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Transregulation of the epidermal growth factor receptor (EGFR) by protein kinase C (PKC) serves as a model for heterologous desensitization of receptor tyrosine kinases, but the underlying mechanism remained unknown. By using c-Cbl-induced ubiquitination of EGFR as a marker for transfer from early to late endosomes, we provide evidence that PKC can inhibit this process. In parallel, receptor down-regulation and degradation are significantly reduced. The inhibitory effects of PKC are mediated by a single threonine residue (threonine 654) of EGFR, which serves as a major PKC phosphorylation site. Biochemical and morphological analyses indicate that threonine-phosphorylated EGFR molecules undergo normal internalization, but instead of sorting to lysosomal degradation, they recycle back to the cell surface. In conclusion, by sorting EGFR to the recycling endosome, heterologous desensitization restrains ligand-induced down-regulation of EGFR.

Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.

Stem cell factor (SCF) and its tyrosine kinase receptor, c-Kit, play a crucial role in regulating migration and proliferation of melanoblasts, germ cells, and hemopoietic cell progenitors by activating a number of intracellular signaling molecules. Here we report that SCF stimulation of myeloid cells or fibroblasts ectopically expressing c-Kit induces physical association with and tyrosine phosphorylation of three signal transducers and activators of transcription (STATs) as follows: STAT1alpha, STAT5A, and STAT5B. Other STAT proteins are not recruited upon SCF stimulation. Recruitment of STATs leads to their dimerization, nuclear translocation, and binding to specific promoter-responsive elements. Whereas STAT1alpha, possibly in the form of homodimers, binds to the sis-inducible DNA element, STAT5 proteins, either as STAT5A/STAT5B or STAT5/STAT1alpha heterodimers, bind to the prolactin-inducible element of the beta-casein promoter. The tyrosine kinase activity of Kit appears essential for STAT activation since a kinase-defective mutant lacking a kinase insert domain was inactive in STAT signaling. However, another mutant that lacked the carboxyl-terminal region retained STAT1alpha activation and nuclear translocation but was unable to fully activate STAT5 proteins, although it mediated their transient phosphorylation. These results indicate that different intracellular domains of c-Kit are involved in activation of the various STAT proteins.

Structural features of v‐kit, the oncogene of HZ4 feline sarcoma virus, suggested that this gene arose by transduction and truncation of cellular sequences. Complementary DNA cloning of the human proto‐oncogene coding for a receptor tyrosine kinase confirmed this possibility: c‐kit encodes a transmembrane glycoprotein that is structurally related to the receptor for macrophage growth factor (CSF‐1) and the receptor for platelet‐derived growth factor. The c‐kit gene is widely expressed as a single, 5‐kb transcript, and it is localized to human chromosome 4 and to mouse chromosome 5. A c‐kit peptide antibody permitted the identification of a 145,000 dalton c‐kit gene product that is inserted in the cellular plasma membrane and is capable of self‐phosphorylation on tyrosine residues in both human glioblastoma cells and transfected mouse fibroblasts. Our results suggest that p145c‐kit functions as a cell surface receptor for an as yet unidentified ligand. Furthermore, carboxy‐ and amino‐terminal truncations that occurred during the viral transduction process are likely to have generated the transformation potential of v‐kit.

The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time‐resolved polarization of phosphorescence of erythrosin‐labeled hormone. The measured rotational correlation times of 16‐20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association.

