Biomedical subjects
Publications and source records attributed to J Schlessinger.
The gene for the fibroblast growth factor receptor BEK was assigned to human chromosome 10 by applying polymerase chain reaction techniques to DNAs from a panel of human x rodent somatic cell hybrids. The gene was further localized to 10q25.3----q26 by in situ hybridization.
For the insulin receptor and the EGF receptor it is believed that ligand occupancy results in interactions within the heterotetrameric alpha 2 beta 2 insulin receptor or between monomeric EGF receptors. These interactions then activate the intracellular receptor tyrosine kinase which induces receptor autophosphorylation and phosphorylation of cellular substrates. In the present study we have approached the nature of this receptor activation and autophosphorylation. We have investigated whether these phenomena occur via an intra--or an intermolecular process. To this end the following receptor model system consisting of two receptors was co-expressed in NIH 3T3 cells: a kinase inactive human insulin receptor (HIR K1018A) and a chimeric (EIR) receptor corresponding to the extracellular and transmembrane domains of the human EGF receptor and the cytosolic domain of the human insulin receptor beta subunit. Using this system we found that stimulation of the cells with EGF induced tyrosine autophosphorylation of the EGF-insulin receptor chimera (150 kd) and tyrosine phosphorylation of the beta-subunit of the kinase-deficient insulin receptor (95 kd). The phosphopeptides of the autophosphorylated cytoplasmic domain of the EGF-insulin receptor chimera were comparable to those of the transphosphorylated beta subunit of the kinase-deficient insulin receptor and the wild type human insulin receptor. When immunoaffinity purified EGF-insulin receptor hybrids and kinase-deficient insulin receptors were used in a cell lysate phosphorylation assay, it was found that addition of EGF produced [32P]-labeling of both receptor species. In conclusion, we have shown that tyrosine transphosphorylation can occur between homologous receptor domains. This transphosphorylation and transactivation could be a possible mechanism for signal amplification.2+ domain could influence interactions between the receptor and cellular structures and, as such, play a key role in signal transduction.
The binding of epidermal growth factor (EGF) to its cell surface receptor (EGF-R) results in a number of intracellular responses including the activation of the receptor intracellular tyrosine kinase. Receptor oligomerization induced by ligand binding has been suggested to play an important role in signal transduction. However, the mechanisms involved in oligomerization and signal transduction are poorly understood. We have produced and purified several milligrams of recombinant extracellular domain of the EGF receptor (EGF-Rx) using the baculovirus/insect cell expression system. The baculovirus-generated EGF-Rx is glycosylated, has had its signal peptide correctly cleaved, and exhibits a dissociation constant for EGF similar to that for solubilized full-length receptor, of about 100 nM. The binding of EGF to EGF-Rx leads to the formation of receptor dimers and higher oligomerization states which are irreversibly captured using the covalent cross-linking agent disuccinimidyl suberate. Interestingly, purified receptor monomers and dimers, stabilized by the cross-linker in the presence of EGF, exhibit increased binding affinity toward EGF as compared with receptor monomers which have not been exposed to EGF. It appears that the high affinity state of receptor can be maintained by the covalent cross-linking agent. These results indicate that in addition to ligand binding, the extracellular domain of EGF receptor possesses the inherent ability to undergo ligand-induced dimerization and that the low affinity state is converted to a high affinity state by EGF.
The chimeric EK-receptor (EK-R), consisting of the epidermal growth factor receptor (EGF-R) extracellular binding domain and p145c-kit cytoplasmic signal-generating sequences, was fully functional in forming high and low affinity EGF binding sites and in ligand-regulated receptor and substrate phosphorylation activities. Relative to EGF-R, EK-R activation stimulated kit-characteristic phosphorylation of human 293 fibroblast substrate polypeptides. Transient coexpression of EK-R with candidate substrates resulted in ligand-induced phosphorylation of phospholipase C gamma and guanosine triphosphatase-activating polypeptide. The RAF-1 serine/threonine kinase was shown to be associated with activated EK-R, but no tyrosine phosphorylation could be detected. The faithfulness of EK-R substrate phosphorylation specificity was confirmed with stem cell factor-stimulated p145c-kit.
Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.
Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains.
