Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3.

Overexpression of ErbB2 and ErbB3 is found in several human cancers, and ErbB2-ErbB3 heterodimers are known as the most potent signaling units among ErbB dimers. While ErbB2 probably undergoes weak endocytosis, ErbB3 is readily internalized even in the absence of added ligand and without requirement for kinase activity. Overexpression of ErbB2 has been demonstrated to inhibit epidermal growth factor-induced internalization and degradation of epidermal growth factor receptor. This happens due to epidermal growth factor receptor-ErbB2 dimerization and can be counteracted by the anti-ErbB2 antibody pertuzumab, which binds the dimerization arm of ErbB2. Pertuzumab does also inhibit ErbB2-ErbB3 dimerization, but to what extent this has effect on constitutive and/or ligand-induced downregulation of ErbB3 is not known. In this study, we demonstrate that expression of ErbB2 as such did not block constitutive internalization of ErbB3, but that heregulin-induced degradation of ErbB3 was significantly slowed in cells expressing high levels of ErbB2. Incubation with pertuzumab did, however, counteract this effect. This indicates that the formation of ErbB2-ErbB3 heterodimers inhibits downregulation of ErbB3 and supports the notion that pertuzumab inhibits ErbB2 dimerization. The inhibitory effect of pertuzumab on ligand-induced ErbB2-ErbB3 heterodimerization was confirmed by the observation that pertuzumab inhibited heregulin-induced phosphorylation of ErbB3 in cells expressing ErbB2 and efficiently reduced heregulin-induced downstream signaling in cells expressing low levels of ErbB2. Altogether the results indicate that pertuzumab can be a valuable therapeutic agent not only in cancers overexpressing ErbB2 but also in cancers co-expressing ErbB2 and ErbB3.

Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes.

The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub(4)) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub(4) chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub(4) was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub(4) was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub(4) was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis.

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis.

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies.

The oncoprotein ErbB3 is endocytosed in the absence of added ligand in a clathrin-dependent manner.

The oncoprotein ErbB3 is overexpressed in several human cancers, for example in pancreatic adenocarcinoma and in ovarian cancers, and ErbB3-containing heterodimers have been demonstrated to be potent signaling units in carcinogenesis. This especially applies to ErbB2-ErbB3 and epidermal growth factor receptor (EGFR)-ErbB3 heterodimers providing anti-apoptotic signaling. Relatively little is understood about the signaling of EGFR-ErbB3 heterodimers and especially about mechanisms involved in downregulation of ErbB3 from the plasma membrane. This is in contrast to EGFR homodimers, for which trafficking has been extensively characterized. In the present study, we have investigated mechanisms involved in endocytosis of ErbB3 in porcine aortic endothelial cells stably expressing either ErbB3 only or stably expressing ErbB3 and EGFR. Our data show that ErbB3 is endocytosed in the absence of added ligand, independently of its tyrosine phosphorylation state and in a clathrin-dependent manner. Functional EGFR-ErbB3 heterodimers were observed to be formed, and dimerization with ErbB3 was observed to negatively affect endocytosis of the EGFR.

Pertuzumab Increases 17-AAG-Induced Degradation of ErbB2, and This Effect Is Further Increased by Combining Pertuzumab with Trastuzumab.

ErbB2 is an important oncogenic protein involved in carcinogenesis of, among others, breast, gastric, and ovarian carcinoma. Over-expression of ErbB2 is found in almost 20% of breast cancers, and this results in proliferative and anti-apoptotic signalling. ErbB2 is therefore an important treatment target. Antibodies recognizing full-length ErbB2 are clinically established, and drugs targeting the ErbB2 stabilizing heat shock protein 90 (Hsp90) are under clinical evaluation. We have investigated effects of the ErbB2-binding antibodies trastuzumab and pertuzumab alone and in combination, as well as the effect of the antibodies in combination with the Hsp90 inhibitor 17-AAG. Our results confirm the notion that combination of different ErbB2-binding antibodies more efficiently down-regulates ErbB2 than does one antibody in isolation. Additionally, our data demonstrate that ErbB2 is most efficiently down-regulated upon incubation with anti-ErbB2 antibodies in combination with Hsp90 inhibitors. The combination of anti-ErbB2 antibodies, and especially the combination of antibodies with 17-AAG, did also increase the inhibition of Akt activation of either agent, which could suggest an anti-proliferative effect. In such case, combining these agents could be beneficial in treatment of tumors not responding to trastuzumab only.

