Expression and functional characterization of platelet-derived growth factor receptor-like gene.

AIM: To investigate the role of platelet-derived growth factor receptor-like gene (PDGFRL) in the anti-cancer therapy for colorectal cancers (CRC). METHODS: PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in CRC and colorectal normal tissues. PDGFRL prokaryotic expression vector was carried out in Escherichia coli (E. coli), and purified by immobilized metal affinity chromatography. The effect of PDGFRL protein on CRC HCT-116 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), clone counting, cell cycle, and wound healing assay. RESULTS: Both RT-PCR and immunohistochemistry showed that the expression of PDGFRL in colorectal normal tissues was higher than in cancer tissues. Recombinant pET22b-PDGFRL prokaryotic expression vector was successfully expressed in E. coli, and the target protein was expressed in the form of inclusion bodies. After purification and refolding, recombinant human PDGFRL (rhPDGFRL) could efficiently inhibit the proliferation and invasion of CRC HCT-116 cells detected by MTT, clone counting and wound healing assay. Moreover, rhPDGFRL arrested HCT-116 cell cycling at the G0/G1 phase. CONCLUSION: PDGFRL is a potential gene for application in the anti-cancer therapy for CRC.

Identification of a candidate integrin-fraction associated with the activated form of the PDGF-receptor.

The activated PDGF-receptor has been shown to coimmunoprecipitate together with alpha v beta 3 integrin out of the 15,000g soluble supernatant of non-ionic detergent cell lysates. I have now further characterized this complex by ultracentrifugation analysis. The ultracentrifugation-conditions were chosen so that the phosphorylated form of the PDGF-receptor was pelleted out of the 15,000g soluble supernatant. Together with the tyrosine-phosphorylated PDGF-receptor small amounts of integrins, cytoskeletal- and extracellular matrix proteins were recovered in the pelleted material. The results show that (i) the candidate-fraction of integrins interacting with the activated PDGF-receptor is small compared to the overall integrin population in the cell lysate, (ii) several proteins known to be present in focal adhesions such as FAK, talin, and vinculin are absent from the integrin-growth factor receptor complexes, while on the other hand (iii) a tyrosine-phosphorylated protein migrating at 120 kDa was highly enriched in the ultracentrifugation-pellet, and finally (iv) non-ionic detergent cell lysates appear to contain quantitatively small fractions of complexed proteins that are qualitatively distinct from their total cellular population. Thus, the separation of protein-complexes from the total cellular proteom may be instrumental for the investigation of cellular protein complexes in general.

Tyrosine kinase activity of purified recombinant cytoplasmic domain of platelet-derived growth factor beta-receptor (beta-PDGFR) and discovery of a novel inhibitor of receptor tyrosine kinases.

Aberrant expression of platelet-derived growth factor and its receptor (PDGFR) has been implicated in various human disorders, including cardiovascular disease and certain types of cancer. Inhibitors of the tyrosine kinase activity of PDGFR are leads in the development of novel agents to combat these diseases. We describe here a novel, potent inhibitor of PDGFR tyrosine kinase, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI). The compound also inhibits signal transduction through fibroblast growth factor receptor 1 (FGFR1), but is not active towards epidermal growth factor receptor (EGFR) or c-Src tyrosine kinase. The activity of DMBI and other tyrosine kinase inhibitors was compared in a cell-based assay as well as in an assay based on purified recombinant platelet-derived growth factor beta-receptor (beta-PDGFR) lacking the transmembrane and ligand-binding domain. We showed that this truncated beta-PDGFR could dimerize, and that dimerization was required for tyrosine kinase activity. Tyrosine kinase activity was modulated by inhibitors of beta-PDGFR autophosphorylation in cells, but not by specific inhibitors of EGFR or c-Src tyrosine kinase. We conclude that beta-PDGFR lacking the transmembrane and ligand-binding domain retains the essential properties of the full-length receptor tyrosine kinase.

Activation of PDGF receptor alpha in vascular smooth muscle cells by mechanical stress.