The receptor-linked tyrosine phosphatase RPTP alpha from human brain (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R., Ravera, M., Ricca, G., Jaye, M., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7000-7004) was expressed in insect cells following infection with recombinant baculovirus. Two major forms of the enzyme, with molecular sizes of 98 kDa and 114 kDa, were detected by immunoblot analysis. This heterogeneity could be ascribed to N-linked glycosylation on the basis of two lines of evidence; namely, blockage of glycosylation with tunicamycin in vivo and removal of carbohydrates by endoglycosidase F in vitro. The 114-kDa form was purified to homogeneity by chromatography on Superose 12 and Mono Q. Compared to the low Mr placenta and T-cell tyrosine phosphatases, RPTP alpha displayed a low optimum pH of 6 and a high Km in the micromolar range toward two artificial substrates (tyrosyl-phosphorylated myelin basic protein and modified lysozyme, respectively). Most effectors had a different and often an opposite influence on phosphatase activity depending on the nature of the substrate and the pH at which the assays were performed. Determination of Km and Vmax values for RPTP alpha suggests that the enzyme could exist in low and high substrate affinity states.
A chimeric receptor consisting of an epidermal growth factor (EGF) receptor ligand-binding domain and platelet-derived growth factor (PDGF) receptor transmembrane and cytoplasmic signalling domains has been constructed and shown to be fully functional in phosphorylation, mitogenesis, transformation, Ca2+ release, and pH change assays. Expression of this receptor in EGF receptor-deficient, PDGF-responsive NIH 3T3 cells allows the activation of PDGF signalling pathways by EGF. This system was used to examine the function of kinase insertion sequences (KIS). While a mutant with a KIS deletion of 83 amino acids displayed a significant but reduced ability to induce mitogenic, transforming, and Ca2+ release responses in transfected cells, deletion of 20 additional amino acids resulted in abolishment of such activities. This differential loss of signalling potential correlated with the reduced or abolished potential of these receptor mutants to phosphorylate cellular substrates such as PLC gamma. Our results suggest an integral role for KIS in PDGF receptor cytoplasmic domain conformation and an involvement in substrate interaction, but provide no evidence for an exclusive role of KIS in the mediation of biological signals.
To study cross-talk between unoccupied epidermal growth factor (EGF) receptors and activated EGF receptor kinases, we have used double-transfected cells, IHE2 cells, expressing both an enzymatically active insulin-EGF chimeric receptor and an inactive kinase EGF receptor mutant. Using immunoaffinity-purified receptors, we show that insulin increased phosphorylation of the insulin-EGF chimeric beta subunit and of the kinase-deficient EGF receptor. Stimulation of intact IHE2 cells with insulin leads to a rapid tyrosine autophosphorylation of the insulin-EGF chimeric beta subunit and to tyrosine phosphorylation of the unoccupied kinase-deficient EGF receptor. Insulin-stimulated transphosphorylation of the kinase-deficient EGF receptor yields the same pattern of tryptic phosphopeptides as those in EGF-induced autophosphorylation of the wild-type human EGF receptor. We conclude that insulin, through activation of the insulin-EGF chimeric receptor, mediates transphosphorylation of the kinase-deficient EGF receptor, further confirming that EGF receptor autophosphorylation may proceed by an intermolecular mechanism. In addition to receptor tyrosine phosphorylation, we find that exposure of cells to insulin results in enhanced phosphorylation on serine and threonine residues of the unoccupied kinase-deficient EGF receptor. These results suggest that insulin-EGF chimeric receptor activation stimulates at least one serine/threonine kinase, which in turn phosphorylates the kinase-deficient EGF receptor. Finally, we show that transphosphorylation and coexpression of an active kinase cause a decrease in the number of cell surface kinase-deficient EGF receptors without increasing their degradation rate.
Cis-diamminedichloroplatinum (II) (cis-Pt) complexed to a carboxymethyl dextran-avidin conjugate was targeted to biotin-monoclonal antibody 108 (b-MAb 108). This MAb recognizes the extracellular domain of the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma (KB) cells over-expressing EGF-R. Cis-Pt-carboxymethyl-dextran-avidin (Pt-dex-Av) containing 60-90 M cis-Pt/M avidin was administered 24 hr following b-MAb108 containing 3-5 M biotin/M MAb. This treatment was potentially more effective in suppressing the growth of established KB tumor xenografts, or in inhibiting the development of lung metastases in nude mice, than free MAb 108, free drug or MAb 108 followed by drug. Replacing b-MAb 108 by unbiotinylated antibody or by b-MAb of a different specificity also yielded lower suppressive effects. The sequential administration of Pt-dex-Av following b-MAb was more effective than introduction of the Pt-dex-Av when already complexed to b-MAb 108. The results presented in this preliminary investigation suggest that Pt-dex-Av is specifically removed from the circulation by b-MAb 108 concentrated at the tumor site.