Substantial evidence exists supporting direct roles for ErbB-2/neu and Src kinase activation in breast cancer. The Csk homologous kinase (CHK) is a recently identified tyrosine kinase which, like Csk, phosphorylates the C-terminal tyrosine of Src kinases, resulting in inactivation of these enzymes. Recently, we observed that CHK is associated with the ErbB-2/neu receptor upon heregulin stimulation of breast cancer cells. Here, we report that CHK expression was observed in 70 out of 80 primary breast cancer specimens but not in normal breast tissues (0/19). Confocal microscopy analysis revealed co-localization of CHK with ErbB-2 in these primary specimens (6/6). In addition, we observed that the cytoplasmic domain of the ErbB-2/neu receptor is sufficient for its interaction with the CHKSH2 domain. Phosphopeptide inhibition of the in vitro interaction of CHKSH2 or native CHK with ErbB-2/neu, as well as site-directed mutagenesis of ErbB-2/neu, indicated that CHKSH2 binds to Tyr1253 of ErbB-2/neu. Interestingly, autophosphorylation at this site confers oncogenicity to this receptor. Moreover, CHK was able to down-regulate ErbB-2/neu-activated Src kinases. Overexpression of CHK in MCF-7 breast cancer cells markedly inhibited cell growth and proliferative response to heregulin as well as decreased colony formation in soft agar. These studies indicate that CHK binds, via its SH2 domain, to Tyr1253 of the activated ErbB-2/neu and down-regulates the ErbB-2/neu-mediated activation of Src kinases, thereby inhibiting breast cancer cell growth. These data strongly suggest that CHK is a novel negative growth regulator in human breast cancer.

The rotational diffusion of epidermal growth factor (EGF)--receptor complexes on living human epidermoid carcinoma cells (A-431) has been measured by phosphorescence emission and anisotropy in the mu s time domain. A biologically active phosphorescent conjugate of EGF, erythrosin-EGF, was applied to living cells. The hormone--receptor complexes were mobile with rotational correlation times in the range 25--90 mu s when labeled and measured at 4 degrees C. Prolonged incubation and exposure to higher temperatures (23 and 37 degrees C) resulted in longer times up to 350 mu s, indicative of the progressive formation of microclusters, estimated to contain 10-50 receptors. Upon internalization of the hormone--receptor complexes, visible patches were observed by fluorescence microscopy, and the rotational correlation times were shorter, indicating a decrease in size of the dynamic unit. The sign of the rotational relaxation also varied with localization and processing of the hormones. The rate of lateral diffusion of EGF--receptor complexes, measured under similar conditions by fluorescence photobleaching recovery, increased with temperature in contrast to the rotational motion.

The ErbB signaling network consists of four transmembrane receptor tyrosine kinases and more than a dozen ligands sharing an epidermal growth factor (EGF) motif. The multiplicity of ErbB-specific ligands is incompletely understood in terms of signal specificity because all ErbB molecules signal through partially overlapping pathways. Here we addressed the action of epiregulin, a recently isolated ligand of ErbB-1. By employing a set of factor-dependent cell lines engineered to express individual ErbBs or their combinations, we found that epiregulin is the broadest specificity EGF-like ligand so far characterized: not only does it stimulate homodimers of both ErbB-1 and ErbB-4, it also activates all possible heterodimeric ErbB complexes. Consistent with its relaxed selectivity, epiregulin binds the various receptor combinations with an affinity that is approximately 100-fold lower than the affinity of ligands with more stringent selectivity, including EGF. Nevertheless, epiregulin's action upon most receptor combinations transmits a more potent mitogenic signal than does EGF. This remarkable discrepancy between binding affinity and bioactivity is permitted by a mechanism that prevents receptor down-regulation, and results in a weak, but prolonged, state of receptor activation.

The multiple isoforms of Neu differentiation factor (NDF/neuregulin) induce a pleiotropic cellular response that is isoform-specific and cell type-dependent. The molecular basis of this heterogeneity was addressed by comparing the two major groups of isoforms, alpha and beta. Both groups bind to the catalytically impaired receptor tyrosine kinase ErbB-3, whose mitogenic stimulation by NDF requires transactivation by other ErbB proteins, either ErbB-1 or ErbB-2. By expressing each pair of receptors in interleukin 3-dependent myeloid cells, we found that both isoforms induced mitogenic signals in cells co-expressing the combination of ErbB-3 with ErbB-2. However, only the beta isoform stimulated cells that expressed both ErbB-3 and ErbB-1, and neither isoform was active on cells expressing ErbB-3 alone. Both isoforms bind to all ErbB-3-expressing cells, albeit with different affinities, but the co-stimulatory mitogenic effect is correlated with the ability of each auxiliary receptor to transphosphorylate ErbB-3. These results imply that NDF isoforms differ in their ability to induce receptor heterodimers; whereas both types of isoforms signal through ErbB-3/ErbB-2 heterodimers, only beta isoforms are able to stabilize ErbB-3/ErbB-1 heterodimers.