Hypertension increases mechanical force on the arterial wall by as much as 30%, resulting in marked alterations in signal transductions and gene expression in vascular smooth muscle cells (VSMCs) that contribute to matrix protein synthesis, cell proliferation, and differentiation. How the mechanical stimuli are converted into a biological signal in cells has yet to be studied. We investigated the role of both cyclic strain and shear stresses in initiating the cellular signaling on cultured VSMCs and found that mechanical forces evoked activation of mitogen-activated protein kinases, followed by enhanced DNA binding activity of transcription factor AP-1. Physical forces rapidly induced phosphorylation of platelet-derived growth factor receptor (PDGFR) alpha, an activated state. When GRB2, an adapter protein, was immunoprecipitated from treated VSMCs followed by Western blot analysis with anti-phosphotyrosine, -PDGFR alpha, and -GRB2 antibodies, respectively, phosphotyrosine positive staining was observed on PDGFR alpha bands of the same blot in stretch-stressed VSMCs, supporting the mechanical stress-induced activation of PDGFR alpha. Conditioned medium from stretch-stressed VSMCs did not result in PDGFR alpha phosphorylation, and antibodies binding to all forms of PDGFs did not block stress-induced PDGFR alpha activation. Thus, mechanical stresses may directly perturb the cell surface or alter receptor conformation, thereby initiating signaling pathways normally used by growth factors.

Association of coatomer proteins with the beta-receptor for platelet-derived growth factor.

The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha- or beta'-COP, showed a normal ligand-induced autophosphorylation. The mutant receptor also behaved like the wildtype receptor with regard to biosynthesis and maturation, and mediated a mitogenic signal. The possible functional importance of the interaction between the PDGF beta-receptor and alpha- and beta'-COP, is discussed.

Isolation and characterization of the platelet-derived growth factor beta receptor promoter.

The PDGFbeta r gene has been implicated in many physiological processes including development and wound healing. Aberrant expression of the receptor is seen in many pathological conditions such as atherosclerosis and inflammatory diseases. To study the mechanisms of PDGFbeta r regulation, we identified the regulatory regions of the gene. We have cloned and characterized the promoter region of the platelet-derived growth factor beta receptor (PDGFbeta r). We isolated a 4.5 Kb genomic fragment which confers PDGFbeta r tissue-specific promoter activity. This fragment can direct transcription of a luciferase reporter gene in a cell-specific manner which correlates well with the known pattern of expression of the PDGFbeta r. The specificity of this clone was demonstrated by its high activity in NIH 3T3 fibroblasts and lack of activity in N-MUNG epithelial cells, a pattern that parallels the expression of the endogenous PDGFbeta r. We have defined a 614 bp region encompassing the 5' untranslated region of the gene which includes the basal promoter region. We generated transgenic mice that carry the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the 4.5 Kb promoter. The expression pattern of the reporter gene was compared to that of the endogenous PDGFbeta r gene. The promoter was able to direct reporter gene expression with the same temporal and spatial pattern as the endogenous PDGFbeta r. The most prominent expression was in condensing mesenchyme of developing blood vessels, bone and tissues adjacent to epithelium. We conclude that this clone contains the regulatory regions sufficient to direct expression of the PDGFbeta r. The further analysis of this promoter will help elucidate the transcriptional regulation of expression of the PDGFbeta r, and provide a useful tool for directing expression of heterologous genes.

Spinal cord oligodendrocytes develop from ventrally derived progenitor cells that express PDGF alpha-receptors.

Platelet-derived growth factor alpha-receptors (PDGFR alpha) are expressed by a subset of neuroepithelial cells in the ventral half of the embryonic day 14 (E14) rat spinal cord. The progeny of these cells subsequently proliferate and migrate into the dorsal parts of the cord after E16. Here, we show that E14 ventral cells are able to generate oligodendrocytes in culture but that dorsal cells acquire this ability only after E16, coinciding with the appearance of PDGFR alpha-immunoreactive cells in the starting population. PDGFR alpha-positive cells in optic nerve and spinal cord cultures co-labelled with antibody markers of oligodendrocyte progenitors. When PDGFR alpha-positive cells were purified from embryonic rat spinal cords by immunoselection and cultured in defined medium, they all differentiated into oligodendrocytes. Very few oligodendrocytes developed in cultures of embryonic spinal cord cells that had been depleted of PDGFR alpha-expressing cells by antibody-mediated complement lysis. These data demonstrate that all PDGFR alpha-positive cells in the embryonic rat spinal cord are oligodendrocyte progenitors and that most or all early-developing oligodendrocytes are derived from these ventrally-derived precursors.

