Developmental expression of platelet-derived growth factor and its receptor in the human placenta

To investigate the role of platelet-derived growth factor (PDGF) during human placental development, expression of the genes encoding PDGF, the PDGF-receptor (PDGF-R) and the c-fos protooncogene was measured. Messenger RNAs for these genes were detected throughout pregnancy and peaked coordinately during the second trimester. An identical pattern of PDGF-R protein expression was confirmed by immunoblotting using a specific PDGF-R antiserum, measurement of PDGF-R kinase activity, and [125I]PDGF binding. These findings show that the components of the PDGF pathway are expressed in a concerted fashion throughout human pregnancy and are present at especially high levels during the midtrimester. Our observations suggest that through autocrine and/or paracrine mechanisms, PDGF is likely to play an important role in placental homeostasis.     

The platelet-derived growth factor receptor alpha is destabilized by geldanamycins in cancer cells

The heat shock protein HSP90 serves as a chaperone for receptor protein kinases, steroid receptors, and other intracellular signaling molecules. Targeting HSP90 with ansamycin antibiotics disrupts the normal processing of clients of the HSP90 complex. The platelet-derived growth factor receptor alpha (PDGFRalpha) is a tyrosine kinase receptor up-regulated and activated in several malignancies. Here we show that the PDGFRalpha forms a complex with HSP90 and the co-chaperone cdc37 in ovarian, glioblastoma, and lung cancer cells. Treatment of cancer cell lines expressing the PDGFRalpha with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) promotes degradation of the receptor. Likewise, phospho-Akt, a downstream target, is degraded after treatment with 17-AAG. In contrast, PDGFRalpha expression is not affected by 17-AAG in normal human smooth muscle cells or 3T3 fibroblasts. PDGFRalpha degradation by 17-AAG is inhibited by the proteasome inhibitor MG132. High molecular weight, ubiquitinated forms of the receptor are detected in cells treated with 17-AAG and MG132. Degradation of the receptor is also inhibited by a specific neutralizing antibody to the PDGFRalpha but not by a neutralizing antibody to PDGF or by imatinib mesylate (Gleevec). Ultimately, PDGFRalpha-mediated cell proliferation is inhibited by 17-AAG. These results show that 17-AAG promotes PDGFRalpha degradation selectively in transformed cells. Thus, not only mutated tyrosine kinases but also overexpressed receptors in cancer cells can be targeted by 17-AAG.     

Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms

Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGFalphaR) is fused to the cytosolic domain of Drosophila Torso (alpha(Tor)) or the mouse fibroblast growth factor receptor 1 (alpha(FR)). alpha(Tor) homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFalphaR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of alpha(Tor) to stimulate the mitogen-activated protein (MAP) kinase pathway to near wild-type levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The alpha(FR) chimeric receptor fails to rescue any aspect of the PDGFalphaR-null phenotype. Instead, alpha(FR) expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The alpha(FR) phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function.     

The role of c-Src in platelet-derived growth factor alpha receptor internalization

We used two approaches to investigate the role of Src family kinases (SFKs) in ligand-stimulated internalization of the platelet-derived growth factor alpha receptor (alphaPDGFR). In cells that normally express SFKs, the internalization rate of the F72/74 mutant alphaPDGFR (which is unable to recruit or activate SFKs) was slower than that of the wild-type (WT) alphaPDGFR. When expressed in cells lacking SFKs (SYF cells), internalization of the WT and mutant receptors was indistinguishable, which indicated that there was not an inherent defect in the mutant receptor's ability to undergo ligand-driven internalization. The internalization difference between the WT and mutant receptors was seen again when c-Src was expressed in the SYF cells. Surprisingly, c-Src slowed the internalization of the mutant receptor but had little effect on WT receptor. We propose the following working hypothesis to explain these findings. In resting cells SFKs suppress internalization of the alphaPDGFR by phosphorylating a hypothetical protein X. This suppression is relieved when cells are exposed to PDGF because SFKs are recruited to the activated WT alphaPDGFR and thereby no longer actively phosphorylate protein X. The internalization of the mutant receptor is slow because it is unable to recruit SFKs and thereby fails to relieve the suppression of internalization. Our findings suggest a role for SFKs in regulating the permissiveness for internalization of the alphaPDGFR.     