Nimotuzumab and cetuximab block ligand-independent EGF receptor signaling efficiently at different concentrations.

The epidermal growth factor receptor (EGFR) is overexpressed in several epithelial tumors such as breast, ovarian, and colon cancers. Nimotuzumab and Cetuximab are antibodies that inhibit ligand binding upon interaction with the EGFR, thereby indirectly inactivating the EGFR kinase. The ability of an antibody to counteract growth depends on its mechanism of action as well as on its binding affinity. Nimotuzumab has lower binding affinity for the EGFR than does Cetuximab. In addition, a mechanistic difference has recently been suggested to explain the different clinical effects of Nimotuzumab and Cetuximab, arguing that Nimotuzumab partly permits kinase activity and downstream signaling under conditions where binding of EGF is inhibited. We have in the current study compared the effects of Nimotuzumab and Cetuximab on binding of EGF as well as on inhibition of constitutive EGFR-ErbB2 dimerization and downstream activation of Erk. Our results demonstrate that (at least in EGFR-overexpressing cells), in contrast to the recently published mechanistic model, Nimotuzumab not only inhibits EGF-stimulated, but also ligand-independent (basal) signaling although at higher concentrations than required with Cetuximab.

CIN85 regulates ubiquitination and degradative endosomal sorting of the EGF receptor.

CIN85 has been demonstrated to interact with a number of proteins involved in endocytosis and intracellular sorting. However, the exact functional role of CIN85 in endocytosis remains unclear. We have investigated whether CIN85 plays a role in EGF-induced EGF receptor (EGFR) internalization, as previously suggested, or whether CIN85 is rather involved in endosomal sorting of the EGFR. When over-expressing a dominant negative interfering CIN85 mutant consisting of three SH3 domains only, we found that internalization of EGF was inhibited. However, when knocking down CIN85 by RNAi, the EGF-EGFR uptake appeared similar to in control cells. Furthermore, in CIN85 depleted cells, EGF-induced ubiquitination of the EGFR was decreased, and degradation of EGF-EGFR complexes was delayed. Our data further demonstrated that depletion of CIN85 increased the recycling of EGF, suggesting that CIN85 plays a role in endosomal sorting of the ubiquitinated EGFR. Our data also demonstrated that CIN85 was constitutively associated with Hrs, and this strengthens the hypothesis of a functional role of CIN85 in endosomal EGFR sorting.

A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner.

The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.

Internalization and intracellular sorting of the EGF receptor: a model for understanding the mechanisms of receptor trafficking.

The epidermal growth factor receptor (EGFR; also known as ErbB1) is one of four related receptor tyrosine kinases. These receptors (EGFR, ErbB2, ErbB3 and ErbB4) are frequently overexpressed in cancer and such overexpression is associated with poor clinical outcome. Understanding the mechanisms involved in growth-factor-receptor downregulation is medically important, as several drugs that interfere with the function and trafficking of ErbB proteins are currently being developed or are already in clinical trials. EGFR has become a model protein for understanding the biology and endocytosis of related growth-factor receptors, and the mechanisms involved in its endocytosis and degradation have been scrutinized for several decades. Nevertheless, the details and principles of these processes are still poorly understood and often controversial. In particular, the literature describing how the ubiquitylation and recruitment of EGFR to clathrin-coated pits are connected is inconsistent and confusing. In this Opinion article, we discuss the impact of signaling motifs, kinase activity and ubiquitylation on clathrin-dependent endocytosis and lysosomal sorting of EGFR. In addition, we discuss potential explanations for contradicting reports, and propose models for the recruitment of ligand-activated EGFR to clathrin-coated pits as well as for lysosomal sorting of ligand-activated EGFR.

Pertuzumab increases epidermal growth factor receptor down-regulation by counteracting epidermal growth factor receptor-ErbB2 heterodimerization.