Tyrphostins. 5. Potent inhibitors of platelet-derived growth factor receptor tyrosine kinase: structure-activity relationships in quinoxalines, quinolines, and indole tyrphostins.

A series of 3-indoleacrylonitrile tyrphostins, 2-chloro-3-phenylquinolines, and 3-arylquinoxalines were prepared and tested for inhibition of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK) activity. The potency of the inhibitors was found to be quinoxalines > quinolines > indoles. Lipophilic groups (methyl, methoxy) in the 6 and 7 positions and phenyl at the 3 position of quinoxalines and quinolines were essential for potency, in contrast to the hydrophilic catechol group in tyrphostins active against EGFR kinase inhibition at different sites. The inhibitors showed selectivity for PDGF and were not active against EGF receptor and HER-2/c-ErbB-2 receptor.

Low M(r) phosphotyrosine protein phosphatase interacts with the PDGF receptor directly via its catalytic site.

Many proteins bind to the activated platelet derived growth factor receptor (PDGF-R) either directly or by means of adapter molecules. Up to now all these proteins were shown to transmit and amplify the signal started with PDGF-R stimulation. In a recent study our group had demonstrated that low M(r) phosphotyrosine protein phosphatase (LMW-PTP) specifically interacts with PDGF-R in NIH3T3 cells. In the present study we have attempted to clarify the modality of interaction, both in vivo and in vitro, of these two proteins, using a catalytically inactive LMW-PTP mutant. Our results indicate that LMW-PTP and PDGF-R interact directly, without the necessity of any adapter protein. This interaction leads to PDGF-R dephosphorylation and, presumably, interrupts one or more of the mitogenic pathways that originate from receptor activation.

Insulin-induced activation of phosphatidylinositol (PI) 3-kinase. Insulin-induced phosphorylation of insulin receptors and insulin receptor substrate-1 displaces phosphorylated platelet-derived growth factor receptors from binding sites on PI 3-kinase.

Phosphatidylinositol (PI) 3-kinase is an enzyme that functions in the signaling pathways downstream from multiple cell surface receptors. The p85 regulatory subunit of PI 3-kinase binds to phosphotyrosine residues of various phosphoproteins including the platelet-derived growth factor (PDGF) receptor, the insulin receptor, and insulin receptor substrate-1 (IRS-1). Using NIH-3T3 cells overexpressing the human insulin receptor, we demonstrate that the p85 regulatory subunit of PI 3-kinase binds to phosphorylated PDGF receptor in cells incubated in the absence of insulin. When insulin is added, p85 is released from phosphorylated PDGF receptors and binds to phosphorylated insulin receptors and insulin receptor substrate-1. Moreover, insulin-induced dissociation of PDGF receptors from binding sites on PI 3-kinase requires a functional insulin receptor and is not prevented by vanadate treatment. In contrast, insulin activation does not displace PDGF receptors from binding sites on Ras GTPase-activating protein. This competition for binding to PI 3-kinase provides a mechanism for cross-talk among signaling pathways initiated by distinct peptide hormones and growth factors such as insulin and PDGF.

Structural role of extracellular domain 1 of alpha-platelet-derived growth factor (PDGF) receptor for PDGF-AA and PDGF-BB binding.

The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.

Mutational analysis of the interaction between the bovine papillomavirus E5 transforming protein and the endogenous beta receptor for platelet-derived growth factor in mouse C127 cells.

