J. Schlessinger - Academia.edu (original) (raw)
Uploads
Papers by J. Schlessinger
Proceedings of the National Academy of Sciences, 2004
Early development of the lens and retina depends upon reciprocal inductive interactions between t... more Early development of the lens and retina depends upon reciprocal inductive interactions between the embryonic surface ectoderm and the underlying neuroepithelium of the optic vesicle. FGF signaling has been implicated in this signal exchange. The docking protein FRS2␣ is a major mediator of FGF signaling by providing a link between FGF receptors (FGFRs) and a variety of intracellular signaling pathways. After FGF stimulation, tyrosine-phosphorylated FRS2␣ recruits four molecules of the adaptor protein Grb2 and two molecules of the protein tyrosine phosphatase Shp2, resulting in activation of the Ras͞extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase͞Akt signaling pathways. In this report, we explore the role of signaling pathways downstream of FRS2␣ in eye development by analyzing the phenotypes of mice that carry point mutations in either the Grb2-(Frs2␣ 4F ) or the Shp2-binding sites (Frs2␣ 2F ) of FRS2␣. Although Frs2␣ 4F/4F mice exhibited normal early eye development, all Frs2␣ 2F/2F embryos were defective in eye development and showed anophthalmia or microphthalmia. Consistent with the critical role of FRS2␣ in FGF signaling, the level of activated extracellular signal-regulated kinase in Frs2␣ 2F/2F embryos was significantly lower than that observed in wild-type embryos. Furthermore, expression of Pax6 and Six3, molecular markers for lens induction, were decreased in the Frs2␣ 2F/2F presumptive lens ectoderm. Similarly, the expression of Chx10 and Bmp4, genes required for retinal precursor proliferation and for lens development, respectively, was also decreased in the optic vesicles of Frs2␣ 2F/2F mice. These experiments demonstrate that intracellular signals that depend on specific tyrosine residues in FRS2␣ lie upstream of gene products critical for induction of lens and retina.
Proceedings of the National Academy of Sciences, 2002
Attenuation of growth factor signaling is essential for the regulation of developmental processes... more Attenuation of growth factor signaling is essential for the regulation of developmental processes and tissue homeostasis in most organisms. The product of Cbl protooncogene is one such regulator, which functions as an ubiquitin ligase that ubiquitinates and promotes the degradation of a variety of cell signaling proteins. Here, we demonstrate that Grb2 bound to tyrosine-phosphorylated FRS2 forms a ternary complex with Cbl by means of its Src homology 3 domains resulting in the ubiquitination of fibroblast growth factor (FGF) receptor and FRS2 in response to FGF stimulation. These observations highlight the importance of FRS2 in the assembly of both positive (i.e., Sos, phosphatidylinositol 3-kinase) and negative (i.e., Cbl) signaling proteins to mediate a balanced FGF signal transduction. However, the partial inhibition of FGF receptor down-regulation in FRS2/ cells indicates that the attenuation of signaling by FGF receptor is regulated by redundant or multiple mechanisms.
Proceedings of the National Academy of Sciences, 1977
Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the l... more Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the lateral mobility and topographical distribution of a major cell surface glycoprotein (CSP). Both endogenous CSP and fluorescent-labeled exogenous CSP bind to the cell surface in a fibrillar pattern and are immobile on the experimental time scale. Azide, vinblastine, and cytochalasin B do not alter the immobility and cell surface distribution of the CSP molecules. Therefore, oxidative phosphorylation and the cytoskeleton do not seem to be responsible for the properties of the bound glycoprotein. The presence of immobile CSP fibrils does not, however, impede the diffusion of a lipid probe, a ganglioside analogue, or various surface antigens. Therefore, the fibrils apparently do not form a "barrier" across the lipid phase of the plasma membrane. In contrast, concanavalin A binds to CSP and is largely immobile in regions rich in CSP. The presence of immobile concanavalin A receptors in areas or on cells lacking CSP indicates that other types of immobile concanavalin A receptors also exist.CSP does not bind to lipid bilayers composed of phosphatidylcholine or oxidized cholesterol. It does bind to dextran-coated bilayers as a diffuse distribution of mobile molecules that can patch after addition of antibodies to CSP. The latter result suggests that CSP molecules do not interact strongly with other CSP molecules under these conditions. Exogenous CSP binds to regions on the cell surface that already bear CSP. In view of the apparent weakness of CSP-CSP interactions on the lipid bilayer, it seems possible that the assembly of CSP fibrils is nucleated by cell surface components in addition to CSP.
