Linkage of tyrosine kinase activity with transforming ability of the p185neu oncoprotein

The neu oncogene encodes a 185 kd glycoprotein (p185neu) with intrinsic tyrosine kinase activity. Sequencing data has demonstrated that oncogenic p185neu differs from c-neu by a single point mutation within the transmembrane region of the glycoprotein. This mutation results in the substitution of a glutamic acid residue for a nonpolar valine residue at amino acid position 664 of the rat neu gene product. Recent studies have demonstrated that this mutation results in specific aggregation of the p185neu oncoprotein mimicking ligand induced dimerization events. The cellular consequences of the aggregated phenotype may include the enzymatic activation of p185neu. We demonstrate that the oncoprotein p185neu possesses higher intrinsic tyrosine kinase activity and that this increase in enzymatic activity is apparent within the plasma membrane, manifesting itself through the increased tyrosine phosphorylation of substrates. Furthermore, the neu oncoprotein itself is also phosphorylated on tyrosine to a higher extent than the proto-oncoprotein. These results strongly link enzymatic activation of p185neu to cellular transformation events. To test directly the effect of p185neu tyrosine kinase activity on cellular transformation we constructed mutant p185neu devoid of ATP binding ability. This mutant protein is expressed at high levels, but is unable to induce the transforming phenotype. The point mutation within the transmembrane region of p185neu mimics aspects of ligand induced activation events including increases in the specific tyrosine kinase activity of the molecule leading to cellular transformation.     

计算机辅助设计HER2/neu酪氨酸激酶小分子抑制剂及其生物活性研究

目的：用计算机辅助设计法寻找HER2/neu受体酪氨酸激酶小分子抑制剂。方法：用MODERLAR软件模拟出HER2/neu和EGFR受体酪氨酸激酶的三维(three dimensional,3D)空间结构;根据HER2/neu酪氨酸激酶ATP结合区的空间结构,搜索数据库,找出候选化合物;用Westernblot方法检测化合物对HER2/neu受体酪氨酸激酶磷酸化的影响;MTT法检测细胞增殖抑制作用。结果：比较HER2/neu和EGFR（靶蛋白）与胰岛素受体酪氨酸激酶、FGFR1受体酪氨酸激酶和Src酪氨酸激酶（模板蛋白）的氨基酸序列发现有35％～41％的相同和52％～55％的相似,用MODELLER程序模拟出HER2/neu和EGFR激酶区域的3D结构。根据HER2/neu受体酪氨酸激酶结构模型,3D数据库的搜索,获得潜在HER2/neu酪氨酸激酶抑制剂化合物,经筛选、优化发现ST2325对HER2/neu磷酸化有明显的抑制作用,其IC50值为6.6μmol/L,且抑制作用是可逆的,对EGFR受体磷酸化没有抑制作用。ST2325对HER2/neu受体表达没有影响。ST2325对HER2/neu过表达的肿瘤细胞MDA MB 453m1的抑制作用更强,而对EGFR过表达的肿瘤细胞MDA MB 468抑制作用相对较弱,其IC50值分别为29.05μmol/L和60.40μmol/L。结论：基于HER2/neu的结构设计,筛选出对HER2/neu酪氨酸激酶有明显选择性抑制作用的ST2325。     

Synergistic inhibition with a dual epidermal growth factor receptor/HER-2/neu tyrosine kinase inhibitor and a disintegrin and metalloprotease inhibitor

The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-alpha, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu-positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points.     

Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors.

Tumors that overexpress HER-2/neu receptor or exhibit enhanced EGFR signaling have been reported to possess constitutively activated Src family kinases, especially pp60c-Src. High levels of pp60c-Src activity have also been reported for cell lines that overexpress the EGFR or the chimeric EGFR-HER-2 receptor. It has therefore been suggested that Src kinases may contribute significantly to the oncogenic phenotype of these cells and to the degree of malignancy of tumors that overexpress EGFR family receptors. In this study we show that the induced expression of c-SRC antisense RNA or the application of a selective Src kinase inhibitor induces growth arrest, programmed cell death and reverses the transformed properties of cells that overexpress EGFR or HER-2 receptors. We show that inhibition of Src kinase expression or activity results in the reduction of Stat3 tyrosine phosphorylation, decline of Bcl-XL expression, and induction of cell death. Using a construct in which the promoter of Bcl-X, which possesses putative Stat3 sites, is tethered to the luciferase reporter gene, we show that inhibition of Src activity or expression induces a decline in Bcl-X expression. We also show that the expression of activated Src induces activation of the Bcl-X promoter. This activation is inhibited by the expression of kinase dead Src or of Stat3beta, the dominant-negative form of Stat3. Taken together, these results support the hypothesis that Src positively regulates the transformed phenotype of cells overexpressing EGFR family kinases. Furthermore, these results also suggest that Src positively regulates Bcl-XL expression via Stat3 activation and thus acts not only as a potent mitogenic signaling element, but also as an anti-apoptotic signaling protein. The combination of both activities probably confers upon activated Src its oncogenic activity. Since Src kinase is activated in many tumors, pp60c-Src kinase inhibitors may prove useful as anti-cancer agents for many types of cancer.     