Epidermal growth factor receptor (EGFR) and ErbB2 readily form heterodimers when both are expressed in the same cell and the EGFR is activated by one of its ligands. Our data show that such heterodimers are constitutively formed also in a ligand-independent manner on overexpression of EGFR and ErbB2 in porcine aortic endothelial cells. Interestingly, cross-linking experiments showed that incubation with the antibody pertuzumab, which has been shown to bind the dimerization arm of ErbB2, resulted in dissolution of EGFR-ErbB2 heterodimers. Incubation with pertuzumab also increased the amount of EGF-induced EGFR homodimers, and under these conditions, endocytosis of radiolabeled EGF was increased. This increase was significant, although slightly more EGF was internalized in cells expressing EGFR only compared with pertuzumab-treated cells expressing both EGFR and ErbB2. By confocal microscopy analysis, more EGF was observed in endosomes on incubation with pertuzumab, and under similar conditions, immunoblotting experiments showed increased EGFR degradation on incubation with both EGF and pertuzumab. These results show that pertuzumab enhanced the endocytic down-regulation of EGFR by counteracting EGFR-ErbB2 heterodimerization. Our previous results showing that ErbB2 counteracts EGFR endocytosis can therefore be explained by tethering of EGFR to ErbB2 at the plasma membrane.

Expression of epidermal growth factor receptor or ErbB3 facilitates geldanamycin-induced down-regulation of ErbB2.

Overexpression of the epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 promotes growth and antiapoptotic signaling. Overexpression of ErbB2 in breast cancer is associated with poor clinical outcome, and ways of down-regulating ErbB2 are important as therapeutic approaches. In contrast to EGFR, ErbB2 has been shown to be endocytosis deficient. However, down-regulation of ErbB2 can be induced by incubation of cells with geldanamycin and geldanamycin derivatives, counteracting the stabilizing function of heat shock protein 90 on ErbB2. In the present study, we have made use of stably transfected isogenic cell lines expressing ErbB2 only or ErbB2 together with EGFR and/or ErbB3. We now show that whereas ErbB2 can be down-regulated by incubation with geldanamycin in cells expressing ErbB2 only, the rate of geldanamycin-induced down-regulation increases significantly when the cells additionally express EGFR and/or ErbB3. This increase does, however, not correlate with activation/phosphorylation of ErbB2. The potential of heterodimer formation in ErbB2-positive breast cancer cells could thus turn out to be prognostically predictive with respect to outcome of treatment with geldanamycin derivatives.

Epsin 1 is involved in recruitment of ubiquitinated EGF receptors into clathrin-coated pits.

Epsin consists of an epsin NH(2)-terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.

Geldanamycin-induced down-regulation of ErbB2 from the plasma membrane is clathrin dependent but proteasomal activity independent.

ErbB2, a member of the epidermal growth factor receptor family, is overexpressed in a number of human cancers. In contrast to the epidermal growth factor receptor, ErbB2 is normally endocytosis resistant. However, ErbB2 can be down-regulated by inhibitors of heat shock protein 90, such as geldanamycin. We now show that geldanamycin induces endocytosis and lysosomal degradation of full-length ErbB2. We further report that the endocytosis of ErbB2 is dynamin and clathrin dependent. When ErbB2 was retained at the plasma membrane due to knockdown of clathrin heavy chain, the intracellular part of ErbB2 was degraded in a proteasomal manner. However, our data strongly suggest that proteasomal activity is not required for geldanamycin-induced endocytosis of ErbB2 in SKBr3 cells. Interestingly, however, proteasomal inhibitors retarded degradation of ErbB2, and electron microscopy analysis strongly suggested that proteasomal activity is required to sort internalized ErbB2 to lysosomes. Because geldanamycin derivatives and inhibitors of proteasomal activity are both used in experimental cancer treatment, knowledge of molecular mechanisms involved in geldanamycin-induced down-regulation of ErbB2 is important for future design of cancer treatment.

Epidermal growth factor receptor and cancer: control of oncogenic signalling by endocytosis.

The epidermal growth factor receptor (EGFR) and other members of the EGFR/ErbB receptor family of receptor tyrosine kinases (RTKs) are important regulators of proliferation, angiogenesis, migration, tumorigenesis and metastasis. Overexpression, mutations, deletions and production of autocrine ligands contribute to aberrant activation of the ErbB proteins. The signalling output from EGFR is complicated given that other ErbB proteins are often additionally expressed and activated in the same cell, resulting in formation of homo-and/or heterodimers. In particular, association of EGFR with ErbB2 prevents its down-regulation, underscoring the importance of the cellular background for EGFR effects. Signalling from ErbB proteins can either be terminated by dissociation of ligand resulting in dephosphorylation, or blunted by degradation of the receptors. Although proteasomal targeting of ErbB proteins has been described, lysosomal degradation upon ligand-induced endocytosis seems to play the major role in EGFR down-regulation. Preclinical and clinical data have demonstrated that EGFR is a central player in cancer, especially in carcinomas, some brain tumours and in non-small cell lung cancer. Such studies have further validated EGFR as an important molecular target in cancer treatment. This review focuses on mechanisms involved in ligand-induced EGFR activation and endocytic down-regulation. A better understanding of EGFR biology should allow development of more tumour-selective therapeutic approaches targeting EGFR-induced signalling.

