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L Claesson-Welsh

Publications and source records attributed to L Claesson-Welsh.

At least 109 records · Page 6Linked to original sources
PubMed1992

The expression of PDGF alpha- and beta-receptors in subpopulations of PDGF-producing cells implicates autocrine stimulatory loops in the control of proliferation in cytotrophoblasts that have invaded the maternal endometrium.

In order to explain the high proliferative potential of human placental cytotrophoblasts, we have addressed the potential involvement of platelet-derived growth factor (PDGF) ligand and receptors. Although PDGF is usually described as a mitogen for cells of mesenchymal origin, we show in this report that extra-villous term placental cytotrophoblasts express the PDGF alpha- and beta-receptor genes, both in vivo and in vitro. In addition, cytotrophoblasts produce significant amounts of PDGF-B protein. By immunohistochemical analysis of receptor expression, we found that the PDGF alpha-receptors could be detected at the cell surface, while the PDGF beta-receptors were only detected intracellularly. In addition, double immunostaining analysis showed that the PDGF alpha- and beta-receptor molecules are expressed in different subpopulations of cytotrophoblasts. The addition of PDGF-AA and PDGF-BB homodimers to cytotrophoblast primary cultures induced a significant increase in DNA synthesis. We conclude, therefore, that PDGF is a growth factor for placental cytotrophoblasts and suggest that the growth of cytotrophoblasts can partly be explained by a PDGF autostimulatory loop, limited by the number of receptor-positive cytotrophoblasts.

Cell Membrane↗
PubMed1992

Transduction of circular membrane ruffling by the platelet-derived growth factor beta-receptor is dependent on its kinase insert.

The platelet-derived growth factor (PDGF) alpha- and beta-receptors both mediate a mitogenic response, but only the beta-receptor mediates circular actin reorganization and chemotaxis. The tyrosine kinase domains of the receptors contain noncatalytic inserts of about 100 residues. In order to determine the role of these domains in the differential signaling of the two receptors, we constructed chimeric PDGF receptors and expressed the constructs in porcine aortic endothelial cells. The chimeric receptors were similar to the wild-type receptors in their ability to induce mitogenicity in response to ligand. Examination of receptor-associated substrates by in vitro kinase assays revealed that phosphoproteins of 72 and 110 kilodaltons were associated with the kinase insert of the alpha-receptor, whereas a phosphoprotein of 130 kilodaltons was associated with the kinase insert of the beta-receptor. Actin reorganization in the form of circular membrane ruffling was seen after ligand stimulation of the beta-receptor and the alpha-receptor containing the beta-receptor kinase insert but not after stimulation of the alpha-receptor or the beta-receptor containing the alpha-receptor kinase insert. These data indicate that the PDGF beta-receptor kinase insert has an essential function in the signal transduction pathway leading to circular membrane ruffling.

Actins↗
PubMed1991

Induction of platelet-derived growth factor alpha- and beta-receptor mRNA and protein by platelet-derived growth factor BB.

We describe that stimulation of human fibroblasts with platelet-derived growth factor BB (PDGF-BB) induces a transient up-regulation of the PDGF alpha- and beta-receptor transcript and protein levels. The effect of PDGF-BB on the receptor transcript levels was more pronounced than those seen when other cytokines were used. Regulation of transcript levels by PDGF-BB was mediated through post-transcriptional mechanisms. No induction could be observed in a nuclear run-on analysis, but cycloheximide treatment attenuated the accumulation of both alpha- and beta-receptor transcripts induced by PDGF-BB. An increase in receptor protein levels was observed using two different experimental approaches. Increased amounts of receptor precursor forms could be immunoprecipitated from metabolically labeled cells, stimulated with PDGF-BB. In a second approach, cells were exposed to different concentrations of PDGF-BB, and, in a subsequent step, ligand binding analysis was performed. In this experiment, an initial down-regulation of receptors was followed by increased levels of the cell surface forms of the receptors. In conclusion, PDGF-BB, but not PDGF-AA, induces increased synthesis of both PDGF alpha- and beta-receptor protein; this constitutes a positive feed-back mechanism, which, for example, could serve to potentiate autocrine stimulation of growth.

Amino Acid Sequence↗
PubMed1991

Identification of a hydrophobic region in the carboxyl terminus of the platelet-derived growth factor beta-receptor which is important for ligand-mediated endocytosis.