Similar early gene responses to ligand-activated EGFR and neu tyrosine kinases in NIH3T3 cells

We have compared the responses of serum-inducible immediate early genes to ligand activation of the epidermal growth factor receptor (EGFR) or a hybrid EGFR/neu receptor containing the intracellular domain of the neu proto-oncogene. Few differences in mRNA induction kinetics were found, emphasizing the functional similarity of these structurally related tyrosine kinases.     

Collagen-homology domain 1 deletion mutant of Shc suppresses transformation mediated by neu through a MAPK-independent pathway.

Shc proteins are implicated in coupling receptor tyrosine kinase to the mitogen-activated protein kinase (MAPK) pathway by recruiting Grb2/SOS to the plasma membrane. To better understand the role of Shc in the oncogenesis by point-mutation activated neu (p185*), we transfected a Shc mutant (ShcdeltaCHI), which lacks the Grb2 binding site Y317 by deletion of collagen-homology domain 1, into p185*-transformed NIH3T3 cells. The cellular transformation phenotypes were found to be largely suppressed by expression of ShcdeltaCH1. Although ShcdeltaCH1 still retained another Grb2 binding site (Y239/240), we did not detect its physical association with Grb2. We also found that ShcdeltaCH1 could associate with p185*; however, this association did not interfere with the endogenous Shc-p185* interaction or the Shc-Grb2 interaction. In addition, p185*-mediated MAPK and Elk activation likewise were not inhibited by ShcdeltaCH1 expression. Taken together, these data demonstrate that ShcdeltaCH1 suppresses the transformation induced by activated neu through a MAPK-independent pathway, indicating that Shc may be involved in other signal pathway(s) critical for cellular transformation in addition to the MAPK pathway.     

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.     

The epidermal growth factor receptor signaling network in head and neck carcinogenesis and implications for targeted therapy

Improved understanding of the molecular signaling pathways that mediate cellular transformation has led to the development of novel strategies for the treatment of cancer. The epidermal growth factor receptor (EGFR), a transmembrane protein with intrinsic tyrosine kinase activity, transduces important signals from the surface of epithelial cells to the intracellular domain. Aberrant signaling through EGFR plays a key role in the carcinogenesis of squamous cell carcinomas of the head and neck (SCCHN). SCCHN tend to express high levels of EGFR, and the degree of expression correlates with poor clinical outcome. Since EGFR is present at much higher levels in cancerous lesions than in normal epithelial tissue, the receptor has been implicated as a highly specific therapeutic target for the treatment of SCCHN. EGFR can be abrogated at the extracellular level using either monoclonal antibodies or toxin conjugates that compete with the natural ligand at the binding site of the receptor, and targeting of the EGFR intracellular domain has been achieved by specific inhibitors of tyrosine kinase activity. Antisense strategies use synthesized DNA or RNA oligonucleotides to block the translation of the mRNA sequences that code for the production of the EGFR or other proteins with a role in EGFR-mediated cell signaling. Clinical evaluation of EGFR-specific monoclonal antibodies and tyrosine kinase inhibitors has demonstrated limited toxicity in SCCHN patients, and concurrent administration with standard cytotoxic therapies has produced additive or synergistic antitumor effects.     

Curcumin inhibits tyrosine kinase activity of p185neu and also depletes p185neu.

