Growth factor receptor signalling: location, location, location.

Ligand binding to plasma membrane receptors initiates a series of events culminating in a variety of changes in cellular phenotypes. Although numerous publications have documented the activation/inactivation of signalling molecules following receptor binding, relatively few investigations have focused on the cellular compartment responsible for either initiating or selecting the particular pathway that mediates the response. Specifically, does receptor signalling occur only at the plasma membrane; is signalling dependent upon the location of defined endosome populations; or are components of both plasma membrane and endosomal activity operative depending upon the particular signalling pathway or cell type? This review addresses aspects of these questions by discussing the evidence supporting or contrasting the interplay between the endocytic and signalling systems for a subset of tyrosine kinase, serine/threonine kinase and G-protein-coupled receptors.

Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-beta.

Transforming growth factor-beta (TGF beta) is a regulator of cellular proliferation which can alter the proteolytic activity of cultured cells by enhancing the secretion of endothelial type plasminogen activator inhibitor and affecting the secretion of plasminogen activators (PAs) in cultured fibroblastic cells. We used the TGF beta-responsive malignant human lung adenocarcinoma cell line A549 to study the relationships between the known TGF beta-induced growth inhibition and the effects of TGF beta on the secretion of PA activity by A549 cells. PA activity was quantitated by caseinolysis assays, and characterized by urokinase mRNA analysis, immunoprecipitation, and zymography assays. PA-inhibitor production was observed in autoradiograms of SDS-polyacrylamide gels and reverse zymography assays. It was found that TGF beta enhanced the production of PA activity by these cells, in accordance with an enhancement of urokinase mRNA levels. A concomitant stimulation of type 1 PA-inhibitor production was also observed in A549 cells in response to TGF beta. In contrast to the observations of A549 cells, TGF beta caused a decrease in the expression of both urokinase and the tissue-type PA mRNA in human embryonic WI-38 lung fibroblasts indicating opposite regulation of the expression of PAs in these cells. The results suggest that TGF beta may play a role in the regulation of the invasive, proteolytically active phenotype of certain lung carcinoma cells.

Pneumocystis carinii inhibits cyclin-dependent kinase activity in lung epithelial cells.

Pneumocystis carinii remains an important cause of pneumonia in patients with AIDS. Attachment of the organism to epithelial cells is a central event in establishing infection, impairing the growth potential of lung epithelial cells and thereby slowing repair. In light of investigations documenting a central role for cyclin-dependent kinases in controlling the cell cycle, we addressed the hypothesis that P. carinii inhibits epithelial cell growth by interfering with host epithelial cyclin-dependent kinase (cdk) activity. We observed that P. carinii significantly impaired growth of cultured mink lung epithelial cells, with effects observed after 48-72 h of treatment. However, the kinase activity associated with p34cdc2 or p33cdk2 was maximally inhibited as early as 24 h after P. carinii exposure. The inhibitory effect on cyclin-dependent kinase activity was mediated by the trophozoite form of P. carinii, in that highly purified trophozoites exerted marked inhibition of p34cdc2 activity. Growth impairment was similarly preceded by P. carinii-induced alteration in the state of epithelial cell p34cdc2 phosphorylation, with no change in p34cdc2 or p33cdk2 protein levels. These data strongly suggest that the antiproliferative activity of P. carinii on respiratory epithelium is mediated in part through modulation of the host cell cycle machinery.

Transforming growth factor beta 1 inhibition of p34cdc2 phosphorylation and histone H1 kinase activity is associated with G1/S-phase growth arrest.

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation. We present data which indicate that epithelial cell proliferation is inhibited when TGF beta 1 is added throughout the prereplicative G1 phase. Cultures become reversibly blocked in late G1 at the G1/S-phase boundary. The inhibitory effects of TGF beta 1 on cell growth occur in the presence of the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Associated with this inhibitory effect is a decrease in the phosphorylation and histone H1 kinase activity of the p34cdc2 protein kinase. These data suggest that TGF beta 1 growth inhibition in epithelial cells involves the regulation of p34cdc2 activity at the G1/S transition.

