Characterization of the interleukin 4 receptor. Structure and signal transduction pathways.
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Animals↗
Biomedical subjects
J H Pierce
Publications and source records attributed to J H Pierce.
Inhibition of platelet-derived growth factor autocrine growth stimulation by a monoclonal antibody to the human alpha platelet-derived growth factor receptor.
A potent neutralizing monoclonal antibody to the human alpha platelet-derived growth factor (PDGF) receptor (alpha PDGFR) was raised by immunizing BALB/c mice with 32D cells expressing the human alpha PDGFR. This monoclonal antibody, designated alpha R1, immunoprecipitated human, monkey, rabbit, pig, dog, and cat, but not hamster, rat, or mouse alpha PDGFRs. Comparison with PR292, a monoclonal antibody previously generated against the alpha PDGFR, showed that both recognized alpha PDGFR extracellular domains, but neither demonstrated reactivity against the beta PDGFR. In vitro binding studies revealed that alpha R1, but not PR292, detection of the alpha PDGFR was blocked by either PDGF AA or PDGF BB. These results strongly suggest that the receptor ligand-binding domain spatially overlapped with the alpha R1 epitope. Monoclonal antibody alpha R1 also inhibited PDGF stimulation of [3H]thymidine uptake by 32D cells expressing the alpha PDGFR (32D alpha R) as well as autocrine growth stimulation of 32D alpha R cells transfected with and expressing PDGF AA or PDGF BB. Therefore, monoclonal antibody alpha R1 may be useful in the detection and growth inhibition of malignancies in which PDGF autocrine stimulation and/or alpha PDGFR overexpression plays an important role(s).
Animals↗
Five PDGF B amino acid substitutions convert PDGF A to a PDGF B-like transforming molecule.
We used site-directed mutagenesis to determine the minimum number of PDGF B residues needed to convert PDGF A to a potently transforming PDGF B-like molecule. Substitution of two PDGF B subdomains, 106-115 and 135-144, were found to be critical. These substitutions were sufficient to broaden the ability of PDGF A to activate beta as well as alpha platelet-derived growth factor (PDGF) receptors and increase its transforming efficiency to that of PDGF B. Within subdomain I, either PDGF B residues Arg-109 and Asn-115 or Arg-109, Leu-110, and Arg-113, in combination with subdomain II PDGF B residues Asn-136, Arg-137, and Arg-142 were identified as being essential. Those mutants with transforming ability comparable with PDGF B showed significantly lower efficiencies of beta receptor triggering. Thus, our studies identify a small number of PDGF B amino acids indispensable for beta PDGF receptor interaction and suggest that a low level of beta PDGF receptor activation is sufficient to dramatically increase PDGF transforming efficiency in NIH 3T3 cells.
3T3 Cells↗
Evidence for a developmental relationship between CD5+ B-lineage cells and macrophages.
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Animals↗
Interleukin (IL)-2 and IL-3 induce distinct but overlapping responses in murine IL-3-dependent 32D cells transduced with human IL-2 receptor beta chain: involvement of tyrosine kinase(s) other than p56lck.
We have established IL-3-dependent 32D myeloid progenitor cells stably expressing the human IL-2 receptor beta chain (IL-2R beta). Whereas parental 32D cells proliferated only in response to IL-3, the transduced cells also proliferated in response to IL-2. Transduced cells expressed high- and intermediate-affinity IL-2Rs, resulting from expression of human IL-2R beta and murine IL-2R alpha chain (IL-2R alpha). IL-2 induced phenotypic changes not induced by IL-3, including the upregulated expression of endogenous murine IL-2R alpha and IL-2R beta and an increase in cell size. Therefore, the transduced IL-2R beta was not merely coupling with the IL-3 signaling pathway. IL-3 augmented several IL-2-induced responses including the up-regulation of IL-2R alpha. Both IL-2- and IL-3-induced proliferation and IL-2 induced IL-2R alpha expression were inhibited by the tyrosine kinase inhibitor herbimycin A. Thus, both IL-2- and IL-3-mediated effects required tyrosine kinase activity. The identity of the tyrosine kinase(s) mediating the IL-2 signals in these cells is not known but cannot be p56lck, a tyrosine kinase found in T cells, since 32D-IL-2R beta cells do not express p56lck.