The functional properties of carboxyl terminally truncated mutants of the platelet-derived growth factor beta-receptor were compared with those of the wild-type receptor and a receptor mutant made kinase negative by a point mutation. A mutant in which 98 amino acids were deleted retained kinase activity and mediated a mitogenic signal, whereas deletion of 141 or 155 amino acids led to loss of kinase activity and ability to mediate a mitogenic signal. The mutant with 155 amino acids deleted, i.e. the entire carboxyl-terminal tail downstream of the kinase domain, did not undergo ligand-mediated internalization and down-regulation, whereas the mutant with 141 amino acids deleted was internalized at a relatively high rate. This indicates that the 14 amino acids immediately downstream of the kinase domain is of importance for the internalization of the platelet-derived growth factor beta-receptor. This region is hydrophobic and shares no similarity to other sequences postulated to mediate endocytotic signals.

Amino Acid Sequence↗
PubMed1991

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.

DNA↗
PubMed1991

Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis.

The proto-oncogene c-kit is allelic with the murine white spotting (W) locus and encodes a transmembrane protein tyrosine kinase that is structurally related to the receptors for platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1). Recently the ligand for the c-kit product, stem cell factor (SCF), was identified in both transmembrane and soluble forms. In order to examine the mechanism for receptor activation by SCF and biological properties of the activated c-kit product, we transfected the wild-type human c-kit cDNA into porcine aortic endothelial cells. We found that the receptor was down-regulated and transmitted a mitogenic signal in response to stimulation with soluble SCF. We also demonstrate that SCF induces dimerization of the c-kit product in intact cells, and that dimerization of the receptor is correlated with activation of its kinase. Activation of the c-kit product by SCF was found to induce circular actin reorganization indistinguishable from that mediated by the PDGF beta-receptor in response to PDGF-BB. Furthermore, soluble SCF was a potent chemotactic agent for cells expressing the c-kit product, a property which might be of importance during embryonic development.

Actins↗
PubMed1991

FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern.

We have previously identified two novel members of the fibroblast growth factor receptor (FGFR) gene family expressed in K562 erythroleukemia cells. Here we report cDNA cloning and analysis of one of these genes, named FGFR-4. The deduced amino acid sequence of FGFR-4 is 55% identical with both previously characterized FGFRs, flg and bek, and has the structural characteristics of a FGFR family member including three immunoglobulin-like domains in its extracellular part. Antibodies raised against the carboxy terminus of FGFR-4 detected 95 and 110 kd glycoproteins with a protein backbone of 88 kd in COS cells transfected with a FGFR-4 cDNA expression vector. The FGFR-4 protein expressed in COS cells could also be affinity-labeled with radioiodinated acidic FGF. Furthermore, ligand binding experiments demonstrated that FGFR-4 binds acidic FGF with high affinity but does not bind basic FGF. FGFR-4 is expressed as a 3.0 kb mRNA in the adrenal, lung, kidney, liver, pancreas, intestine, striated muscle and spleen tissues of human fetuses. The expression pattern of FGFR-4 is distinct from that of flg and bek and the yet additional member of the same gene family, FGFR-3, which we have also cloned from the K562 leukemia cells. Our results suggest that FGFR-4 along with other fibroblast growth factor receptors performs cell lineage and tissue-specific functions.

Amino Acid Sequence↗
PubMed1991

Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor.

The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex.

Animals↗
PubMed1991

cDNA cloning and expression of a human FGF receptor which binds acidic and basic FGF.

We have isolated and characterized a cDNA clone, phFGFR, encoding a human fibroblast growth factor (FGF) receptor. phFGFR contains an open reading frame which encodes an 820 amino acid polypeptide with three immunoglobulin-like domains in the extracellular part and an intracellular split tyrosine kinase domain. Transient expression in COS-1 cells and immunoprecipitation using an antiserum raised against a C-terminal peptide, gave rise to two components, representing mature (130 kDa) and precursor (115 kDa) forms of the phFGFR encoded polypeptide, which was denoted hFGFR-1. Crosslinking of iodinated acidic FGF (aFGF) and basic FGF (bFGF) to transiently expressing COS-1 cells revealed a major band of 95 kDa, which was competed for by both aFGF and bFGF. From Scatchard analyses, the Kd:s for binding of aFGF and bFGF to hFGFR-1 were estimated to 25 pM and 41 pM, respectively. Thus, phFGFR encodes a human FGF receptor with high affinity for both aFGF and bFGF.

Amino Acid Sequence↗
PubMed1991

Human malignant mesothelioma cell lines express PDGF beta-receptors whereas cultured normal mesothelial cells express predominantly PDGF alpha-receptors.