Proceedings of the National Academy of Sciences, 1978
Highly fluorescent analogs of insulin and e idermal growth factor were repared by the covalent at... more Highly fluorescent analogs of insulin and e idermal growth factor were repared by the covalent attachment of these peptides to a--actalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The a-lactalbumin was specifically linked to the lysine residue of insulin or to the a-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptorhormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormonereceptor complexes can move laterally in the plane of theplasma membrane with a diffusion coefficient of (3-5) X 10-cm2/ sec.
Proceedings of the National Academy of Sciences, 1978
Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are... more Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are mobile on the cell surface, with similar diffusion coefficients, D (3-5) X 10-10 cm2/sec at 230C. Increasing the temperature to 370C results in rapid receptor immobilization. The immobilization is attributed to aggregation of hormone-receptor complexes, their internalization, or a combination of both processes.
Proceedings of the National Academy of Sciences, 1983
The addition of epidermal growth factor (EGF) to cultured rat pituitary cells (GH3) leads to incr... more 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 (A431) 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.
Proceedings of the National Academy of Sciences, 1976
We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor com... more We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor complexes on the plasma membrane of cultured myoblasts of rat. Transport rates were measured by observing the recovery of fluorescence in a small region of the cell surface initially photobleached irreversibly by an intense, focused laser light pulse. Under different conditions we measured effective diffusion coefficients of the receptor complexes in the range 8 x 10(-12) less than D less than 3 x 10(-11) cm2/sec which is two orders of magnitude lower than we found for a fluorescent lipid probe, D approximately (8 +/- 3) x 10(-9) cm2/sec. This large difference and the presence of apparently immobile concanavalin A receptors suggests that factors beyond the fluoidity of the phospholipid bilayer membrane matrix control the rate of lateral transport of the complexes. Effective mobilities of the complexes decrease with increases in the valence, dose, and occupation time of the lectin on the membrane. These properties imply an aggregation of the lectin-receptor complexes. Mobilities are not influenced by azide, colchicine or preincubation at low temperature. Cytochalasin B and low temperatures, during the time of measurement, decrease the lateral transport rate.
Proceedings of the National Academy of Sciences, 2008
The mechanism of PDGF-receptor beta (PDGFRbeta) activation was explored by analyzing the properti... more The mechanism of PDGF-receptor beta (PDGFRbeta) activation was explored by analyzing the properties of mutant receptors designed based on the crystal structure of the extracellular region of the related receptor tyrosine kinase KIT/stem cell factor receptor. Here, we demonstrate that PDGF-induced activation of a PDGFRbeta mutated in Arg-385 or Glu-390 in D4 (the fourth Ig-like domain of the extracellular region) was compromised, resulting in impairment of a variety of PDGF-induced cellular responses. These experiments demonstrate that homotypic D4 interactions probably mediated by salt bridges between Arg-385 and Glu-390 play an important role in activation of PDGFRbeta and all type III receptor tyrosine kinases. We also used a chemical cross-linking agent to covalently cross-link PDGF-stimulated cells to demonstrate that a Glu390Ala mutant of PDGFRbeta undergoes typical PDGF-induced receptor dimerization. However, unlike WT PDGFR that is expressed on the surface of ligand-stimulated cells in an active state, PDGF-induced Glu390Ala dimers are inactive. Although the conserved amino acids that are required for mediating D4 homotypic interactions are crucial for PDGFRbeta activation, these interactions are dispensable for PDGFRbeta dimerization. Moreover, PDGFRbeta dimerization is necessary but not sufficient for tyrosine kinase activation.
Peptides, 1990
Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3... more Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain.
Journal of Cell Science, 1985
The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was stu... more The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.
