Lena Claesson-Welsh

Proteolytic processing regulates receptor specificity and activity of VEGF‐C

The recently identified vascular endothelial growth factor C (VEGF‐C) belongs to the platelet‐derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial‐specific receptor tyrosine kinases VEGFR‐3 and VEGFR‐2. The VEGF homology domain spans only about one‐third of the cysteine‐rich VEGF‐C precursor. Here we have analysed the role of post‐translational processing in VEGF‐C secretion and function, as well as the structure of the mature VEGF‐C. The stepwise proteolytic processing of VEGF‐C generated several VEGF‐C forms with increased activity towards VEGFR‐3, but only the fully processed VEGF‐C could activate VEGFR‐2. Recombinant ‘mature’ VEGF‐C made in yeast bound VEGFR‐3 (KD = 135 pM) and VEGFR‐2 (KD = 410 pM) and activated these receptors. Like VEGF, mature VEGF‐C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF‐C formed mostly non‐covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF‐C activity, and reveal novel structure–function relationships in the PDGF/VEGF family.

PDGF stimulates an increase in GTP–Rac via activation of phosphoinositide 3-kinase

BACKGROUNDnPhosphoinositide 3-kinases (PI 3-kinases) are thought to play an important role in coordinating the responses elicited by a variety of growth factors, oncogene products and inflammatory stimuli. These responses include activation of membrane ruffling, chemotaxis, glucose transport, superoxide production, neurite outgrowth and pp70 S6 kinase. Some of these responses are also known to be regulated by Rac, a small GTP-binding protein related to Ras. Neither the transducing elements upstream of Rac, nor those downstream of PI 3-kinase, have been defined.nnnRESULTSnWe show here that platelet-derived growth factor (PDGF) can stimulate an increase in the level of GTP-Rac by at least two distinct mechanisms: firstly, by increased guanine nucleotide exchange; and secondly, by inhibition of a Rac GTPase activity. The first of these mechanisms is essential for the activation of Rac, and we show that it is dependent upon PDGR-stimulated synthesis of phosphatidylinositol (3,4,5)-trisphosphate.nnnCONCLUSIONSnThese results suggest that Rac activation lies downstream of PI 3-kinase activation on a PDGF-stimulated signalling pathway. Furthermore, as Rac has been implicated in at least two diverse cellular responses that are also though to require activation of PI 3-kinase--a reorganization of the actin cytoskeleton known as membrane ruffling and the neutrophil oxidative burst--these results suggest that Rac may be a major effector protein for the PI 3-kinase signalling pathway in many cell types.

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.

TGF-β1 binding protein: A component of the large latent complex of TGF-β1 with multiple repeat sequences

Abstract TGF-β occurs in a latent complex of high M r . We report the cDNA cloning and an initial structural and functional characterization of a component of the large latent TGF-β1 complex, denoted TGF-β1 binding protein (TGF-β1-BP). Most of the sequence of fibroblast TGF-β1-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. β-hydroxylated asparagine residues were identified in two of the EGF-like repeats. TGF-β1-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-β1-BP gene or that TGF-β1-BP undergoes cell-specific proteolysis. TGF-β1-BP was found not to bind and inactivate TGF-β1; its role in the latent complex is discussed.

Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling

BACKGROUNDnThere is substantial evidence that phosphoinositide 3-kinase (PI 3-kinase) is a critical component of signalling pathways used by the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a second-messenger in cells but its putative targets are still unknown.nnnRESULTSnA particular rearrangement of actin filaments, which results in membrane ruffling, is elicited by the activation of PDGF beta-receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF beta-receptor activation is inhibited by three independent manipulations of PI 3-kinase activity: firstly, by the deletion of tyrosine residues in the PDGF beta-receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase.nnnCONCLUSIONSnThese results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated membrane ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules.