Curcumin, a natural compound present in turmeric, possessing both anti-inflammatory and antioxidant effects, has been studied vigorously as a chemopreventative in several cancer models. The erbB2/neu gene-encoded p185neu tyrosine kinase is a potent oncoprotein. Overexpression of p185neu in breast cancer is known to be a poor prognostic factor. We investigated the effect of curcumin on p185neu tyrosine kinase and on the growth of breast cancer cell lines. Curcumin dose-dependently inhibited p185neu autophosphorylation and transphosphorylation in vitro and depleted p185neu protein in vivo. It dissociated the binding of p185neu with GRP94 (glucose-regulated protein), a molecular chaperone, and enhanced the depletion of p185neu. The amount of p185neu protein on the cell membrane was drastically decreased after curcumin treatment. These data demonstrated a new mechanism of the anti-tyrosine kinase activity of curcumin. The growth of several breast cancer cell lines was inhibited; the IC50 ranged from 7 to 18 microM, which, however, did not correlate with the expression level of p185neu. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in p185neu-overexpressing cell lines by 5 microM curcumin (% of control, basal level versus overexpression: 59.3 versus 16.7%; P < 0.001 by Student's t test). Because curcumin effectively inhibited p185neu tyrosine kinase activity by depleting p185neu and potently suppressed the growth of multiple breast cancer cell lines, its therapeutic potential in advanced breast cancer is worthy of further investigation.     

Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance.     

A 25 kDa polypeptide is the ligand for p185neu and is secreted by activated macrophages.

Medium conditioned by mouse peritoneal macrophages, activated by muramyl dipeptide (MDP), was used as a possible source of p185neu-specific ligand. MDP-activated macrophage-conditioned medium (MDP-CM) was shown to induce p185neu down-regulation in NEU-expressing NIH3T3 cells in a dose-dependent and temperature-sensitive manner. To exclude the possibility of an indirect action of proteins/metabolites present in MDP-CM on p185neu turnover, a ligand-trapping approach was used. Secreted NEU protein possessing only the extracellular domain but lacking transmembrane and protein kinase domains was expressed in HeLa cells and then purified from conditioned medium, using affinity chromatography on WGA-Sepharose. Co-incubation of the truncated, soluble NEU protein preparation with MDP-CM abolished MDP-CM-induced p185neu down-regulation and reduced self-phosphorylation. It is concluded that a putative p185neu-specific ligand is produced by macrophages activated by MDP. Using MDP-CM, the presence of a 25 kDa polypeptide distinct from EGF, PDGF, FGF, IGF, TGF-alpha and TGF-beta and TNF-alpha, could be demonstrated by decorating a Western blot with soluble NEU and anti-NEU antibodies. Thus, a 25 kDa (non-reduced) p185neu ligand has been described.     

Nordihydroguaiaretic Acid (NDGA) Inhibits the IGF-1 and c-erbB2/HER2/neu Receptors and Suppresses Growth in Breast Cancer Cells

Summary Nordihydroguaiaretic acid (NDGA) is a phenolic compound isolated from the creosote bush Larrea divaricatta that has anti-cancer activities both in vitro and in vivo. We can now attribute certain of these anti-cancer properties in breast cancer cells to the ability of NDGA to directly inhibit the function of two receptor tyrosine kinases (RTKs), the insulin-like growth factor receptor (IGF-1R) and the c-erbB2/HER2/neu (HER2/neu) receptor. In MCF-7 human breast cancer cells, low micromolar concentrations of NDGA inhibited activation of the IGF-1R, and downstream phosphorylation of both the Akt/PKB serine kinase and the pro-apoptotic protein BAD. In mouse MCNeuA cells, NDGA also inhibited ligand independent phosphorylation of HER2/neu. To study whether this inhibitory effect in cells was due to a direct action on these receptors, we studied the IGF-1-stimulated tyrosine kinase activity of isolated IGF-1R, which was inhibited by NDGA at 10 μM or less. NDGA was also effective at inhibiting autophosphorylation of the isolated HER2/neu receptor at similar concentrations. In addition, NDGA inhibited IGF-1 specific growth of cultured breast cancer cells with an IC50 of approximately 30 μM. NDGA treatment (intraperitoneal injection 3 times per week) also decreased the activity of the IGF-1R and the HER2/neu receptor in MCNeuA cells implanted into mice. This inhibition of RTK activity was associated with decreased growth rates of MCNeuA cells in vivo. These studies indicate that the anti-breast cancer properties of NDGA are related to the inhibition of two important RTKs. Agents of this class may therefore provide new insights into potential therapies for this disease.     

