Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF

Although all EGF receptors in EGF receptor-expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high-affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high-affinity state, we studied EGF-binding properties of an EGF receptor mutant that lacks the actin-binding site. Interestingly, we found that cells expressing this mutant receptor still display both high- and low-affinity classes of EGF receptors, indicating that the actin-binding domain does not determine the high-affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF.     

Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma.

Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal-transducing proteins such as phospholipase C-gamma (PLC gamma), phosphatidylinositol-3'-kinase (PI3'-K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain-containing substrates within the Trk cytoplasmic domain suggests a non-essential function of PI3'-K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co-operative function of the PLC gamma-mediated pathway for neuronal differentiation of PC12 cells.     

The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3''-kinase from T lymphocytes.

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.     

Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.

Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.     

EGF induces differentiation of an IL-3-dependent cell line expressing the EGF receptor.

Interleukin 3 (IL-3) is a T cell-derived lymphokine that supports the growth and development of hematopoietic cells. Tyrosine phosphorylation has been suggested to play an important role in IL-3-dependent cell proliferation. To test whether a growth factor receptor carrying a tyrosine kinase can be functional in IL-3 dependent cells, we used a retroviral vector to introduce the human EGF receptor into a murine IL-3-dependent pre-mast cell line, IC2. The EGF receptors expressed on the infected clones bind EGF with both high and low affinities. EGF stimulates the infected cells for a short term growth response. In the presence of IL-3 and EGF, infected clones differentiate into more mature mast cells characterized by increases in intracellular granulation and histamine content. This differentiation is reversible when EGF is removed. EGF induces tyrosine phosphorylation of several cellular proteins and the expression of oncogenes c-fos and c-myc, in a manner analogous to IL-3 stimulation. These results indicate that the EGF receptor is functional in the pre-mast IC2 cells; EGF can support short-term proliferation and activates the signals that induce cell differentiation. Thus, EGF receptor-expressing IC2 cells provide a unique cellular system for in vitro study of mast cell differentiation.     

Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1.

The novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV-1 to T cells. DC-SIGN is also important in the initiation of immune responses by regulating DC-T cell interactions through intercellular adhesion molecule 3 (ICAM-3). We have characterized the mechanism of ligand binding by DC-SIGN and identified the crucial amino acids involved in this process. Strikingly, the HIV-1 gp120 binding site in DC-SIGN is different from that of ICAM-3, consistent with the observation that glycosylation of gp120, in contrast to ICAM-3, is not crucial to the interaction with DC-SIGN. A specific mutation in DC-SIGN abrogated ICAM-3 binding, whereas the HIV-1 gp120 interaction was unaffected. This DC-SIGN mutant captured HIV-1 and infected T cells in trans as efficiently as wild-type DC-SIGN, demonstrating that ICAM-3 binding is not necessary for HIV-1 transmission. This study provides a basis for the design of drugs that inhibit or alter interactions of DC-SIGN with gp120 but not with ICAM-3 or vice versa and that have a therapeutic value in immunological diseases and/or HIV-1 infections.     

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor. We have compared the phosphopeptide maps of human EGF receptor, and two deletion mutants lacking 63 and 126 amino acids from the carboxyl-terminal tail with the phosphopeptide maps of HER/neu and a chimeric EGF receptor containing the carboxyl-terminal tail of HER2/neu. HER2/neu is highly homologous to the EGF receptor, and it probably functions as a growth factor receptor for as yet unidentified growth factor. On the basis of this analysis, we have concluded that all autophosphorylation sites of EGF receptor and HER2/neu are located in their carboxyl-terminal tails. Utilizing the EGF receptors with carboxyl-terminal deletions, we were also able to identify tyr1086 as an additional autophosphorylation site of EGF receptor. Direct microsequencing of a phosphorylated tryptic peptide from the human EGF receptor confirmed this assignment.     