Proceedings of the National Academy of Sciences, 1995
Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in m... more Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these
Proceedings of the National Academy of Sciences, 2004
Early development of the lens and retina depends upon reciprocal inductive interactions between t... more Early development of the lens and retina depends upon reciprocal inductive interactions between the embryonic surface ectoderm and the underlying neuroepithelium of the optic vesicle. FGF signaling has been implicated in this signal exchange. The docking protein FRS2␣ is a major mediator of FGF signaling by providing a link between FGF receptors (FGFRs) and a variety of intracellular signaling pathways. After FGF stimulation, tyrosine-phosphorylated FRS2␣ recruits four molecules of the adaptor protein Grb2 and two molecules of the protein tyrosine phosphatase Shp2, resulting in activation of the Ras͞extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase͞Akt signaling pathways. In this report, we explore the role of signaling pathways downstream of FRS2␣ in eye development by analyzing the phenotypes of mice that carry point mutations in either the Grb2-(Frs2␣ 4F ) or the Shp2-binding sites (Frs2␣ 2F ) of FRS2␣. Although Frs2␣ 4F/4F mice exhibited normal early eye development, all Frs2␣ 2F/2F embryos were defective in eye development and showed anophthalmia or microphthalmia. Consistent with the critical role of FRS2␣ in FGF signaling, the level of activated extracellular signal-regulated kinase in Frs2␣ 2F/2F embryos was significantly lower than that observed in wild-type embryos. Furthermore, expression of Pax6 and Six3, molecular markers for lens induction, were decreased in the Frs2␣ 2F/2F presumptive lens ectoderm. Similarly, the expression of Chx10 and Bmp4, genes required for retinal precursor proliferation and for lens development, respectively, was also decreased in the optic vesicles of Frs2␣ 2F/2F mice. These experiments demonstrate that intracellular signals that depend on specific tyrosine residues in FRS2␣ lie upstream of gene products critical for induction of lens and retina.
Proceedings of the National Academy of Sciences, 2002
Attenuation of growth factor signaling is essential for the regulation of developmental processes... more Attenuation of growth factor signaling is essential for the regulation of developmental processes and tissue homeostasis in most organisms. The product of Cbl protooncogene is one such regulator, which functions as an ubiquitin ligase that ubiquitinates and promotes the degradation of a variety of cell signaling proteins. Here, we demonstrate that Grb2 bound to tyrosine-phosphorylated FRS2 forms a ternary complex with Cbl by means of its Src homology 3 domains resulting in the ubiquitination of fibroblast growth factor (FGF) receptor and FRS2 in response to FGF stimulation. These observations highlight the importance of FRS2 in the assembly of both positive (i.e., Sos, phosphatidylinositol 3-kinase) and negative (i.e., Cbl) signaling proteins to mediate a balanced FGF signal transduction. However, the partial inhibition of FGF receptor down-regulation in FRS2/ cells indicates that the attenuation of signaling by FGF receptor is regulated by redundant or multiple mechanisms.
Proceedings of the National Academy of Sciences, 1977
Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the l... more Fluorescence photobleaching recovery and immunofluorescence methods have been used to study the lateral mobility and topographical distribution of a major cell surface glycoprotein (CSP). Both endogenous CSP and fluorescent-labeled exogenous CSP bind to the cell surface in a fibrillar pattern and are immobile on the experimental time scale. Azide, vinblastine, and cytochalasin B do not alter the immobility and cell surface distribution of the CSP molecules. Therefore, oxidative phosphorylation and the cytoskeleton do not seem to be responsible for the properties of the bound glycoprotein. The presence of immobile CSP fibrils does not, however, impede the diffusion of a lipid probe, a ganglioside analogue, or various surface antigens. Therefore, the fibrils apparently do not form a "barrier" across the lipid phase of the plasma membrane. In contrast, concanavalin A binds to CSP and is largely immobile in regions rich in CSP. The presence of immobile concanavalin A receptors in areas or on cells lacking CSP indicates that other types of immobile concanavalin A receptors also exist.CSP does not bind to lipid bilayers composed of phosphatidylcholine or oxidized cholesterol. It does bind to dextran-coated bilayers as a diffuse distribution of mobile molecules that can patch after addition of antibodies to CSP. The latter result suggests that CSP molecules do not interact strongly with other CSP molecules under these conditions. Exogenous CSP binds to regions on the cell surface that already bear CSP. In view of the apparent weakness of CSP-CSP interactions on the lipid bilayer, it seems possible that the assembly of CSP fibrils is nucleated by cell surface components in addition to CSP.