Animals↗
Mitogenic signal transduction: a common target for oncogenes that induce resistance to ionizing radiations.
We hypothesized that resistance to ionizing radiations accompanying neoplastic transformation caused by some oncogenes was due to common biochemical pathways affecting the mechanism of mitogenic signal transduction. In order to verify this hypothesis, we studied the formation of mitogenic second messengers in cells transformed by oncogenes that induce radioresistance. We observed an increase of diacylglycerol which activates protein kinase C, an increase of phosphatidylcholine metabolism, with a concomitant decrease of inositol lipid metabolism. Our data show that sensitivity to ionizing radiations was inversely related to the intracellular level of diacylglycerol; study of signalling alterations in spontaneous tumors could provide predictive indications about the responsiveness of neoplasia to radiation therapy.
3T3 Cells↗
A deletion in the extracellular domain of the alpha platelet-derived growth factor (PDGF) receptor differentially impairs PDGF-AA and PDGF-BB binding affinities.
32D cells transfected with the human alpha platelet-derived growth factor receptor (alpha PDGFR) bind PDGF-AA, -AB, and -BB isoforms with high affinity, and the binding of each can be efficiently competed by all three isoforms. In an effort to develop better understanding of spatial relationships of binding sites for PDGF-AA and -BB, we constructed an alpha PDGFR mutant which deleted amino acids 150-189 within its extracellular domain. This mutant showed a marked decrease in high affinity binding sites for PDGF-AA without comparable alteration in affinity for PDGF-BB. These findings imply that the high affinity binding sites for PDGF-AA and PDGF-BB in the alpha PDGFR extracellular domain are not structurally coincident.
Amino Acid Sequence↗
A single amino acid substitution is sufficient to modify the mitogenic properties of the epidermal growth factor receptor to resemble that of gp185erbB-2.
The epidermal growth factor (EGF) receptor (EGFR) and the erbB-2 gene product, gp185erbB-2, exhibit distinct abilities to stimulate mitogenesis in different target cells. By using chimeric molecules between these two receptors, we have previously shown that their intracellular juxtamembrane regions are responsible for this specificity. Here we describe a genetically engineered EGFR mutant containing a threonine for arginine substitution at position 662 in the EGFR juxtamembrane domain, corresponding to threonine 694 in gp185erbB-2. This mutant, designated EGFRThr662, displayed affinity for EGF binding and catalytic properties that were indistinguishable from those of the wild type EGFR. However, EGFRThr662 behaved much as gp185erbB-2 in a number of bioassays which readily distinguish between the mitogenic effects of EGFR and gp185erbB-2. Moreover, significant differences were detected in the pattern of intracellular proteins phosphorylated on tyrosine in vivo by EGFR and EGFRThr662 in response to EGF. Thus, small differences in the primary sequence of two closely related receptors have dramatic effects on their ability to couple with mitogenic pathways.
3T3 Cells↗
IL-4 activates a distinct signal transduction cascade from IL-3 in factor-dependent myeloid cells.
Interleukin-4 (IL-4) was shown to induce a potent mitogenic response in the IL-3-dependent myeloid progenitor cell line, FDCP-2. Although IL-4 could not sustain long-term growth of FDCP-2 cells, it enhanced their growth in serum-free medium containing IL-3. IL-4 triggered prominent tyrosine phosphorylation of a substrate(s) migrating at 170 kDa and less striking phosphorylation of several other proteins, including the IL-4 receptor. By contrast, IL-3 induced distinct tyrosine phosphorylation of proteins migrating at 145, 97, 70, 55 and 52 kDa in the same cell line. IL-4 treatment of FDCP-2 cells caused a dramatically strong association of phosphatidylinositol 3-kinase (PI 3-kinase) both with the 170 kDa tyrosine phosphorylated substrate and with the IL-4 receptor itself. By contrast, IL-3 triggered only weak association of PI 3-kinase activity with the 97 kDa substrate. While IL-4 did not affect cellular raf, IL-3 stimulation did induce a shift in its mobility presumably due to serine/threonine phosphorylation. Taken together, our results indicate that IL-4 and IL-3 activate distinct phosphorylation cascades in the same cell background; this may reflect a difference in the biological function of these two cytokines.