The bovine papillomavirus E5 protein is a 44-amino-acid membrane-associated protein that forms a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor in rodent and bovine fibroblasts, resulting in sustained receptor activation and cell transformation. We report here that high-level expression of the E5 protein caused a reduction in the level of the mature form of the PDGF beta receptor in acutely and stably transformed mouse C127 cells. To explore in more detail the interaction of the E5 protein and the PDGF beta receptor, we tested the abilities of various E5 point mutants to bind the PDGF receptor, to induce PDGF receptor down-regulation and tyrosine phosphorylation, and to transform cells. A transformation-competent mutant, like the wild-type E5 protein, bound the receptor and induced receptor tyrosine phosphorylation and down-regulation. Transformation-defective E5 proteins either failed to interact with the endogenous PDGF beta receptor in mouse fibroblasts or underwent an aberrant interaction with the receptor. Mutation of glutamine at position 17, aspartic acid at position 33, or both carboxyl-terminal cysteine residues required for E5 homodimerization interfered with stable complex formation with the PDGF receptor, tyrosine phosphorylation and down-regulation of the receptor, and cell transformation. Point mutations at several other carboxyl-terminal positions generated transformation-defective E5 proteins that formed a complex with the PDGF receptor and induced receptor tyrosine phosphorylation but did not induce PDGF receptor down-regulation. Either PDGF receptor activation is not sufficient for transformation of C127 cells or the receptors that are tyrosine phosphorylated in response to these mutant E5 proteins are not fully activated and therefore are not able to deliver a mitogenic signal.

Ligand-induced polyubiquitination of receptor tyrosine kinases.

The platelet-derived growth factor beta-receptor undergoes polyubiquitination as a consequence of ligand binding. In the present study, we have examined the ligand-induced receptor ubiquitination also in the other receptor tyrosine kinase (structurally different) subfamilies by immunoblotting with anti-ubiquitin antiserum. In addition to the platelet-derived growth factor alpha- and beta-receptors, all the monomeric receptor tyrosine kinases examined, such as the receptors for epidermal growth factor (subfamily I), colony stimulating factor-1 (subfamily III), and fibroblast growth factor (subfamily IV), were found to be ubiquitinated after ligand stimulation. However, the insulin receptor (subfamily II), which is a tetrameric molecule, was not. These data suggest that the ligand-induced polyubiquitination of the receptor is a general phenomenon observed in most of the monomeric receptor tyrosine kinases.

Activation of phospholipase C gamma in Schizosaccharomyces pombe by coexpression of receptor or nonreceptor tyrosine kinases.

The fission yeast Schizosaccharomyces pombe has no detectable endogenous receptor tyrosine kinases or associated signalling apparatus, and we have used this cell system to reconstitute mammalian platelet-derived growth factor beta (PDGF beta) receptor-linked activation of phospholipase C gamma 2 (PLC gamma 2). The PDGF beta receptor migrates as a glycosylated protein of 165 kDa associated exclusively with membrane fractions. No tyrosine autophosphorylation was detected when PDGF beta was expressed alone. PLC gamma 2 appears as a 140-kDa protein distributed between particulate and soluble fractions which exhibits characteristic selectivity for phosphatidylinositol 4,5-bisphosphate and is sensitive to powerful activation by Ca2+. When coexpressed, both PDGF beta and PLC gamma 2 undergo tyrosine phosphorylation, and this is accompanied by a > 26-fold increase in [3H]inositol 4,5-biphosphate ([3H]IP2) and [3H]inositol 1,4,5-triphosphate [3H]IP3 production. Treatment with the tyrosine phosphatase inhibitor pervanadate further increased PLC gamma 2 tyrosine phosphorylation as well as [3H]IP2 and [3H]IP3 generation. Phosphorylated PLC gamma 2 was found predominantly in membrane fractions. To test a nonreceptor tyrosine kinase, we then expressed the human proto-oncogene c-src together with its negative regulator Csk. These were immunodetectable as bands at 60 kDa (c-Src) and 50 kDa (Csk) and distributed between membrane and cytosolic fractions. When yeast coexpressing c-Src, Csk, and PLC gamma 2 was incubated with pervanadate, PLC gamma 2 was tyrosine phosphorylated and [3H]IP2 and [3H]IP3 production increased 11.0- and 7.0-fold, respectively. Csk expressed alone with PLC gamma 2 was ineffective. Similar PLC gamma 2 activation was observed upon in vitro mixing with the extracts expressing either c-Src or the PDGF beta receptor. In summary, this is the first report of a reconstitution of mammalian tyrosine kinase-linked effector activation in yeast cells and also the first demonstration of direct PLC gamma 2 activation by the proto-oncogene c-src. These observations indicate that S. pombe provides a powerful cell system in which to study critical molecular interactions and activities underlying receptor and nonreceptor tyrosine kinase-dependent cell signaling.