Myc is an essential negative regulator of platelet-derived growth factor beta receptor expression

Platelet-derived growth factor BB (PDGF BB) is a potent mitogen for fibroblasts as well as many other cell types. Interaction of PDGF BB with the PDGF beta receptor (PDGF-betaR) activates numerous signaling pathways and leads to a decrease in receptor expression on the cell surface. PDGF-betaR downregulation is effected at two levels, the immediate internalization of ligand-receptor complexes and the reduction in pdgf-betar mRNA expression. Our studies show that pdgf-betar mRNA suppression is regulated by the c-myc proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppress pdgf-betar mRNA and protein. Suppression of pdgf-betar mRNA in response to Myc is specific, since expression of the related receptor pdgf-alphar is not affected. We further show that Myc suppresses pdgf-betar mRNA expression by a mechanism which is distinguishable from Myc autosuppression. Analysis of c-Myc-null fibroblasts demonstrates that Myc is required for the repression of pdgf-betar mRNA expression in quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-mediated repression of pdgf-betar mRNA levels plays an important role in the regulation of basal pdgf-betar expression in proliferating cells. Thus, our studies suggest an essential role for Myc in a negative-feedback loop regulating the expression of the PDGF-betaR.     

Regulatory mechanisms for the expression and activity of platelet-derived growth factor receptor

PDGF is one of the most potent serum mitogens, and the signalling mechanism by way of its receptor tyrosine-kinase has been extensively studied since its first purification in 1979. The identification of homology between the simian sarcoma virus oncogene, v-sis, and the B-chain of PDGF, as well as the frequent over-expression of both the ligands and receptors in various tumours and stroma led to the proposal of the PDGF-mediated autocrine and paracrine hypothesis. Consistent with the important roles of PDGF in the growth and survival of cells, the expression and activity of PDGF receptors are tightly controlled by both positive and negative feedback mechanisms at different levels. The deregulation of the control system can result in serious pathological conditions such as chronic inflammation and tumours. Understanding the molecular mechanisms for the regulatory system and the signalling pathway of PDGF is essential in order to find effective therapies in the diseases where PDGF is involved.     

Modulation of muscarinic-receptor expression in human embryonic lung fibroblasts by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is known to have regulatory control of a large number of cellular components, including various receptors. We show that muscarinic acetylcholine receptors of the m2 subtype on CCL 137 human fibroblasts in culture are affected by PDGF treatment. A time-dependent down-regulation is observed in steady-state RNA levels, followed by a decrease in ligand-binding capacity. Minimum RNA levels are attained at 11 h; minimum binding capacity is observed after 24 h of treatment. To our knowledge, this is the first example of negative gene control by PDGF.     

Insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the platelet-derived growth factor receptor

R^? cells are 3T3-like cells derived from mouse embryos in which the insulin-like growth factor I (IGF-I) receptor (IGF-IR) genes have been disrupted by targeted homologous recombination. These cells cannot grow in serum-free medium supplemented by the growth factors that sustain the growth of other 3T3 cell lines, and cannot be transformed by oncogenes that easily transform wild type mouse embryo cells. We have used these cells to study the role of the IGF-IR in the growth and transformation of cells overexpressing the platelet-derived growth factor (PDGF)-b?b? receptor. We report that an overexpressed PDGF-b?b? receptor fails to induce mitogenesis or transformation in cells lacking the IGF-IR, while capable of doing so in cells expressing the IGF-IR. We conclude that the ability of the activated PDGF-b?b? receptor to stimulate cell proliferation and transformation requires a funcitional IGF-IR. © 1995 Wiley-Liss, Inc.     

Isolation and characterization of the alpha platelet-derived growth factor receptor from rat olfactory epithelium.

We have cloned and characterized a new member of the receptor tyrosine kinase family. The cDNA clone, isolated from a rat olfactory cDNA library, has considerable homology to the family of receptors that includes the colony-stimulating factor 1 receptor, the c-kit proto-oncogene, and the platelet-derived growth factor (PDGF) receptors. Analysis of DNA sequence homology, ligand-binding, and ligand-stimulated phosphorylation data suggests that this clone encodes the rat PDGF-A/B or alpha-receptor. Comparison of its sequence to those of other receptors allows us to postulate a mechanism for receptor dimerization and activation. The expression of the rat alpha-PDGF receptor in nonneuronal cells of the olfactory epithelium and in the olfactory bulb is consistent with a role for PDGF in glial cell generation.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

Pleiotrophin gene expression is highly restricted and is regulated by platelet-derived growth factor.

Pleiotrophin (PTN) is a growth and neurite extension promoting polypeptide which is highly expressed in brain and in tissues derived from mesenchyme. The PTN gene is developmentally regulated and is closely related to the MK and RI-HB genes, both of which are developmentally regulated and induced by retinoic acid. We now have screened 17 cell lines and report that expression of the PTN gene in these cells is restricted to embryo fibroblasts and intestinal smooth muscle cells. However, NIH 3T3 cells stimulated by the platelet-derived growth factor (PDGF) express a marked increase in levels of PTN mRNA whereas retinoic acid failed to increase levels of PTN mRNA in NIH 3T3 cells or in F9 embryonal carcinoma cells within 72 hours of exposure. The results suggest that expression of the PTN gene is highly restricted and that the PTN gene is a new member of the PDGF-induced cytokine family.     

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.     

Purification of the receptor for platelet-derived growth factor from porcine uterus

The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor.     

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.