In human malignant mesothelioma cell lines elevated expression of the platelet-derived growth factor (PDGF) beta-chain (c-sis) gene was previously reported, while normal mesothelial cells barely express this gene. Expression of the PDGF A-chain gene was only slightly elevated in these cell lines compared with normal mesothelial cells. For a putative autocrine function of the produced PDGF, in these cells expression of PDGF receptors is a prerequisite. In this paper we report on the expression of PDGF alpha- and beta-receptors in normal and malignant mesothelial cells. Cultured normal mesothelial cells expressed PDGF alpha-receptor mRNA and protein and had weak to undetectable levels of the PDGF beta-receptor mRNA and protein. In contrast, malignant mesothelioma cell lines were found to express PDGF beta-receptor mRNA and protein, while PDGF alpha-receptor expression was not detectable by Northern blotting and immunoprecipitation. Binding experiments with [125I]-PDGF-AA and [125I] PDGF-BB to normal and malignant mesothelial cell lines confirmed these observations. These results suggest that autocrine stimulation of growth may occur both in cultured normal mesothelial cells (PDGF-AA acting via the alpha-receptor) and in malignant mesothelioma cell lines (PDGF-BB acting via the beta-receptor).

Blotting, Northern↗
PubMed1990

Expression of messenger RNAs for platelet-derived growth factor and its receptors in human sarcoma cell lines.

Growth factors of the platelet-derived growth factor (PDGF) family have been thought to possess autocrine functions in certain neoplasms of mesenchymal and glial origin. This notion has been based on observations that these tumors express PDGF genes and produce PDGF-like growth factors. Corresponding data on PDGF receptor expression in sarcoma cell lines is essentially lacking. The cloning of cDNA for 2 distinct PDGF receptors with different abilities to recognize the members of the PDGF family and availability of recombinant PDGF for binding studies have recently made it possible to study the expression of both receptor types in tumor cell lines. We present here a study on 8 human sarcoma cell lines, and show a large variability and independency in the expression of the 2 PDGF receptor types as well as of the genes encoding the corresponding ligands.

Humans↗
PubMed1990

TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences.

TGF-beta occurs in a latent complex of high Mr. We report the cDNA cloning and an initial structural and functional characterization of a component of the large latent TGF-beta 1 complex, denoted TGF-beta 1 binding protein (TGF-beta 1-BP). Most of the sequence of fibroblast TGF-beta 1-BP is made up of cysteine-rich repeats of two different kinds; there are 16 EGF-like repeats and three repeats with a distant resemblance to EGF, but of a distinct type hitherto not found in any other protein. beta-hydroxylated asparagine residues were identified in two of the EGF-like repeats. TGF-beta 1-BP purified from human platelets is considerably smaller than the fibroblast form (125-160 kd vs. 170-190 kd), suggesting that there is alternative splicing of the TGF-beta 1-BP gene or that TGF-beta 1-BP undergoes cell-specific proteolysis. TGF-beta 1-BP was found not to bind and inactive TGF-beta 1; its role in the latent complex is discussed.

Amino Acid Sequence↗
PubMed1990

Structural and functional aspects of platelet-derived growth factor and its receptors.

Platelet-derived growth factor (PDGF) is a dimeric molecule that exists as homodimers or heterodimers of related polypeptide chains (A and B). Two types of PDGF receptor have been identified. The PDGF alpha-receptor binds all three isoforms with high affinity whereas the beta-receptor binds only PDGF-BB with high affinity, PDGF-AB with low affinity and does not appear to bind PDGF-AA. The alpha- and beta-receptors are structurally related, each having an intracellular protein tyrosine kinase domain. Ligand-induced functional activation of the receptors appears to involve receptor dimerization. Binding of PDGF to its receptor is followed by internalization and degradation of the ligand-receptor complex. Experiments with mutant receptors have shown that ligand-induced internalization is not absolutely dependent on the kinase activity of the beta-receptor. The v-sis oncogene of simian sarcoma virus (SSV) is a retroviral version of the PDGF B chain gene and SSV-transformation is mediated by an autocrine PDGF-like growth factor. Formal evidence that the expression of the PDGF beta-receptor is sufficient to confer susceptibility to SSV-transformation has been obtained using porcine endothelial cells expressing a recombinant human beta-receptor. PDGF is a chemotactic agent for several cell types. Recent experiments have shown that the PDGF beta-receptor mediates a chemotactic response and that this effect requires an intact protein tyrosine kinase activity.

Animals↗
PubMed1990

B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase.