Dysregulation of Ack1 inhibits down-regulation of the EGF receptor.

The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of (125)I-EGF internalization, whereas internalization of (125)I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized (125)I-EGF was increased, while degradation of (125)I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis.

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.

Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor.

Sorting of endocytosed EGF receptor (EGFR) to internal vesicles of multivesicular bodies (MVBs) depends on sustained activation and ubiquitination of the EGFR. Ubiquitination of EGFR is mediated by the ubiquitin ligase Cbl, being recruited to the EGFR both directly and indirectly through association with Grb2. Endosomal sorting of ubiquitinated proteins further depends on interaction with ubiquitin binding adaptors like Hrs. Hrs localizes to flat, clathrin-coated domains on the limiting membrane of endosomes. In the present study, we have investigated the localization of EGFR, Cbl and Grb2 with respect to coated and non-coated domains of the endosomal membrane and to vesicles within MVBs. Both EGFR, Grb2, and Cbl were concentrated in coated domains of the limiting membrane before translocation to inner vesicles of MVBs. While almost all Hrs was in clathrin-positive coats, EGFR and Grb2 in coated domains only partially colocalized with Hrs and clathrin. The extent of colocalization of EGFR and Grb2 with Hrs and clathrin varied with time of incubation with EGF. These results demonstrate that both clathrin-positive and clathrin-negative electron dense coats exist on endosomes and are involved in endosomal sorting of the EGFR.

Ubiquitin binding in endocytosis--how tight should it be and where does it happen?

Ubiquitin is an important tag in membrane transport. From studies in yeast, monoubiquitin has been considered sufficient to elicit uptake of cell surface transporters and receptors into endosomes. Two articles in the current issue of Traffic (Hawryluk et al. and Barriere et al.) indicate that stronger binding is required to retain and concentrate cargo in endocytic microdomains of the plasma membrane. High avidity interactions can be obtained by tandemly arrayed ubiquitin interaction motifs (UIM), in proteins such as the endocytic adaptors epsin and Eps15, interacting with polyubiquitin or by UIM-containing proteins binding several ubiquitins brought together through oligomerization of receptors. A controversial issue has been where such interactions take place. One view is that the association of epsin with ubiquitinated cargo is negatively regulated by its interaction with clathrin (Chen H and De Camilli P. Proc Natl Acad Sci USA 2005;102:2766-2771). This contention is now challenged by the articles of Hawryluk et al. and Barriere et al. Hawryluk et al. demonstrate that epsin and Eps15 consistently co-localize with clathrin but never with caveolin.

Activation of the epidermal growth factor (EGF) receptor induces formation of EGF receptor- and Grb2-containing clathrin-coated pits.

In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the mu2 or alpha subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the alpha subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the alpha subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.

The inhibitory effect of ErbB2 on epidermal growth factor-induced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptor-ErbB2 oligomeric complexes at the plasma membrane.

By constructing stably transfected cells harboring the same amount of epidermal growth factor (EGF) receptor (EGFR), but with increasing overexpression of ErbB2, we have demonstrated that ErbB2 efficiently inhibits internalization of ligand-bound EGFR. Apparently, ErbB2 inhibits internalization of EGF-bound EGFR by constitutively driving EGFR-ErbB2 hetero/oligomerization. We have demonstrated that ErbB2 does not inhibit phosphorylation or ubiquitination of the EGFR. Our data further indicate that the endocytosis deficiency of ErbB2 and of EGFR-ErbB2 heterodimers/oligomers cannot be explained by anchoring of ErbB2 to PDZ-containing proteins such as Erbin. Instead, we demonstrate that in contrast to EGFR homodimers, which are capable of inducing new clathrin-coated pits in serum-starved cells upon incubation with EGF, clathrin-coated pits are not induced upon activation of EGFR-ErbB2 heterodimers/oligomers.