There is a growing list of oncogenes encoding transmembrane tyrosine kinases that have structures reminiscent of growth factor receptors. In most cases, the ligands for these putative receptors are unknown. Using the neu oncogene as a model system, we have developed several experimental approaches for the detection of such hypothetical ligands. The following lines of evidence collectively imply that a candidate ligand of the neu-encoded oncoprotein is secreted by ras-transformed fibroblasts: Medium conditioned by ras transformants is able to induce down-modulation of the neu-encoded p185 and to activate its intrinsic tyrosine kinase activity in vitro. In addition, a rapid increase in the phosphorylation in vivo of tyrosine residues of the neu-encoded protein is induced by the conditioned medium. Finally, transfer of the neu gene into hematopoietic cells renders them mitogenically responsive to the conditioned medium. The possibility of indirect activation of the oncoprotein through other known receptors, especially the receptor for the epidermal growth factor, was experimentally excluded.

Antibodies generated against two synthetic peptides corresponding to two defined regions on the transforming protein of Rous sarcoma virus, pp60src, interact specifically with the epidermal growth factor (EGF)-receptor kinase. An antibody directed against a synthetic peptide corresponding to the major phosphorylation site of pp60src interacts specifically with EGF receptor and immunoprecipitates a functional EGF-receptor kinase. The second antibody, which binds close to a region on the src molecule that is required for its kinase activity, also binds to EGF-receptor kinase and prevents the autophosphorylation of the receptor molecules. Neither antibody binds to intact cells, but they do recognize various forms of the solubilized receptor. It is concluded that at least two cytoplasmic domains of the EGF receptor are antigenically and presumably also structurally related to specific domains on pp60src.

The ErbB signaling module consists of four receptor tyrosine kinases and several dozen ligands that activate specific homo- and heterodimeric complexes of ErbB proteins. Combinatorial ligand/receptor/effector interactions allow large potential for signal diversification. Here we addressed the possibility that turn-off mechanisms enhance the diversification potential. Concentrating on ErbB-1 and two of its ligands, epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), and the Neu differentiation factor (NDF/neuregulin) and one of its receptors, ErbB-3, we show that ligand binding variably accelerates endocytosis of the respective ligand-receptor complex. However, unlike the EGF-activated ErbB-1, which is destined primarily to degradation in lysosomes, NDF and TGF-alpha direct their receptors to recycling, probably because these ligands dissociate from their receptors earlier along the endocytic pathway. In the case of NDF, structural, as well as biochemical, analyses imply that ligand degradation occurs at a relatively late endosomal stage. Attenuation of receptor down-regulation by this mechanism apparently confers to both NDF and TGF-alpha more potent and prolonged signaling activity. In conclusion, alternative endocytic trafficking of ligand-ErbB complexes may tune and diversify signal transduction by EGF family ligands.

Cyanogen bromide-cleaved epidermal growth factor (CNBr-EGF) binds to EGF receptors with reduced affinity compared to the native hormone but fails to induce DNA synthesis. However, at similar receptor occupancy, CNBr-EGF is as potent as EGF in activating early cell responses to the hormone. The phosphorylation of membrane proteins, the stimulation of Na+-K+-ATPase as reflected by the ouabain-sensitive uptake of 86Rb of fibroblasts, changes in the organization of microfilaments and in cell-morphology, and the activation of the enzyme ornithine-decarboxylase are all induced by CNBr-EGF as well as EGF Our results are consistent with the notion that EGF-induced phosphorylation could act as a "second messenger" for the action of various EGF-induced responses such as activation of Na+-K+-ATPase, changes in the cytoskeleton and cell morphology, and the activation of the enzyme ornithine decarboxylase. However, the stimulation of phosphorylation of membrane proteins and other early responses are either not required or necessary but insufficient for the induction of DNA synthesis. Suboptimal concentrations of EGF together with CNBr-EGF stimulate DNA synthesis in human fibroblasts. Other growth factors such as insulin, fibroblast growth factor, and prostaglandin F2 alpha, which potentiate the mitogenic response of EGF, do not effect the response to CNBr-EGF. This suggests that the restoration of the mitogenic properties of CNBr-EGF by suboptimal doses of EGF occurs at the level of EGF receptors or during their processing.