Overview of monoclonal antibodies and small molecules targeting the epidermal growth factor receptor pathway in colorectal cancer

The epidermal growth factor receptor (EGFR) provides survival signals and is overexpressed in the majority of colorectal cancers. As more is learned about the molecular details of EGFR signaling, antibodies can be designed to interfere with specific domains of the EGFR molecule. In this review, we analyze preclinical and current clinical data on EGFR-targeting molecules and their potential role in the treatment of colorectal cancer. Cetuximab binds to domain III of EGFR and hinders ligand binding. It is now approved by the US Food and Drug Administration for metastatic colorectal cancer treatment. Panitumumab is another widely studied anti-EGFR antibody with similar properties. Bispecific antibodies are modified immunoglobulin molecules containing 2 different binding specificities. These antibodies can redirect the immune response against tumor cells by tethering effector cells such as CD3e-expressing T cells or CD16-expressing natural killer cells and granulocytes to the surface of cancer cells. Tyrosine kinase inhibitors are quinazoline-derived, low molecular weight synthetic molecules that can block the intracellular tyrosine kinase domain of several receptors, including EGFR, Erb2, and vascular endothelial growth factor receptor, and thereby inhibit ligand-induced receptor phosphorylation and abrogate the biologic effect of EGFR signaling. The presence of skin rash and EGFR gene amplification have been advanced as possible predictors of clinical effectiveness of targeted anti-EGFR therapies.     

Epidermal growth factor receptor inhibitors in cancer treatment

The epidermal growth factor receptor (EGFR) is a cellular transmembrane receptor with tyrosine kinase enzymatic activity which plays a key role in human cancer. EGFR-dependent signaling is involved in cancer cell proliferation, apoptosis, angiogenesis, invasion and metastasis. Targeting the EGFR is a valuable molecular approach in cancer therapy. Several anti-EGFR drugs are in Phase III clinical development as single agent or in combination with other anticancer modalities. Cetuximab (Erbitux), a chimeric human-mouse monoclonal immunoglobin (Ig)G1 antibody, which blocks ligand binding and functional activation of the EGFR, is currently registered in the USA, Switzerland and the European Union for the treatment of advanced, irinotecan-refractory colorectal cancer. Gefitinib, (Iressa), a small molecule EGFR-selective inhibitor of tyrosine kinase activity which blocks EGF autophosphorylation and activation, has been the first EGFR-targeting drug to be registered in 28 countries worldwide, including the USA, for the third-line treatment of chemoresistant non-small cell lung cancer patients. This review will focus on the preclinical background and on the clinical data with the anti-EGFR drugs in most advanced clinical development. Furthermore, a series of open clinical issues for the development of optimal strategies of using EGFR-targeting agents will be discussed.     

SUCI02选择性抑制HER-2/neu受体酪氨酸激酶磷酸化及其对HER-2/neu过表达乳腺癌细胞生长的影响

背景与目的：HER-2／neu受体过度表达与肿瘤的发生发展、对化疗的敏感性以及患者预后有关;我们通过大量筛选,发现SUCI02[N-(4-乙氧苯基)-2-羟基酸胺]能抑制HER2／neu受体酪氨酸激酶磷酸化。本研究拟探讨SUCI02对HER-2／nell过度表达的肿瘤细胞生长的影响。方法：用免疫沉淀、免疫印迹法检测：HER-2／neu受体酪氨酸激酶磷酸化、酪氨酸激酶蛋白水平的变化;MTT法检测SUCI02对乳腺癌细胞的抑制作用。结果：SUCI02能抑制HER-2／neu受体的自身磷酸化,在乳腺癌MDA—MB．453m1细胞中,SUCI02对HER-2／neu受体自身磷酸化的IC50为4．34μg／ml,对HER-2／neu的表达没有任何影响。在用SUCI02处理MDA-MB-453ml细胞30min后,用培养液洗掉药物,继续培养不同时间,结果可见洗掉药物后30min,SUCI02对HER-2／neu受体磷酸化的抑制就开始恢复。SUCI02处理MDA-MB-453ml细胞后其下游靶分子MAPK和AKT激活明显受抑制,呈现剂量依赖性。SUCI02对EGFR受体酪氨酸磷酸化在最高剂量用到40μg／ml下没有明显的抑制作用。应用MTT法检测了SUCI02对过度表达HER-2／neu的MDA-MB-453ml细胞的生长抑制作用,同时应用过表达EGFR的乳腺癌MDA—MB-468细胞作为对照,结果可见SUCI02对HER-2／neu过表达的肿瘤细胞相对于过表达EGFR的肿瘤细胞有更明显的抑制作用。结论：SUCI02选择性抑制HER-2／neu受体酪氨酸激酶磷酸化,阻断其下游信号途径,对过度表达HER-2／neu的肿瘤细胞具有更强的生长抑制作用。     