Proceedings of the National Academy of Sciences, 1978
Highly fluorescent analogs of insulin and e idermal growth factor were repared by the covalent at... more Highly fluorescent analogs of insulin and e idermal growth factor were repared by the covalent attachment of these peptides to a--actalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The a-lactalbumin was specifically linked to the lysine residue of insulin or to the a-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptorhormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormonereceptor complexes can move laterally in the plane of theplasma membrane with a diffusion coefficient of (3-5) X 10-cm2/ sec.
Proceedings of the National Academy of Sciences, 1978
Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are... more Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are mobile on the cell surface, with similar diffusion coefficients, D (3-5) X 10-10 cm2/sec at 230C. Increasing the temperature to 370C results in rapid receptor immobilization. The immobilization is attributed to aggregation of hormone-receptor complexes, their internalization, or a combination of both processes.
Proceedings of the National Academy of Sciences, 1983
The addition of epidermal growth factor (EGF) to cultured rat pituitary cells (GH3) leads to incr... more 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 (A431) 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.
Proceedings of the National Academy of Sciences, 1976
We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor com... more We report measurements of the lateral mobility of fluorescent labeled concanavalin A receptor complexes on the plasma membrane of cultured myoblasts of rat. Transport rates were measured by observing the recovery of fluorescence in a small region of the cell surface initially photobleached irreversibly by an intense, focused laser light pulse. Under different conditions we measured effective diffusion coefficients of the receptor complexes in the range 8 x 10(-12) less than D less than 3 x 10(-11) cm2/sec which is two orders of magnitude lower than we found for a fluorescent lipid probe, D approximately (8 +/- 3) x 10(-9) cm2/sec. This large difference and the presence of apparently immobile concanavalin A receptors suggests that factors beyond the fluoidity of the phospholipid bilayer membrane matrix control the rate of lateral transport of the complexes. Effective mobilities of the complexes decrease with increases in the valence, dose, and occupation time of the lectin on the membrane. These properties imply an aggregation of the lectin-receptor complexes. Mobilities are not influenced by azide, colchicine or preincubation at low temperature. Cytochalasin B and low temperatures, during the time of measurement, decrease the lateral transport rate.
Proceedings of the National Academy of Sciences, 2008
The mechanism of PDGF-receptor beta (PDGFRbeta) activation was explored by analyzing the properti... more The mechanism of PDGF-receptor beta (PDGFRbeta) activation was explored by analyzing the properties of mutant receptors designed based on the crystal structure of the extracellular region of the related receptor tyrosine kinase KIT/stem cell factor receptor. Here, we demonstrate that PDGF-induced activation of a PDGFRbeta mutated in Arg-385 or Glu-390 in D4 (the fourth Ig-like domain of the extracellular region) was compromised, resulting in impairment of a variety of PDGF-induced cellular responses. These experiments demonstrate that homotypic D4 interactions probably mediated by salt bridges between Arg-385 and Glu-390 play an important role in activation of PDGFRbeta and all type III receptor tyrosine kinases. We also used a chemical cross-linking agent to covalently cross-link PDGF-stimulated cells to demonstrate that a Glu390Ala mutant of PDGFRbeta undergoes typical PDGF-induced receptor dimerization. However, unlike WT PDGFR that is expressed on the surface of ligand-stimulated cells in an active state, PDGF-induced Glu390Ala dimers are inactive. Although the conserved amino acids that are required for mediating D4 homotypic interactions are crucial for PDGFRbeta activation, these interactions are dispensable for PDGFRbeta dimerization. Moreover, PDGFRbeta dimerization is necessary but not sufficient for tyrosine kinase activation.
Peptides, 1990
Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3... more Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain.
Journal of Cell Science, 1985
The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was stu... more The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas.
Proceedings of the National Academy of Sciences, 1995
Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in m... more Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these