Animals↗
Transduction of a signal for arachidonic acid metabolism by untriggered CSF-1 receptor induces an opposite effect to that induced by CSF-1 receptor and its ligand: separate regulation of phospholipase A2 and cyclooxygenase by CSF-1 receptor/CSF-1.
The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E2 (PGE2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE2 production. Addition of ST638, a tyrosine kinase inhibitor, to cells transfected with c-fms in the absence of CSF-1 inhibited PGE2 production within 10-60 min. Its addition to the same cells in the presence of CSF-1 induced an opposite effect, but required longer treatment (24 h). In either cell type, AA release was not affected by this agent. These data indicate that CSF-1 may regulate cyclooxygenase activity. The different effect of CSF-1 receptor on PGE2 production in the presence or absence of CSF-1 and the opposite effect of a tyrosine kinase inhibitor on PGE2 suggest that both the receptor alone or the receptor-ligand complex may transduce an active, but different, signal through tyrosine phosphorylation. CSF-1 receptor and CSF-1 may exert separate, but related, effects on phospholipase A2 and cyclooxygenase activity which, in concert, or along with other tyrosine kinases, regulate prostaglandin production.
Animals↗
The expression of the mouse VpreB/lambda 5 locus in transformed cell lines and tumors of the B lineage differentiation pathway.
The expression of RNA transcripts from two pre B lymphocyte related genes, VpreB and lambda 5, has been studied in a series of transformed cell lines which appear frozen at different states of B lineage differentiation, from early progenitors to surface Ig positive B cells. In the HAFTL-1 cell line, which arose from fetal liver by transformation with a retrovirus containing the Hras oncogene, Northern analysis of poly A+ mRNA as well as in situ hybridization of RNA in single cells revealed that lambda 5 and VpreB are already expressed at the progenitor stage and increase in expression as the progenitors differentiate to precursor (preB) cells, or are turned off as the progenitors differentiate to myeloid cells. Continued rearrangements of Ig genes in pre B cell lines leading to Ig expression on the surface of NFS-5 pre B cells do not influence the continued expression of VpreB and lambda 5. Surface Ig-positive B lineage cell lines also express the pre B-related genes. Both Ly1+ as well as Ly1- pre B cells are VpreB- and lambda 5-positive. Lipopolysaccharide (LPS) stimulation of 70Z/3 pre B cells does not turn off lambda 5 expression. It therefore appears that, at least in transformed cell lines, the expression of VpreB and lambda 5 is not directly regulated by the expression of microH, kappa L, or lambda L chains, LPS reactivity, or the Ly1 surface antigen. Fusion of plasmacytoma cells with normal pre B cells to generate pre B hybridomas leads to down-regulation of VpreB/lambda 5 expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Activation of the colony-stimulating factor 1 receptor leads to the rapid tyrosine phosphorylation of GTPase-activating protein and activation of cellular p21ras.
We have previously reported that platelet-derived growth factor (PDGF) induced tyrosine phosphorylation of GTPase-activating protein (GAP) in intact quiescent fibroblasts under conditions in which insulin and basic fibroblast growth factor (bFGF) were ineffective (Molloy et al., 1988). In the present study, we have provided evidence that colony-stimulating factor 1 (CSF-1) is capable of inducing tyrosine phosphorylation of GAP and its associated cellular proteins, p62 and p190, in NIH3T3 cells overexpressing the human CSF-1 receptor (CSF-1R). However, the extent of GAP tyrosine phosphorylation induced by CSF-1 was approximately 10% of that induced by PDGF-BB in the NIH3T3 fibroblasts. Despite this significant difference, both PDGF-BB and CSF-1 increased the activation of p21ras, the extent of which correlated well with the mitogenic response induced by each growth factor in these cells. Taken together, our findings provide evidence for a possible role of tyrosine phosphorylation of GAP and GAP-associated phosphoproteins in regulating transduction of CSF-1-induced mitogenic signals through p21ras activation.
3T3 Cells↗
Expression of M-CSF and its receptor (C-FMS) during factor-independent cell line evolution from hematopoietic progenitor cells cocultivated with gamma irradiated marrow stromal cell lines.