PDGF binding to its receptor promotes the association with and stimulates the phosphorylation of PLC-gamma 1 at tyrosine and serine residues. Also, PDGF induces an increase in the hydrolysis of inositol phospholipids by PLC. How PDGF activates PLC was investigated by substituting phenylalanine for tyrosine at PLC-gamma 1 phosphorylation sites 771, 783, and 1254 and expressing the mutant enzymes in NIH 3T3 cells. Phenylalanine substitution at Tyr-783 completely blocked the activation of PLC by PDGF, whereas mutation at Try-1254 inhibited and mutation at Tyr-771 enhanced the response. Like the wild type, PLC-gamma 1 substituted with phenylalanine at Tyr-783 became associated with the PDGF receptor and underwent phosphorylation at serine residues in response to PDGF. These results suggest that PLC-gamma 1 is the PLC isozyme that mediates PDGF-induced inositol phospholipid hydrolysis, that phosphorylation on Tyr-783 is essential for PLC-gamma 1 activation. These results provide direct evidence that growth factor receptors activate the function of intracellular protein by tyrosine phosphorylation.
Activation of phospholipase C (PLC), leading to a rise in cytosolic Ca2+, and of phospholipase A2 (PLA2) leading to a release of arachidonic acid, are among the early transmembrane signalling events that have been demonstrated in response to occupancy of the epidermal growth factor (EGF) receptor. The tyrosine kinase activity of the receptor has been shown to be necessary for both of these responses. This requirement for the tyrosine kinase activity could conceivably implicate a role for receptor autophosphorylation in the activation of PLA2. We now demonstrate that coupling of the EGF receptor to PLA2 was not impaired in a deletion mutant (CD126) devoid of the 126 amino acids from the C-terminus which include four major autophosphorylation sites. Functional coupling of the EGF receptor to PLA2 was demonstrated using three different experimental designs: (1) release of [14C]arachidonic acid from prelabelled intact cells. (2) release of [3H]arachidonic acid from prelabelled cells permeabilized with glass beads, and (3) direct measurement of PLA2 enzymic activity in cell-free extracts using an 'in vitro' assay employing exogenous phospholipid substrate. Functional coupling of the EGF receptor to PLA2 occurred despite the absence of a demonstrable Ca(2+)-signalling response and the detection of diminished but persistent PLC-gamma phosphorylation on tyrosine residues in the CD126 deletion mutants. These results point to a clear distinction in the biochemical mechanism and role for receptor autophosphorylation in functional coupling of the EGF receptor to PLA2 activation versus Ca2+ signalling.
Increased levels of epidermal growth factor receptor (EGFR) have been shown on squamous cell carcinomas. Recently, we described a squamous cell carcinoma (MH-85) derived from the oral cavity which was associated with several paraneoplastic syndromes including hypercalcemia and cachexia. This tumor induced the same paraneoplastic syndromes in nude mice (BALB/c, nu/nu, male, 4-6 weeks old). Scatchard analysis revealed that there are two classes of EGFR in MH-85. The dissociation constant and number of binding sites for the high affinity receptors were 38 pM and 5 x 10(4)/cell, respectively, and 2.2 nM and 6 x 10(5) cell, respectively, for the low affinity receptors. Growth of MH-85 in culture was stimulated by epidermal growth factor (EGF) and inhibited by monoclonal antibody 108 to human EGFR, which recognizes the extracellular domain of the EGF receptor. Surgical removal of submandibular glands from male nude mice resulted in a dramatic decrease in plasma EGF levels and a significant reduction of tumor growth, hypercalcemia, and cachexia. When EGF (5 micrograms/mouse, every 2 days for 6 weeks, i.p.) was administered to these sialoadenectomized animals, tumor growth increased, with a parallel increase in hypercalcemia. When monoclonal antibody 108 (1 mg/mouse, i.p.) was given 1, 5, and 10 days after MH-85 tumor implantation, tumor formation was retarded, which resulted in delayed onset of hypercalcemia and cachexia. Moreover, when the antibody was injected 6 times in nude mice exhibiting large tumors and profound hypercalcemia and cachexia, there was a striking decrease in tumor growth, which was accompanied with a reversal of hypercalcemia and cachexia. These results indicate that growth of the human squamous cell carcinoma MH-85 is dependent on the EGFR pathway and that subsequent development of hypercalcemia and cachexia is dependent on tumor growth. They also suggest that agents which interfere with the EGFR pathway may have therapeutic potential as anticancer agents in some human tumors.
To determine the domains of the low-affinity nerve growth factor (NGF) receptor required for appropriate signal transduction, a series of hybrid receptors were constructed that consisted of the extracellular ligand-binding domain of the human epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and cytoplasmic domains of the human low-affinity NGF receptor (NGFR). Transfection of these chimeric receptors into rat pheochromocytoma PC12 cells resulted in appropriate cell surface expression. Biological activity mediated by the EGF-NGF chimeric receptor was assayed by the induction of neurite outgrowth in response to EGF in stably transfected cells. Furthermore, the chimeric receptor mediated nuclear signaling, as evidenced by the specific induction of transin messenger RNA, an NGF-responsive gene. Neurite outgrowth was not observed with chimeric receptors that contained the transmembrane domain from the EGFR, suggesting that the membrane-spanning region and cytoplasmic domain of the low-affinity NGFR are necessary for signal transduction.
