF receptor and the rates of specific receptor/ligand binding and trafficking processes. Our studies show that the ability to predict cell proliferation as a function of serum growth factors such as EGF could lead to the designed development of cells with optimized growth responses. This approach may also aid in elucidation of mechanisms underlying loss of normal cell proliferation control in malignant transformation, by demonstrating that receptor trafficking dynamics may in some cases play as important a role as intrinsic signal transduction in determining the overall resulting mitogenic response.     

Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation

Previous studies showed that the epidermal growth factor receptor (EGFR) can be transactivated by platelet-derived growth factor (PDGF) stimulation and that EGFR transactivation is required for PDGF-stimulated cell migration. To investigate the mechanism for cross talk between the PDGF beta receptor (PDGFbetaR) and the EGFR, we stimulated rat aortic vascular smooth muscle cells (VSMC) with 20 ng of PDGF/ml. Transactivation of the EGFR, defined by receptor tyrosine phosphorylation, occurred with the same time course as PDGFbetaR activation. Basal formation of PDGFbetaR-EGFR heterodimers was shown by coimmunoprecipitation studies, and interestingly, disruption of this receptor heterodimer abolished EGFR transactivation. Breakdown of the heterodimer was observed when VSMC were pretreated with antioxidants or with a Src family kinase inhibitor. Disruption of heterodimers decreased ERK1 and ERK2 activation by PDGF. Although PDGF-induced PDGFbetaR activation was abolished after pretreatment with 1 microM AG1295 (a specific PDGF receptor kinase inhibitor), EGFR transactivation was still observed, indicating that PDGFbetaR kinase activity is not required. In conclusion, our data demonstrate that the PDGFbetaR and the EGFR form PDGFbetaR-EGFR heterodimers basally, and we suggest that heterodimers represent a novel signaling complex which plays an important role in PDGF signal transduction.