Reduced activation of RAF-1 and MAP kinase by a fibroblast growth factor receptor mutant deficient in stimulation of phosphatidylinositol hydrolysis.

Signaling via the fibroblast growth factor receptor 1 (FGFR1, flg) was analyzed in Ba/F3 hematopoietic cells expressing either wild-type or a mutant FGF receptor (Y766F) unable to activate phospholipase C-gamma (PLC-gamma) and stimulate phosphatidylinositol (PI) hydrolysis. Stimulation of cells expressing wild-type or mutant FGFR with acidic FGF (aFGF) caused similar activation of Ras. However, an approximately 3-fold reduced activation of Raf-1 and MAP kinase was observed in aFGF-stimulated cells expressing mutant receptors as compared to cells expressing wild-type FGF receptors. Comparison of phosphopeptide maps of Raf-1 immunoprecipitated from the two cell types activated by either aFGF or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) suggests that Raf-1 is phosphorylated by both Ras-dependent and PLC-gamma-dependent mechanisms. In spite of the differential effect on Raf-1 and MAP kinase activation, aFGF stimulated similar proliferation of cells expressing wild-type or mutant receptors indicating that Ras-dependent activation of Raf-1 and MAP kinase is sufficient for transduction of FGFR mitogenic signals. Ras may also activate signal transduction pathways that are complementary or parallel to the MAP kinase pathway to stimulate cell proliferation.

Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation.

Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (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). Using the polymerase chain reaction technique, simultaneous expression of both isoforms was demonstrated in human T-cell and vascular smooth muscle libraries, as well as in the A431 human epidermal cancer cell line. Following transient expression in COS-1 cells, each isoform gave rise to two proteins of 100 and 130 kDa, respectively. Endoglycosidase treatment showed that the 100-kDa species corresponded to a molecule exclusively glycosylated on N-residues, whereas the 130-kDa species contained both, N- and O-linked carbohydrates. Pulse-chase experiments demonstrated that the smaller RPTP alpha protein is a precursor of the larger one. A high affinity antibody was generated that recognizes the immature protein only; however, both proteins can be detected by Western blot analysis after a simple chemical hydrolysis. Following Superose 12 chromatography, the 100- and 130-kDa species of RPTP alpha emerged as 200- and 340-kDa proteins, respectively. Both species exhibited similar enzymatic activities as determined with a peptide substrate in immunoprecipitates.

Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding.

Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation.

A novel receptor tyrosine phosphatase-sigma that is highly expressed in the nervous system.

A novel transmembrane receptor protein tyrosine phosphatase-sigma (RPTP-sigma) was cloned from a rat brain stem cDNA library. The extracellular segment of one form of RPTP-sigma contains 824 amino acids and is composed of three immunoglobulin-like and five fibronectin type III (FNIII)-like repeats. The 627-amino acid cytoplasmic region of RPTP-sigma consists of two catalytic domains oriented in tandem. Northern blot analyses indicate that RPTP-sigma is highly expressed in the brain as two major transcripts of 5.7 and 6.9 kilobases (kb). The 5.7-kb transcript is expressed exclusively in the brain while the 6.9-kb species can be detected in the lung and heart, but at significantly lower levels. In situ hybridization studies confirm that RPTP-sigma is localized predominantly in the nervous system and can be detected in the rat as early as embryonic day 12. During embryonic development, RPTP-sigma is expressed extensively in the central and peripheral nervous systems, including the trigeminal and dorsal root ganglia as well as the retina. In adult rat brain, expression is restricted primarily to the olfactory tubercule, cerebellum, and hippocampus. Within the latter structure, RPTP-sigma is present in the pyramidal cell layer and granular layer of the dentate gyrus. Transfection of RPTP-sigma cDNA into human embryonic kidney 293 cells results in the synthesis of a protein with an apparent molecular mass of 200 kDa as detected by immunoprecipitation and immunoblot analyses using polyclonal antibodies against the FNIII-like repeats present in the extracellular domain of RPTP-sigma. The gene for RPTP-sigma has been mapped to distal chromosome 17 in the mouse.

Retroviral gene transfer of epidermal growth factor receptor into HL60 cells results in a partial block of retinoic acid-induced granulocytic differentiation.