Modulation of transforming growth factor type beta action by activated ras and c-myc.

Transfection of C3H/10T1/2 cells with a c-myc gene resulted in the acquisition of responsiveness to transforming growth factor type beta. Cells transfected with an activated H-ras gene or an H-ras and c-myc gene, however, exhibited a transformed morphology and spontaneous soft-agar growth, a phenotype induced reversibly by transforming growth factor type beta in responsive fibroblasts.

Distinct endocytic responses of heteromeric and homomeric transforming growth factor beta receptors.

Transforming growth factor beta (TGF beta) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGF beta receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGF beta receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF beta receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGF beta receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGF beta receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGF beta receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGF beta receptors and that TGF beta receptor heteromers and homomers show distinct trafficking behavior.

Transforming growth factor beta receptor signaling and endocytosis are linked through a COOH terminal activation motif in the type I receptor.

Transforming growth factor beta (TGF-beta) coordinates a number of biological events important in normal and pathophysiological growth. In this study, deletion and substitution mutations were used to identify receptor motifs modulating TGF-beta receptor activity. Initial experiments indicated that a COOH-terminal sequence between amino acids 482-491 in the kinase domain of the type I receptor was required for ligand-induced receptor signaling and down-regulation. These 10 amino acids are highly conserved in mammalian, Xenopus, and Drosophila type I receptors. Although mutation or deletion of the region (referred to as the NANDOR BOX, for nonactivating non-down-regulating) abolishes TGF-beta-dependent mitogenesis, transcriptional activity, type I receptor phosphorylation, and down-regulation in mesenchymal cultures, adjacent mutations also within the kinase domain are without effect. Moreover, a kinase-defective type I receptor can functionally complement a mutant BOX expressing type I receptor, documenting that when the BOX mutant is activated, it has kinase activity. These results indicate that the sequence between 482 and 491 in the type I receptor provides a critical function regulating activation of the TGF-beta receptor complex.

Mechanisms of transforming growth factor-beta receptor endocytosis and intracellular sorting differ between fibroblasts and epithelial cells.

Transforming growth factor-betas (TGF-beta) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-beta type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-beta receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells.

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.

Growth factors and cancer.

Growth factors, defined as polypeptides that stimulate cell proliferation, are major growth-regulatory molecules for cells in culture and probably also for cells in vivo. Nontransformed cells show an absolute requirement for growth factors for proliferation in culture and generally more than one growth factor is required. Under usual culture conditions, growth factors are more rapidly depleted than other media components and thus become rate limiting for proliferation. The loss of or decreased requirement for specific growth factors is a common occurrence in neoplastically transformed cells and may lead to a growth advantage, a cardinal feature of cancer cells. Recent work with transforming growth factors, the platelet-derived growth factor, and oncogenes has produced some insight into the mechanisms through which alterations in growth factor-receptor-response pathways could lead to a growth advantage. Evidence has been derived for autocrine secretion in which the cell produces its own growth factor. Many transformed mesenchymal cells produce PDGF (the product of the c-sis proto-oncogene) and certain transformed cells both produce and respond in a growth-stimulatory manner to TGF beta. With TGF beta, which is a growth inhibitor for certain epithelial and other cell types, the loss of the normal inhibitory response in transformed cells could have the same result as the activation of a growth-stimulatory response. Two proto-oncogenes, erbB and fms, encode growth factor receptors. In the erbB case, the viral erbB aberrant receptor produced is truncated and appears to be constitutively activated without the need for a growth factor. Recent studies suggest that the p21 product of the ras oncogene may be an obligatory intermediate in transducing the growth factor signal. Activation of ras may, therefore, activate the growth factor pathway without the need for either a growth factor or its receptor. The transcription of myc and fos is induced by growth factor stimulation of quiescent cells. The protein products of both are nuclear associated and conceivably could be involved in regulating other genes important in the control of cell proliferation. Activation or inappropriate expression of either myc or fos could produce the same end result as stimulation of a growth factor pathway leading to a growth advantage. Study of the molecular mechanism(s) of growth factor action has just begun. The excitement and attention focused on cellular oncogenes in recent years is now turning toward growth factors, not only as they concern the control of normal cell growth but also the involvement of growth factor-initiated pathways in the etiology of cancer.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of transforming growth factor beta 1 action by multiple transducing pathways: evidence for both G protein-dependent and -independent signaling.