Identification and characterization of binding sites for human urotensin-II in Sprague-Dawley rat renal medulla using quantitative receptor autoradiography

Urotensin-II (U-II), a ligand for the G-protein-coupled receptor UT, has been characterized as the most potent mammalian vasoconstrictor identified to date. Although circulating levels of U-II are altered in lower species (e.g., fish) upon exposure to hypo-osmotic stress, little is known about the actions of this cyclic undecapeptide within the kidney, an organ that plays a pivotal role in the control of cardiovascular homeostasis, influencing both cardiac preload (plasma volume) and after load (peripheral resistance). The present study reports the identification of specific, high affinity [125I]hU-II binding sites in Sprague-Dawley rat kidney outer medulla by autoradiography and also through membrane radioligand binding (Kd 1.9 +/- 0.9 nM and Bmax 408 +/- 47 amol mm(-2) and Kd 1.4 +/- 0.3 nM and Bmax 51.3 +/- 7.8 fmol mg(-1) protein, respectively). Differences were observed in the binding characteristics within rat strains. Compared to the Sprague-Dawley, Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rat kidney outer medulla displayed low density < 20 fmol mg(-1) protein and low affinity (> 1 microM) [125I]hU-II binding sites. Thus, the relative contribution of specific U-II binding sites to the physiological actions of U-II in the control of cardiorenal homeostasis is worthy of further investigation.     

Identification of GABA receptor binding sites in rat and rabbit uterus

The present study provides evidence for the presence of gamma-aminobutyric acid (GABA) and L-glutamate decarboxylase in the uteri of the rat and the rabbit. Furthermore, it has been demonstrated for the first time that the uterine tissue contains high-affinity GABAA receptor binding sites in a high density. The present findings indicate that GABA may have a role in the uterine functions.     

Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.     

Antigen-mediated migration of murine pro-B Ba/F3 cells via an antibody/receptor chimera

Cell migration is one of the fundamental cellular responses governing development, homeostasis and disorders of the body. Therefore, artificial control of cell migration holds great promise for the treatment of many diseases. In this study, we developed an artificial cell migration system based on chimeric receptors that can respond to an artificial ligand that is quite different from natural chemoattractants. Chimeric receptors consisting of an anti-fluorescein single-chain Fv tethered to the extracellular D2 domain of erythropoietin receptor (EpoR) and the transmembrane/cytoplasmic domains of EpoR, gp130, interleukin-2 receptor, c-Kit, c-Fms, epidermal growth factor receptor (EGFR) or insulin receptor were expressed in the murine Ba/F3 pro-B cell line. Migration assays revealed that chimeric receptors containing the cytoplasmic domain of c-Kit, c-Fms or EGFR transduced migration signals in response to fluorescein-conjugated bovine serum albumin (BSA-FL). Furthermore, based on the cell migration in response to BSA-FL, we successfully selected genetically modified cells from mixtures of gene-transduced and untransduced cells. This study represents the first demonstration of cell migration in response to an artificial ligand that is quite different from natural chemoattractants, suggesting its potential application to immunotherapies and tissue engineering.     

Phosphorylation of the PDGF receptor beta subunit creates a tight binding site for phosphatidylinositol 3 kinase.

The beta subunit of the platelet derived growth factor receptor (PDGFR) coprecipitates with a phosphatidyl-inositol 3 kinase activity (PI3K) following stimulation of cells by PDGF. Mutagenesis of a tyrosine (Y) phosphorylation site, Y751, in the PDGFR, greatly reduces PI3K, consistent with the possibility that phosphorylation of Y751 signals association of PI3K. To test this we have reconstituted the binding of the PDGFR beta subunit and PI3K in vitro. Binding is rapid, saturable and requires phosphorylation of the PDGFR at Y751, but does not require PDGF-dependent phosphorylation of PI3K. To test which portions of the PDGFR are important for binding, we used an antibody to a small region of the receptor that includes Y751. This antibody blocked in vitro binding of PI3K to the receptor, while an antiserum to the C-terminus of the receptor had no effect on binding of PI3K. In addition, we found that PDGF stimulation of a cell results in the association of essentially all the PI3K activity with cellular PDGFRs. These data suggest that PI3K is a specific ligand for PDGF receptors that are phosphorylated at Y751.     

EGF receptor activity and mitogenic response of Balb/3T3 cells expressing Ras and Myc oncogenes. EGF receptor activity in oncogene transformed cells.

EGF receptors are found on the surface of most cells, usually with high and low binding affinities. To investigate functional relationships between EGF (EGF-like growth factors) and oncogenes we have characterized the expression of the epidermal growth factor receptor (EGFr) in H-Ras, v-Myc, and H-Ras-v-Myc transformed Balb/3T3 cells. H-Ras cells show a marked decrease in the number of EGFr molecules per cell compared to parental cells. v-Myc and H-Ras-v-Myc transformed cells express an intermediate level of receptors. The majority of the EGF receptors on the parental and oncogene transformed cells bind EGF with low affinity and this low affinity receptor is down-regulated by oncogene transformation. v-Myc expression, in the H-Ras-v-Myc transformed cells, abrogates the receptor down-regulation seen with H-Ras transformation. The mechanism of abrogation is not a result of a change in the p21-Ras concentration in the H-Ras-v-Myc transformed cells. In addition, the mitogenic response to EGF was examined. H-Ras and H-Ras-v-Myc transformed cells do not respond to EGF mitogenically. In contrast, EGF stimulates DNA synthesis in parental cells and v-Myc transfected cells; this result suggests that growth promoting signals from the EGF receptor may not be required in H-Ras transformed cells.     