A novel method has been developed to allow cloning of protein targets for receptors with tyrosine kinase activity. By utilizing the carboxy-terminal tail of EGF receptor (EGFR) as a probe to screen lambda gt11 expression libraries, several EGFR-binding proteins have been cloned; two have been analyzed and contain unique SH2 and SH3 domains. One gene (GRB-1) has been fully sequenced, is expressed in various tissues and cell lines, and has a molecular mass of 85 kd. Interestingly, GRB-1 encodes the human counterpart of the PI3 kinase-associated protein p85. Advantages of this technique include the ease of cloning tyrosine kinase receptor targets present at low levels and the ability to identify proteins that are related in their capacity to bind activated receptors but contain no significant DNA sequence homology. This method, termed CORT (for cloning of receptor targets), offers a general approach for the identification and cloning of various receptor targets.
To test the feasibility of using the human epidermal growth factor receptor (EGFR) as a model for growth factor receptor action in human hematopoietic cells, we infected Burkitt lymphoma cells (Namalwa) with a recombinant amphotrophic retrovirus containing a thymidine kinase promoter-driven human EGFR complementary DNA and the neomycin resistance gene. Neomycin-resistant cells expressing surface EGFR were selected by cell sorting using anti-EGFR monoclonal antibody 225. The selected cells expressed a Mr 170,000 protein immunoprecipitated by monoclonal antibody 225 and apparently identical to EGFR from A431 carcinoma cells. Infected Namalwa cells expressed 42,000 epidermal growth factor (EGF) binding sites/cell, and Scatchard analysis showed two affinities (Kd approximately 5 nM and approximately 0.5 nM). EGFR autophosphorylation was detected using antiphosphotyrosine antibodies after 5 min exposure to EGF. EGF binding induced rapid EGFR internalization (t1/2 = 9 min) and mobilization of transferrin receptors to the cell surface within 1 min. In fetal bovine serum-containing and serum-free cultures, EGF did not stimulate Namalwa cell proliferation. However, in the presence of 1.25% dimethyl sulfoxide (DMSO), EGF caused a dose-dependent increase in DNA synthesis. DMSO induced a cell cycle block in G1, which was partially reversed by EGF. DMSO induced changes in some B-cell markers suggesting cellular differentiation and increased surface EGF receptor number. Cells grown in DMSO and EGF were established as an EGF-dependent cell line for greater than 12 weeks, whereas cells grown in DMSO without EGF died within 1-2 weeks. Namalwa cells expressing EGFR demonstrated more rapid tumor growth in athymic mice. These studies demonstrate expression of functional EGFR mediating early biochemical and growth responses in a human hematopoietic cell, and indicate that EGFR can be used as an effective model in human hematopoietic cells. Results using DMSO are consistent with studies in other human leukemia cells indicating that agents inducing differentiation can restore growth factor dependence in previously factor-independent leukemia cells.
Recent evidence shows that different fibroblast growth factors (FGF) bind with similar high affinities to two FGF receptors (FGFR) called flg and bek. In order to explore the mechanism of FGFR tyrosine autophosphorylation, we have generated cell lines which co-express a kinase-negative mutant of FGFR and an active form of FGFR. The following transfected NIH 3T3 cells were generated: (i) cells which express a shorter truncated form of bek (two Ig domains) together with a kinase-negative mutant of full length bek (bek K517A), (ii) cells which express wild-type bek together with kinase-negative flg (flg K514A) and (iii) cells co-expressing wild-type flg together with bek K517A. Immunoprecipitations with either bek-or flg-specific antisera followed by immunoblotting indicated that the double transfectants express the desired receptor species. The addition of acidic FGF (aFGF) to the various cell lines followed by immunoprecipitation with anti-FGFR antibodies and immunoblotting with anti-phosphotyrosine specific antibodies indicated that aFGF induces tyrosine phosphorylation of the kinase-negative FGFR mutants. These results show that tyrosine autophosphorylation of the kinase-negative FGFR is mediated by a transphosphorylation mechanism and that both homologous (bek----bek) and heterologous (bek----flg and flg----bek) transphosphorylation occurs in living cells. Recent evidence shows that tyrosine autophosphorylation of receptors with tyrosine kinase activities is essential for mediating interactions with signaling molecules. Therefore, heterologous transphosphorylation could amplify the response of cells to various forms of FGFs and their cognate receptors.
Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand-binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high-affinity binding site typical of human EGFR.