The kinase domain and membrane localization determine intracellular interactions between epidermal growth factor receptors.

Receptor tyrosine kinases play a central role in cellular growth, differentiation, and oncogenesis. All of these responses are triggered by growth factors interacting with the extracellular domain of transmembrane-spanning receptors, leading to dimerization and activation of an intrinsic tyrosine-specific kinase activity by an allosteric mechanism. Precise mechanisms of receptor dimerization remain poorly understood, and current models suggest that the ligand binding domain plays a major determining role. To examine the role of the intracellular domain in the association of juxtaposing receptor molecules, the full-length epidermal growth factor receptor was transiently co-expressed in human 293 fibroblasts with a truncated receptor that lacks the extracellular domain. After metabolic labeling with [35S]methionine, the association of these receptor constructs was monitored by co-immunoprecipitation with an extracellular domain-specific antibody. Specific interactions found between these receptors were independent of ligand binding or an intact ATP-binding site. Truncated receptors that had sequences necessary for membrane localization, and that were capable of interacting with full-length receptor tyrosine kinase, also displayed constitutive kinase activity as well as the capacity to transphosphorylate kinase-negative receptors. Receptor co-immunoprecipitation occurred between constructs that comprise the intracellular domains of the epidermal growth factor and beta-platelet-derived growth factor receptors, and HER-2. Subsequent deletion analysis has identified the major region of epidermal growth factor receptor intracellular interaction to be within the kinase domain.

p53 expression during normal tissue regeneration in response to acute cutaneous injury in swine.

The present studies investigated the in vivo expression of the p53 suppressor gene and protein product in response to acute cutaneous injury in swine, along with the parallel expression of the c-sis/PDGF-B mitogen and its receptor beta (PDGF-R beta). p53 expression was shown to be suppressed during the period of active cellular proliferation in the injured tissue and to reemerge during the stages of healing. In contrast, c-sis/PDGF-B and PDGF-R beta were expressed during the early phase of active cellular proliferation and they were suppressed upon healing. This inverse relationship between mitogenic growth factors and p53 suggests the presence of well-controlled physiologic mechanisms that regulate in vivo the processes of normal tissue repair in response to injury. At the stages of tissue regeneration, these mechanisms include both the expression of growth factors that promote cell proliferation and the suppression of p53 that downregulates proliferation. At the stages of healing, the expression of the mitogenic growth factors is suppressed and that of p53 reemerges, reaching its peak at the time of complete epithelialization and healing of the injured tissue. These studies are the first to link the response of p53 protein to physiologic processes of tissue regeneration in vivo.

Activation of phospholipase C-gamma 1 through transfected platelet-derived growth factor receptor enhances interleukin 2 production upon antigen stimulation in a T-cell line.