Heregulin-induced activation of ErbB3 by EGFR tyrosine kinase activity promotes tumor growth and metastasis in melanoma cells

ErbB3 receptor tyrosine kinase has been shown to induce tumor progression in several types of cancer through heterodimerization with ErbB2. However, the role of ErbB3 and its ligand heregulin (HRG) in tumor metastasis remains poorly understood. In the present study, we tried to clarify their contributions to the metastasis of ErbB3-overexpressing B16-BL6 melanoma cells. Stimulation with HRG induced phosphorylation of ErbB3 and metastatic properties including MMP-9 expression, invasion, adhesion and experimental lung metastasis in vivo. These cellular responses were blocked by inhibiting the tyrosine kinase activity of EGFR with PD153035. In addition, phosphorylation of EGFR was rapidly induced by HRG, suggesting that EGFR is a possible heterodimeric counterpart of ErbB3. RNA interference demonstrated that subcutaneous tumor growth and angiogenesis was attenuated by inactivation of ErbB3 in cancer cells. Although experimental pulmonary metastasis was not affected by the knockdown of ErbB3, spontaneous metastasis was, even when primary tumors in the foot pad were amputated at a similar size. These results indicate that HRG-induced activation of ErbB3 via EGFR promotes tumor growth and metastasis of melanoma cells.     

Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials

The epidermal growth factor receptor (EGFR) is a growth factor receptorthat induces cell differentiation and proliferation upon activation throughthe binding of one of its ligands. The receptor is located at the cellsurface, where the binding of a ligand activates a tyrosine kinase in theintracellular region of the receptor. This tyrosine kinase phosphorylates anumber of intracellular substrates that activates pathways leading to cellgrowth, DNA synthesis and the expression of oncogenes such as fos and jun.EGFR is thought to be involved the development of cancer, as the EGFRgene is often amplified, and/or mutated in cancer cells.In this review we will focus on: (I) the structure and function of EGFR,(II) implications of receptor/ligand coexpression and EGFR mutations oroverexpression, (III) its effect on cancer cells, (IV) the development of themalignant phenotype and (V) the clinical aspects of therapeutic targeting ofEGFR.     

Signaling-inactive epidermal growth factor receptor/ligand complexes in intact carcinoma cells by quinazoline tyrosine kinase inhibitors

Several inhibitors of EGF receptor (EGFR) tyrosine kinase activity have been developed that compete with ATP at its binding site such as the quinazolines PD 153035 and ZD 1839 or the 4,5-dianilino-phthalimides DAPH1 and DAPH2. When tested on human A431 cells, the quinazolines completely blocked EGF-induced receptor phosphorylation at 100 nM, whereas it was inhibited by DAPH1 and DAPH2 by only 20% at 3 microM. Quinazoline-treated A431 as well as tumor cells expressing less EGFR (A549, MDA MB 231, and T47D) bound 3- to 6-fold more (125)I-labeled EGF than untreated intact control cells. Scatchard analysis revealed the disappearance of low- and high-affinity EGFR on A431 cells upon PD 153035 treatment. A single receptor class of intermediate ligand binding affinity emerged and its number corresponded to the sum of the two classes. DAPH1 and DAPH2 did not change ligand binding properties of EGFR. PD 153035 exerted the most potent effects on EGF binding to A431 or on inhibiting EGF-stimulated growth of rat MTLn3 cells at low ligand concentrations. Cross-linking of EGFR on PD 153035-treated A431 cells indicated the formation of inactive dimers that further increased upon addition of EGF. Chemical cross-linking of (125)I-labeled EGF to PD 153035-treated A431 cells revealed increased binding to monomeric and dimeric EGFR. Thus, the quinazolines sequestered EGFR plus the ligand into inactive receptor/ligand complexes. This novel mode of action of quinazoline tyrosine kinase inhibitors may be the basis for their extraordinary potency especially in conditions when the ligand is present in limiting amounts.