The effects of cholera toxin (CT) on transforming growth factor beta 1-stimulated protooncogene expression, [gamma-35S]GTP binding, GTPase activity and growth under anchorage-independent and -dependent conditions were studied in AKR-2B fibroblast cells. CT was shown to inhibit TGF beta 1-stimulated c-sis and c-myc mRNA expression. Actinomycin D decay and nuclear runon experiments demonstrated that this inhibition was not due to an increased decay of protooncogene message, but to a decreased transcriptional activation. These inhibitory effects were not secondary to changes in the ability of TGF beta 1 to bind to its receptor(s) since radioreceptor assays and affinity labeling studies demonstrated that CT had no effect on TGF beta 1 binding. ADP ribosylation of AKR-2B plasma membranes with [alpha-32P]NAD+ revealed a Mr 45,000 protein as the major CT substrate. The labeling of this Mr 45,000 protein in membranes could be inhibited by prior pretreatment of the cells with increasing concentrations of CT. Treatment of membranes with nanogram concentrations of CT abolished the increase in [gamma-35S]GTP binding following addition of TGF beta 1 as well as decreased basal binding. Similarly, CT pretreatment of membranes inhibited TGF beta 1-stimulated GTPase activity. Unexpectedly however, the stimulatory effects of TGF beta 1 on anchorage-independent growth in soft agar were unaffected by CT. Only pertussis toxin was able to inhibit TGF beta 1-induced colony formation in soft agar in a dose-dependent manner. Furthermore, differential effects of both CT and pertussis toxin were observed on TGF beta 1-stimulated monolayer growth; CT was inhibitory, whereas pertussis toxin was without effect. These results suggest that the diverse biological effects of TGF beta 1 are mediated through multiple intracellular pathways distinguishable by their toxin sensitivities.

Mid-G1 arrest and epidermal growth factor independence of ras-transfected mouse cells.

Transfection of C3H/10T1/2 cells with either a c-myc or an activated c-Ha-ras gene decreased the cellular dependence for serum-derived factors to proliferate in monolayer. The c-myc-transfected cells did, however, require a high plasma concentration for significant growth, while the ras transfectants grew extremely well in either low or high concentrations of either plasma or serum. Stimulation of quiescent cultures with purified growth factors demonstrated that c-myc transfection did not alter qualitatively or quantitatively the requirement for both epidermal growth factor (EGF) and insulin to progress to DNA synthesis. Cells transfected with either a ras gene alone or a combination of ras plus c-myc lost their dependence on EGF for DNA synthesis; cultures became committed to S phase in serum-free medium supplemented with insulin alone. The ras transfectants arrested in mid-G1, 6 h prior to S phase. The EGF independence of the ras transfectants is consistent with the mid-G1 arrest of these cells at a point(s) distal to the primary action of EGF in early G0-G1.

Dissociation of p34cdc2 complex formation from phosphorylation and histone H1 kinase activity.