Porcine aorta endothelial cells are devoid of receptors for platelet-derived growth factor (PDGF). We have transfected such cells with cDNA for the PDGF B-type receptor, both the wild-type receptor and a mutant form of the receptor (K634A), in which the putative nucleotide-binding lysine of the protein-tyrosine domain has been changed to alanine. Immunoprecipitation studies of metabolically labeled cells showed that both types of receptors were synthesized and processed to the mature form of Mr 190,000. In cells expressing the wild-type receptor, PDGF-BB, the natural ligand for the B-type receptor, induced membrane ruffling and reorganization of actin. Such a response has previously been seen in cells expressing the natural PDGF B-type receptor in response to PDGF-BB. No such effect was induced in nontransfected cells or in cells expressing the K634A mutant receptor. PDGF was also shown to be chemotactic for cells expressing the wild-type receptor, whereas no chemotactic response was elicited in control cells or in cells expressing the K634A mutant receptor. Our study thus provides formal evidence that the PDGF B-type receptor mediates a motility response including actin reorganization and chemotaxis. Furthermore, the results establish a role for the receptor-associated protein-tyrosine kinase in the transduction of the chemotactic signal.

Animals↗
PubMed1990

Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF beta receptor in isolated pancreatic islet cells stimulates DNA synthesis.

Suspensions rich in pancreatic beta cells were transfected by means of electroporation or by using the liposome technique with DNA constructs coding for the B chain of platelet-derived growth factor (PDGF) and the PDGF alpha and beta receptors to induce a mitotic response in this slowly replicating cell type. Transfection with the B-chain construct induced synthesis of the PDGF B-chain homodimer (PDGF-BB) as assessed by the presence of 125I-labeled PDGF-BB competing activity in the conditioned medium of the transfected islet cells. Moreover, islet cells transfected with the PDGF beta-receptor construct exhibited increased immunofluorescence staining with a PDGF beta-receptor antibody. These cells also displayed increased 125I-labeled PDGF-BB binding compared with control transfected cells. Cotransfection with the B-chain construct or the addition of 10% fetal bovine serum or purified PDGF all induced DNA synthesis in islet cells transfected with the PDGF beta-receptor construct. Islet cells transfected with the PDGF alpha-receptor construct did not respond with stimulation of [3H]thymidine incorporation to any of the PDGF isoforms (PDGF-AA, -AB, or -BB). Cotransfection of the PDGF alpha- and beta-receptor constructs resulted in a loss of the DNA synthesis response to PDGF. The beta cells exhibited elevated levels of [3H]inositol trisphosphate after transfection with the B-chain and beta-receptor constructs, indicating activation of phospholipase C. Islet cells transfected with the different receptor constructs exhibited different patterns of tyrosine phosphorylation upon ligand activation. The results demonstrate that pancreatic islet cells can be stimulated to increase DNA synthesis by transfection with the PDGF beta-receptor gene, whereas cotransfection with the alpha-receptor gene may attenuate the growth response.

Animals↗
PubMed1990

Expression of PDGF A-chain and beta-receptor genes during rat myoblast differentiation.

L6J1 rat myoblasts and rat skeletal muscle were studied for expression of mRNAs encoding PDGF A-chain, PDGF B-chain, PDGF alpha-receptor, and PDGF beta-receptor during in vitro and in vivo myoblast differentiation. RNA blot hybridizations demonstrated expression of the PDGF A-chain gene and the PDGF beta-receptor gene in L6J1 myoblasts and in crude muscle tissue isolated from developing rats. Transcripts of the PDGF A-chain were identified at all examined stages of in vitro and in vivo myogenic differentiation. Expression of the PDGF beta-receptor gene decreased in differentiated myotubes of L6J1 cells and in rat adult muscle tissue. Receptor binding assays demonstrated specific binding of PDGF-BB, but not -AA, to exponentially proliferating L6J1 myoblasts and to terminally differentiated L6J1 myotubes. The binding per cell nucleus was higher in exponentially proliferating myoblasts than in differentiated L6J1 myotubes. In serum free medium PDGF-BB was shown to increase c-fos protooncogene immunoreactivity in L6J1 myoblasts. In the presence of 0.5% FCS, PDGF-BB increased DNA synthesis in L6J1 myoblasts, while PDGF-AA showed no such effect. Differentiation, as monitored by myotube formation, was reduced in PDGF-BB-treated cultures. The possible role of PDGF in myoblast proliferation and differentiation is discussed.

Animals↗
PubMed1990

Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction.

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

Animals↗