Gamma irradiation of plateau-phase clonal bone marrow stromal cell lines produces factor-independent growth of cocultivated clonal interleukin-3/granulocyte-macrophage colony-stimulating factor-dependent hematopoietic progenitor cell lines. The process is associated with three biologic changes including: (i) adherence of hematopoietic cells to stromal cells forming 'cobblestone islands'; (ii) an intermediate stage [during which the cells show proliferation in suspension in the presence in leukemogenic stromal factor (LSF), a factor similar to macrophage colony-stimulating factor (M-CSF) released by irradiated stromal cells, and transient hematopoietic cell surface expression of MAC-1, and c-fms (M-CSF receptor)]; and (iii) a third stage of factor-independence. A monoclonal antibody to M-CSF receptor inhibited proliferation of intermediate stage but not all factor-independent cell subclones. In the present studies, a subclonal factor-independent malignant subline of FDC-P1JL26 derived by cocultivation with gamma-irradiated stromal cells as well as the parent clone and intermediate stage cells were shown to express significant levels of M-CSF polyA+ mRNA and M-CSF of at least two sizes (23 and 15 kDa) as detected by 35S-methionine labelling and immunoprecipitation with polyclonal anti-M-CSF antiserum. There was no significant difference in intracellular M-CSF protein size between cells at each of the three stages of biologic change. This M-CSF was not detected on the cell surface by fluorescence-activated cell sorting (FACS). In contrast, c-fms expression at the cell surface was detected by FACS analysis and c-fms polyA+ mRNA was only detected during the intermediate stage of induction of factor-independence. FDC-P1JL26 parent cells, the subclone stimulated by LSF, and the factor-independent subclone, showed little or no detectable autophosphorylation of the c-fms receptor at tyrosine. There was no detectable rearrangement of the M-CSF or c-fms genes by Southern analysis between clonal lines during the three stages. While we cannot rule out an autocrine mechanism or mutated c-fms receptor mechanism, the data also suggest that evolution of hemopoietic cell factor-independence during cocultivation with irradiated stromal cells may involve a mechanism distal to the c-fms receptor/M-CSF interaction.
Animals↗
Role of alpha beta receptor heterodimer formation in beta platelet-derived growth factor (PDGF) receptor activation by PDGF-AB.
We investigated the ability of highly purified recombinant platelet-derived growth factor (PDGF) AB to interact with the products of alpha and beta receptor genes expressed in cells independently or concurrently. Although PDGF-AB lacked any detectable ability to bind or activate beta receptors in cells expressing only this receptor, efficient beta receptor activation by this ligand was readily observed in cells coexpressing alpha platelet-derived growth factor receptors (alpha PDGFRs). beta receptor activation induced by PDGF-AB was shown to be dependent upon in vivo physical association of this receptor with alpha PDGFRs. Moreover, cross-linking analysis established the existence of PDGF-AB-induced beta PDGFR dimers in vivo. All of these findings argue that initial PDGF-AB interaction with the alpha PDGFR induces conformational changes in the ligand or receptor that facilitates efficient recruitment of beta PDGFR by this PDGF isoform.
Animals↗
Mitogenic signal transduction in normal and transformed 32D hematopoietic cells.
We studied mitogenic signal transduction in normal and oncogene-transformed 32D cells, a murine hematopoietic cell line that is normally dependent on interleukin-3 (IL3) for proliferation and survival. The formation of second messengers was measured in normal cells stimulated with IL3, and in cells transfected with foreign growth factor receptor genes and stimulated with appropriate growth factors. We also measured the steady-state level of second messengers in 32D cells transformed by erbB, abl, and src oncogenes which abrogate growth factor requirement. We found that IL3 stimulated the formation of diacylglycerol independently of inositol lipid turnover, but concomitantly with increased turnover of phosphatidylcholine. Epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) stimulated the 'classical' turnover of inositol lipids with formation of diacylglycerol and calcium-mobilizing inositol phosphates. Colony stimulating factor-1 triggered inositol lipid turnover, although to a much lower extent than EGF and PDGF. Transformed cells showed elevated levels of diacylglycerol together with increased turnover of phosphoinositides and phosphatidylcholine. Taken together these results indicate that different growth factors and oncoproteins associate with multiple signalling pathways in 32D cells.
Animals↗
Ligand stimulation of transfected and endogenous growth factor receptors enhances cytokine production by mast cells.