The cell cycle inhibitor mimosine was used to examine the activation of the p34cdc2 protein kinase in S phase of the cell cycle. Addition of mimosine to cycling epithelial cells halted cell cycle traverse in S phase, coincident with an inhibition of p34cdc2 histone H1 kinase activity. Mimosine treatment did not alter p34cdc2 synthesis or turnover; however, overall phosphorylation of p34cdc2 was decreased to near undetectable levels. Although activity of p34cdc2 was inhibited, the ability of the protein to form high molecular weight complexes, a phenomenon associated with kinase activation in vivo, was not affected. These results indicate that p34cdc2 complex formation can occur in the absence of phosphorylation and that phosphorylation of p34cdc2 is then required to activate these preformed complexes.

Histologic studies of the intraocular toxicity of imatinib mesylate in rabbits.

PURPOSE: To evaluate histologic signs of toxicity of the protein tyrosine kinase inhibitor, imatinib mesylate, in rabbit eyes. METHODS: Twenty Dutch-belted rabbits underwent intravitreal injections of 0.1 ml solutions of imatinib mesylate. Ten rabbits were killed and enucleated 1 week after injection of imatinib mesylate (1.65 mg (four eyes), 165 microg (four eyes), and 16.5 microg (two eyes)). Ten rabbits injected with imatinib mesylate (165 microg (five eyes) and 825 microg (five eyes)) were enucleated 1 month later. Eyes were fixed in 10% formalin and stained with haematoxylin and eosin for microscopic examination. RESULTS: All four eyes injected with 1.65 mg of imatinib mesylate and enucleated at 1 week demonstrated ocular toxicity. All four eyes injected with 165 microg and enucleated at 1 week showed no ocular toxicity. One of the two eyes injected with 16.5 microg and enucleated at 1 week revealed focal areas of subretinal fluid and retinal undulations, suggestive of retinal oedema. None of the 10 eyes injected with imatinib mesylate at either the 165 or 825 microg dose and enucleated at 1 month showed ocular toxicity. CONCLUSIONS: Imatinib mesylate at 1.65 mg caused extensive retinal toxicity in rabbit eyes. In contrast, lower doses did not appear to cause toxicity, but may be associated with retinal oedema.

Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s).

Transforming growth factor beta (TGF beta 1) is a potent regulator of DNA synthesis and cellular proliferation. In this study, we investigated whether the growth stimulatory signal of TGF beta 1 is transduced intracellularly by guanine nucleotide regulatory proteins (G-proteins). In plasma membranes from AKR-2B cells, TGF beta 1 increased binding of the radiolabelled, non-hydrolysable GTP analogue, guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]), in a dose-dependent manner. Maximal effects occurred between 0.4 and 1.0 nM-TGF beta 1. Specific binding of GTP[35S] occurred with a Kd of 3.2 x 10(-8) M which was not affected by addition of TGF beta 1. Instead, TGF beta 1 increased the number of available binding sites for GTP[35S] from 16.2 +/- 1.2 to 21.6 +/- 2.1 pmol/mg of protein. GTP[35S] binding was both nucleotide- and growth-factor-specific. Only guanine nucleotides were able to compete for binding, and of the growth factors tested (epidermal growth factor, platelet-derived growth factor, insulin, TGF beta 1 and TGF beta 2) only TGF beta 1 affected GTP[35S] binding. TGF beta 1 increased GTPase activity, as determined by the release of 32PO4(3-) from GTP gamma[32P], from 116 +/- 5.5 to 175 +/- 4.3 pmol/mg of protein following a 15 min incubation. Pretreatment of the membranes with pertussis toxin inhibited both TGF beta 1-stimulated binding of GTP[35S] as well as TGF beta 1-stimulated GTPase activity. These inhibitory actions of pertussis toxin were associated with toxin-induced ADP-ribosylation of a 41 kDa protein. Furthermore, the stimulatory effects of TGF beta 1 on c-sis mRNA expression were shown to be pertussis-toxin sensitive and could be mimicked by direct activation of G-proteins with AIF4-. These results demonstrate that in AKR-2B cells a pertussis-toxin-sensitive guanine nucleotide regulatory protein(s) is coupled to TGF beta 1 receptor binding.