A method is described for a rapid two-step purification of the membrane receptor for epidermal growth factor (EGF) from cultured human A-431 cells. After solubilization of the cells with Triton X-100, the receptor is immobilized on an immunoaffinity column containing a monoclonal antibody directed against the receptor. In the second step of purification, the receptor, eluted from the antibody column, is adsorbed and specifically eluted from a lectin-agarose column. The molecular species obtained is mainly the 170,000-dalton EGF receptor polypeptide. The activity of the pure receptor depends on the conditions used for the desorption from the immunoaffinity beads. High-yield elution is obtained with acidic buffer and the receptor so purified specifically binds EGF, but is devoid of the kinase activity. When the elution is done with alkaline buffers or with buffer containing urea, a fully active receptor kinase is purified (yield of 10%). The pure receptor binds 125I-EGF with a Kd of 4 X 10(-8) M and retains EGF-sensitive protein kinase activity which phosphorylates tyrosine residues on the receptor itself. An additional protocol is described for large-scale purification (yield of 55%) of EGF receptor for the analysis of its primary structure. In this procedure, the EGF receptor is first purified by immunoaffinity chromatography which is followed by preparative gel electrophoresis of the 32P internally labeled receptor to remove minor protein contaminants.

Each of six peptides derived from the human epidermal growth factor (EGF) receptor very closely matches a part of the deduced sequence of the v-erb-Btransforming protein of avian erythroblastosis virus (AEV). In all, the peptides contain 83amino acid residues, 74of which are shared with v-erb-B. The AEV progenitor may have acquired the cellular gene sequences of a truncated EGF receptor (or closely related protein) lacking the external EGF-binding domain but retaining the transmembrane domain and a domain involved in stimulating cell proliferation. Transformation of cells by AEV may result, in part, from the inappropriate acquisition of a truncated EGF receptor from the c-erb-Bgene.

A brush-border membranal proteinase, which specifically clips the catalytic subunit of cAMP-dependent protein kinase, is shown to cleave the receptor for the epidermal growth factor (EGF) (Mr = 170,000) into two fragments of Mr = 140,000 and 30,000. The 140-kDa fragment retains its EGF-binding site and its EGF-dependent protein tyrosine kinase activity on exogenous substrates, but it loses its capacity to undergo self-phosphorylation. It is shown to be distinct from the 150-kDa fragment of the EGF receptor obtained by the Ca2+-activated neutral proteinase. The membranal proteinase strictly recognizes the native structure of the receptor and fails to cleave either the denatured receptor or its 150-kDa degradation product. Thus the membranal proteinase acts as a conformation-recognizing probe for both the protein-tyrosine kinase domain of the EGF receptor and the catalytic subunit of cAMP-dependent protein-Ser/Thr kinase, suggesting that the known sequence homology between these two kinases is also reflected in their conformation. The well defined 140-kDa fragment described here is useful for structure-function studies of the EGF receptor.

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A-431 cells and selected according to their ability to inhibit the binding of radiolabeled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerful tool to study the structure, processing, and mode of action of EGF receptors.

The addition of epidermal growth factor (EGF) to cultured rat pituitary cells (GH3) leads to increased synthesis of prolactin and to partial inhibition of cell proliferation. Monoclonal antibodies generated against EGF receptor from human epidermoid carcinoma (A-431) cells were used to characterize the EGF receptor kinase system of GH3 cells and to investigate the role of the hormone-receptor complex in the expression of the prolactin gene in these cells. The EGF receptor of GH3 cells is a 170,000-dalton protein associated with a protein kinase. It is similar but not identical to the EGF receptor identified in other tissues. The immunoprecipitated membrane receptor is phosphorylated on both serine and tyrosine residues. The monoclonal antibody denoted 2G2-IgM binds to EGF receptor on GH3 cells. Like EGF, the monoclonal antibody induced the synthesis of prolactin and morphological changes in these cells. Hence, EGF receptor in GH3 cells, when properly triggered, contains all of the biological attributes necessary for the induction of EGF-induced gene expression and morphological changes in GH3 cells.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.