IL-3 dependent mast cell lines produce cytokines in response to Fc receptor cross-linkage or to ionomycin. In this study we have observed that cells pre-cultured in IL-3 produce 10-100 times more cytokine after receptor cross-linkage in comparison with IL-4 pre-cultured cells. Although several hematopoietin receptors, including those for IL-3, IL-4 and EPO, do not contain tyrosine kinase domains, their occupancy with ligand causes tyrosine phosphorylation of specific cellular substrates. Therefore, the contribution of tyrosine kinase activation to the ability of an IL-3 dependent mast cell line, CFTL-15, to produce cytokines was analyzed. The CFTL-15 cells were transfected with growth factor receptors containing ligand-inducible tyrosine kinase domains (EGFR and PDGFR, and CSF-IR) or with the EPOR. All of the transfectants were able to proliferate in response to IL-3 or to their respective growth factor and to produce IL-3 in response to IgE receptor cross-linkage. Stimulation of the EGFR and PDGFR transfectants with their respective ligands resulted in the production of IL-3, IL-6, and GM-CSF. Stimulation of the CSF-1R or EPOR transfectants with growth factor alone failed to induce cytokine production. However, in co-stimulation assays each of the growth factors enhanced the amount of cytokine produced in response to Fc epsilon RI cross-linkage. The ability of these stimuli to induce tyrosine phosphorylation in the transfectants was analyzed. Fc epsilon RI cross-linkage in the transfectants routinely induced the tyrosine phosphorylation of 145, 86 and 72 kDa proteins, with occasional phosphorylation of 55, 52, and 40 kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Selective inhibition of PGE2 production in cells transfected with c-fms encoded CSF-1 receptor genes by the tyrosine kinase inhibitor, ST638.
In this study, we investigated the effect of ST638, a novel tyrosine kinase inhibitor, on TPA and ionomycin-stimulated PGE2 production after transfection of c-fms encoded CSF-1 receptor (CSF-1R) DNA (with or without transforming activity) into the myeloid progenitor cell line, 32D. ST638 inhibited prostaglandin E2 (PGE2) production induced after transfection of normal c-fms into 32D cell line, but failed to inhibit PGE2 production induced in 32D cells transformed with c-fms containing a point mutation at tyrosine 969 in the intracellular domain (substitution with phenylalanine) and at leucine 301 in the extracellular domain (substitution with serine). The selective effect on PGE2 production in normal c-fms-bearing cells by ST638 may indicate the presence of different induction pathways, one sensitive to ST638 and the other not. Alternatively, the phosphorylation site at tyr969 in c-fms may be a site of ST638 action, and upon its removal from the transforming c-fms, ST638 loses its inhibitory effect.
Arachidonic Acid↗
The v-abl, c-fms, or v-myc oncogene induces gamma radiation resistance of hematopoietic progenitor cell line 32d cl 3 at clinical low dose rate.
A variety of viral and cellular oncogenes have been described with differing mechanisms of action but with the common property of inducing morphologic alteration of cells in culture. Subclonal lines of oncogene expressing cells have been shown to produce tumors in vivo. Expression of the N-ras oncogene in embryo fibroblast NIH/3T3 cells has been demonstrated to increase radioresistance in vitro, and these results have been confirmed and extended to human cell lines expressing the c-raf oncogene. In the present report, we have examined the effects of expression of the c-fms, v-abl, or v-myc oncogene in a clonal hematopoietic progenitor cell line 32D cl 3. The 32D cell line is nonmalignant in vivo and is dependent upon a source of Interleukin-3 (IL-3) for growth in vitro. The radiation survival of 32D cl 3 cells transfected and expressed in the c-fms oncogene showed significant increase in the radioresistance at both 5 cGy/min and 116 cGy/min. A clone of 32D cl 3 transfected and expressing the v-myc oncogene demonstrated increased radioresistance at both dose rates. Results of split dose experiments suggested significant repair of sublethal irradiation damage of 32D-v-abl cells. Results were compared with expression of the same v-abl oncogene in the NIH/3T3 embryo fibroblast cell line. The data demonstrate that gamma irradiation resistance is significantly increased by each oncogene expressed in 32D cl 3 cells. The data on cell line 32D cl 3 may correlate with the radioresistance of v-abl expressing human hematopoietic cell malignancies treated by irradiation therapy.
Animals↗