Chimeric granulocyte/macrophage colony-stimulating factor/transforming growth factor-beta (TGF-beta) receptors define a model system for investigating the role of homomeric and heteromeric receptors in TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) belongs to a family of ligands that regulate cell growth and differentiation. The most commonly observed receptors are referred to as the type I, type II, and type III (betaglycan) TGF-beta receptors. Two receptor models have been presented to account for the various cellular responses to TGF-beta. The first proposes that all TGF-beta signaling results from the formation of a heteromeric type I/type II complex, while the second suggests that distinct type I or type II TGF-beta receptor combinations mediate aspects of TGF-beta signaling. We have addressed this general question relating to TGF-beta signaling by constructing chimeric receptors consisting of the extracellular domain of the granulocyte/macrophage colony-stimulating factor (GM-CSF) alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF-beta receptor. Since high affinity GM-CSF binding requires dimerization of the alpha and beta ligand binding subunits, the response elicited by defined type I and/or type II TGF-beta receptor cytoplasmic domain homomers or heteromers can be examined. We show in mesenchymal AKR-2B cells that while TGF-beta-dependent transient luciferase activity, endogenous gene activity, and long-term biological responses are similarly induced by activating the chimeric heteromeric receptors with GM-CSF as the endogenous TGF-beta receptor, chimeric homomeric type I/type I or type II/type II receptors are signaling-incompetent.

Inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta is coupled through a pertussis-toxin-sensitive substrate.

Transforming growth factor beta 1 (TGF beta 1) inhibits the proliferative response of mink lung epithelial cells (CCL64) to serum and to epidermal growth factor (EGF). This response to TGF beta 1 can be inhibited by prior exposure of the cells to nanogram concentrations of pertussis toxin (PT), suggesting the involvement of a guanine-nucleotide-binding regulatory protein (G-protein) in mediating TGF beta 1-induced growth inhibition. To characterize further this G-protein dependence, we have isolated, by chemical mutagenesis, a CCL64 variant (CCL64-D1) that is resistant to TGF beta 1. Whereas in the parental CCL64 cells TGF beta 1 stimulates both GTP[35S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and GTPase activity, in the CCL64-D1 variants TGF beta 1 is without effect. Quantitative immunoblotting with antisera for G-protein alpha- and beta-subunits, as well as PT-catalysed ADP-ribosylation analyses, revealed no appreciable changes in the level of G-protein expression in the CCL64-D1 variants compared with parental cells. In contrast with another TGF beta-resistant clone, MLE-M, which we show lacks detectable type I receptor protein, the CCL64-D1 cells retain all three TGF beta cell-surface binding proteins. On the basis of these studies, we propose that a necessary component of TGF beta 1-mediated growth inhibition in CCL64 epithelial cells is the coupling of TGF beta 1 receptor binding to G-protein activation.

Distinct pathways regulate transforming growth factor beta 1-stimulated proto-oncogene and extracellular matrix gene expression.

The effect of pertussis toxin (PT) on transforming growth factor beta 1 (TGF beta 1)-induced proto-oncogene expression was investigated in AKR-2B fibroblasts. PT substantially abolished c-sis and c-myc mRNA expression following TGF beta 1 stimulation. This inhibitory effect was specific for TGF beta 1-stimulated proto-oncogene expression and associated with the ADP-ribosylation of a 41-kDa substrate. Actinomycin D decay and nuclear run-on experiments demonstrated that the inhibitory effects of PT are a result of decreased transcriptional activation and not to an increased decay of proto-oncogene message. PT did not, however, affect TGF beta 1-stimulated fibronectin and collagen mRNA accumulation nor did it have any inhibitory effect on TGF beta 1-induced morphological transformation. These data indicate that TGF beta 1-stimulated gene expression is coupled to multiple pathways distinguished by their sensitivity to PT.