The cytoplasmic domain of CD28 is both necessary and sufficient for costimulation of interleukin-2 secretion and association with phosphatidylinositol 3''-kinase.

T-cell activation requires two signaling events. One is provided by the engagement of the T-cell antigen receptor, and the second represents a costimulatory signal provided by antigen-presenting cells. CD28 mediates a costimulatory signal by binding its ligands, B7-1 and B7-2, on antigen-presenting cells, but the signaling pathway activated by CD28 has not been identified. A homologous molecule, CTLA-4, expressed on activated T cells, also binds to B7-1 and B7-2, but whether it has a signaling function is not known. We performed a structure-function analysis of CD28 to identify the functional domain which activates signal transduction. Truncation of the 40-amino-acid CD28 cytoplasmic tail abrogated costimulatory signaling. Chimeric constructs containing the extracellular and transmembrane regions of CD8 linked to the cytoplasmic region of CD28 had a costimulatory signaling function. Similar chimeras containing the cytoplasmic tail of CTLA-4 did not signal. Thus, the cytoplasmic region of CD28, but not CTLA-4, is sufficient to mediate costimulatory signaling. In addition, after CD28 stimulation, the p85 subunit of phosphatidylinositol 3'-kinase and phosphatidylinositol 3'-kinase activity were found in CD28 immunoprecipitates. The CD8-CD28 chimera, which has a costimulatory signaling function, associates with p85, while the nonfunctioning CD8-CTLA-4 chimera and a CD8-zeta chimera do not associate with p85. These results suggest that phosphatidylinositol 3'-kinase is specifically activated by CD28 and may mediate proximal events in the costimulatory signaling pathway regulated by CD28.     

Identification of three distinct receptor binding sites of murine interleukin-11

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. A complex of IL-11 and the IL-11 receptor (IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling. In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays. The location of these residues, as predicted from structural studies and a model of IL-11, reveals that mIL-11 has three distinct receptor binding sites. These are structurally and functionally analogous to the previously defined receptor binding sites I, II, and III of interleukin-6 (IL-6). This supports the hypothesis that IL-11 signals via the formation of a hexameric receptor complex and indicates that site III is a generic feature of cytokines that signal via association with gp130.     

Identification of ribosome binding sites in Escherichia coli using neural network models.

This study investigated the use of neural networks in the identification of Escherichia coli ribosome binding sites. The recognition of these sites based on primary sequence data is difficult due to the multiple determinants that define them. Additionally, secondary structure plays a significant role in the determination of the site and this information is difficult to include in the models. Efforts to solve this problem have so far yielded poor results. A new compilation of E. coli ribosome binding sites was generated for this study. Feedforward backpropagation networks were applied to their identification. Perceptrons were also applied, since they have been the previous best method since 1982. Evaluation of performance for all the neural networks and perceptrons was determined by ROC analysis. The neural network provided significant improvement in the recognition of these sites when compared with the previous best method, finding less than half the number of false positives when both models were adjusted to find an equal number of actual sites. The best neural network used an input window of 101 nucleotides and a single hidden layer of 9 units. Both the neural network and the perceptron trained on the new compilation performed better than the original perceptron published by Stormo et al. in 1982.     

Identification of leukotriene D4 receptor binding sites in guinea pig lung homogenates using [3H]leukotriene D4

We have characterized [3H]leukotriene D4 binding to guinea pig lung homogenates. Both biphasic dissociation kinetics and curvilinear Scatchard plots indicated the presence of [3H]leukotriene high and low affinity states of the binding sites. The rank order of potency for the competition study was leukotriene C4 = leukotriene D4 greater than leukotriene E4 much greater than arachidonic acid, and for their contractile effect on lung strips was leukotriene C4 = leukotriene D4 = leukotriene E4 much greater than arachidonic acid. FPL-55712 was the only other agent tested that inhibited binding. These results suggest that binding of [3H]leukotriene D4 to the homogenate is consistent with its binding to specific leukotriene D4 receptor sites.