HL60 cells are devoid of endogenous epidermal growth factor receptor (EGFR). They respond to retinoic acid and undergo terminal granulocytic differentiation. EGFR complementary DNA was introduced into HL60 cells by retroviral gene transfer. Scatchard plot showed that the binding characteristics are identical to those of A431 cells. HL60-EGFR cells were estimated to express 34,000 EGFR/cell (Kd = 5 nM). The tyrosine phosphorylation upon ligand binding is the first step of signal transduction. The dominant phosphotyrosyl proteins in epidermal growth factor-stimulated HL60-EGFR cells include a 170 kDa protein (EGFR itself), and 125 and 53 kDa proteins. The EGFR signal results in the induction of 92 kDa gelatinase/matrix metalloproteinase in HL60-EGFR cells, thereby providing evidence of the function of the exogenous EGFR and a semiquantitative measure of the EGFR signal. These HL60-EGFR cells offer a unique opportunity to examine the potentially important role of EGFR (c-erbB) in maintaining homeostasis between self-renewal and differentiation. c-erbB has been shown to play a physiological role in the self-renewal of the very early avian stem cells which do express EGFR. The v-erbB (double truncated EGFR) has been shown to cause avian erythroblastosis. We found that these HL60-EGFR cells responded to retinoic acid differently from the HL60-control cells. A partial block of only 45% granulocytic differentiation and concomitant proliferation was noted, consistent with a shift of balance between self-renewal and differentiation toward the former.

A 40-kDa epidermal growth factor/transforming growth factor alpha-binding domain produced by limited proteolysis of the extracellular domain of the epidermal growth factor receptor.

Elucidation of the three-dimensional structure of the complex of the epidermal growth factor (EGF) and its receptor is essential for understanding the molecular mechanisms of the EGF-receptor interaction and EGF-induced receptor-receptor interaction. NMR is useful to investigate interactions in solution between macromolecules at atomic resolution, but has a limitation in molecular masses of target proteins: less than 300 residues. We have prepared a fragment with apparent molecular mass of 40 kDa in SDS gels from the soluble extracellular domain of the EGF receptor (sEGFR, 619 residues) by sequential limited proteolysis with proteinase K and bromelain. This fragment is a monomeric structural domain consisting of 202 amino acid residues (Cys302-Arg503) and 18-kDa sugar chains, and binds EGF and transforming growth factor-alpha (TGF alpha). This 40-kDa domain has a dissociation constant of about 1 microM for human TGF alpha, which is similar to that of the parental sEGFR. sEGFR oligomerizes in response to EGF and TGF alpha, while the 40-kDa domain does not, suggesting that the sequences other than this domain is required for receptor oligomerization. The 40-kDa ligand-binding domain described in this report is suitable for analysis by various physico-chemical approaches such as NMR.

Cloning and expression of the human substance K receptor and analysis of its role in mitogenesis.

The primary structure of the human substance K receptor was established from the sequences of complementary DNA clones isolated from a human jejunal complementary DNA library. It consists of 398 amino acids, including seven putative transmembrane regions. The gene for the human substance K receptor was localized to chromosome region 10p13-10q23, a region with frequent chromosomal abnormalities. The human substance K receptor was expressed in transfected NIH-3T3 cells lacking endogenous substance K receptors, and Scatchard analysis of 125I-labeled substance K binding indicates approximately 100,000 receptors/cell with a single dissociation constant of 12 nM. Covalent cross-linking experiments utilizing 125I-substance K and three different chemical cross-linking reagents (disuccinimidyl suberate, disuccinimidyl tartrate, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl) demonstrate an apparent molecular weight of 45,000, consistent with little or no N-linked glycosylation. The binding of substance K to its receptor on transfected cells led to a rapid increase in the production of total inositol phosphates and the release of Ca2+ from internal stores. Growth of the cells transfected with the human substance K receptor is stimulated by the addition of substance K to the medium to a level similar to 10% serum. Therefore, the human substance K receptor can function as a growth factor receptor when expressed in mouse 3T3 cells.

Expression of functional epidermal growth factor receptors in a human hematopoietic cell line.

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.

Generation of recombinant cytoplasmic domain of epidermal growth factor receptor with intrinsic protein tyrosine kinase activity.

We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.

Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.

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.