Phospholipase C-gamma 1 (PLC gamma 1) plays an important role in the signal transduction pathway by producing second messengers. However, the activation mechanism of PLC gamma 1 and the role of the phosphatidylinositol pathway for interleukin 2 (IL-2) production in T lymphocytes remain to be determined. To analyze the functional role of this pathway in T cells, we expressed an epidermal growth factor receptor (EGF) or platelet-derived growth factor (PDGF) receptor (EGF-R or PDGF-R), both of which are known to directly activate PLC gamma 1 in fibroblasts, into a murine T-cell hybridoma. Both receptors were expressed on the cell surface and caused tyrosine phosphorylation of multiple substrates, including the receptor itself, upon ligand binding. While EGF stimulation did not either cause phosphorylation of PLC gamma 1 or induce Ca2+ mobilization in the EGF-R transfectant in this system, PDGF treatment induced tyrosine phosphorylation of PLC gamma 1 and Ca2+ mobilization in the PDGF-R transfectant. Stimulation through PDGF-R enhanced IL-2 production upon antigen stimulation of the transfectants, although PDGF treatment alone did not induce IL-2 production. These results suggest that activation of the phosphatidylinositol pathway affects the downstream pathway to IL-2 production but is not sufficient to produce IL-2 and that cooperation with signals from tyrosine kinase cascades is required for IL-2 production.

Monoamine-activated alpha 2-macroglobulin binds trk receptor and inhibits nerve growth factor-stimulated trk phosphorylation and signal transduction.

Monoamine-activated alpha 2-macroglobulin (alpha 2M) has been shown to inhibit beta-nerve growth factor (NGF)-promoted neurite outgrowth and the survival of embryonic sensory and forebrain neurons, whereas normal alpha 2M has little or no such activity. The objective of this study is to elucidate the mechanism of inhibition by monoamine-activated alpha 2M. Methylamine-activated alpha 2M (MA-alpha 2M) and serotonin-activated alpha 2M (5HT-alpha 2M) dose dependently inhibit NGF-promoted neurite outgrowth of the pheochromocytoma PC12 cell and its subline PC12(6-24) which overexpresses human trk protooncogene product, but have no effect on their viability, and this inhibition can be blocked by high concentrations of NGF. The binding of MA-alpha 2M to trk, which is a part of high-affinity NGF receptor, was studied with PC12(6-24) cells and NIH-3T3 fibroblasts expressing trk (trk-3T3). In each case MA-alpha 2M readily forms stable complexes with trk in vivo, whereas normal alpha 2M does not. Both 5HT-alpha 2M and MA-alpha 2M also dose dependently block NGF-promoted autophosphorylation of trk in vivo, whereas normal alpha 2M and plasmin-reacted alpha 2M are inactive or much less active. MA-alpha 2M also blocks NGF-promoted incorporation of 32P from [32P]ATP into trk receptors in vitro. Neither MA-alpha 2M, 5HT-alpha 2M, nor normal alpha 2M, however, blocks either platelet-derived growth factor-stimulated or epidermal growth factor-stimulated tyrosine phosphorylation of the respective receptors. Tyrosine phosphorylation of two of the intracellular substrates, phospholipase C-gamma 1 and extracellular signal-regulated kinase-2, in the NGF-promoted pathways is also dose dependently blocked by MA-alpha 2M. However, by comparison MA-alpha 2M is more effective in inhibiting the activation of phospholipase C-gamma 1 than trk. We conclude that monoamine-activated alpha 2M may block neurite outgrowth and neuronal survival by its specific binding to NGF receptors, thus inhibiting the NGF-promoted activation of intracellular second messenger pathways.

The growth regulation of neurofibroma cells in neurofibromatosis type-1: increased responses to PDGF-BB and TGF-beta 1.

In neurofibromatosis type-1 (NF-1), abnormal growth regulation may be related to the formation of multiple neurofibromas. We investigated the growth responses of neurofibroma-derived cells (NF cells) and control skin fibroblasts to various growth factors. The responses to platelet-derived growth factor (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta 1) in NF cells were significantly greater than those in control fibroblasts. The increased response to PDGF-BB in NF cells was accompanied by an increased number of PDGF beta receptors, which was demonstrated by both 125I PDGF-BB binding assay and immunoblotting analysis. The increased response to TGF-beta 1 was assumed to be mediated through PDGF-like protein induction; TGF-beta 1-treated NF cells produced greater amounts of 36-kD PDGF-like protein than TGF-beta 1-treated control fibroblasts. These observations suggest that certain growth factors, e.g., PDGF-BB and TGF-beta, may play some role in the development of neurofibromas in NF-1.