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

INDUCTION OF B-TYPE RECEPTORS FOR PLATELET-DERIVED GROWTH FACTOR IN VASCULAR INFLAMMATION: POSSIBLE IMPLICATIONS FOR DEVELOPMENT OF VASCULAR PROLIFERATIVE LESIONS

Expression of B-type receptors for platelet-derived growth factor (PDGF) in frozen sections of blood vessels from tissues affected by abnormal vascular cell proliferation was investigated by immunohistochemical techniques and compared with expression of these receptors in blood vessels of normal tissues. Receptors were not expressed, or expressed at low levels, in vessels of normal tissues. In contrast, a pronounced expression of PDGF B-type receptors was seen on vascular smooth muscle cells in atherosclerotic plaques, rejected kidneys, and chronic synovitis. These observations suggest induction of PDGF B-type receptors on vascular smooth muscle cells in inflamed tissues, which would render such cells responsive to growth stimulation by PDGF released from captured platelets, or produced locally (eg, by inflammatory cells or smooth muscle cells). Autocrine or paracrine stimulation of cell growth caused by the effect of PDGF on cells with induced receptors may be important in the formation of the proliferative lesions found in atherosclerosis and in certain forms of chronic inflammation.

PDGF alpha- and beta-receptors activate unique and common signal transduction pathways.

We have examined the signal transduction pathways of the PDGF alpha‐ and beta‐receptors, in order to characterize the specificity of each receptor type in the signaling. Porcine aortic endothelial cell lines expressing equal levels of either PDGF alpha‐ or beta‐receptors were established. The alpha‐ and beta‐receptor cells responded mitogenically to stimulation with the proper PDGF isoforms. Three aspects of actin reorganization were examined after ligand stimulation: loss of stress fibres, appearance of edge ruffles and formation of circular membrane ruffles. The beta‐receptor cells showed a response to ligand stimulation which included all three features. The alpha‐receptor cells exhibited edge ruffles and loss of stress fibres, but circular ruffles could not be found in several independent alpha‐receptor cell lines. The beta‐receptor cells, but not the alpha‐receptor cells, were able to migrate chemotactically towards a concentration gradient of ligand. The molecular basis for the differences in signalling were explored by comparing the pattern of increased phosphorylation of potential substrates for the alpha‐ and beta‐receptors in [32P]orthophosphate labelled intact cells and using an in vitro kinase assay. Certain of the observed substrates were common for the two receptors, whereas others were specific for either one. We conclude that certain of the known PDGF induced cellular effects are transduced only by the beta‐receptor; the presence of alpha‐receptor‐specific substrates suggests that there are also alpha‐receptor‐specific signals, which have yet to be identified.

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

Two novel sites of autophosphorylation were localized to the C‐terminal tail of the PDGF beta‐receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF‐BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C‐gamma (PLC‐gamma) compared to the wild type PDGF beta‐receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC‐gamma could be detected. These data show that tyrosine phosphorylation of PLC‐gamma is dependent on autophosphorylation of the PDGF beta‐receptor at Tyr1009 and Tyr1021.

Shc and a Novel 89-kDa Component Couple to the Grb2-Sos Complex in Fibroblast Growth Factor-2-stimulated Cells

A major pathway for mitogenicity is gated via the small GTP-binding protein Ras. Receptor tyrosine kinases couple to Ras through the Src homology 2 (SH2) domain protein Grb2. The activated fibroblast growth factor receptor-1 (FGFR-1) expressed in L6 myoblasts did not bind Grb2 directly, but indirectly, through the small adaptor protein Shc, which was tyrosine-phosphorylated in response to fibroblast growth factor-2 (FGF-2) stimulation. A FGFR-1 mutant in which Tyr, a known autophosphorylation site, was changed to Phe, mediated less efficient tyrosine phosphorylation of Shc. FGF-2 stimulation of mutant FGFR-1-expressing cells still allowed formation of complexes containing Shc, Grb2, and the nucleotide exchange factor Sos and mediation of a mitogenic signal. Another pool of Grb2 was found in complex with a tyrosine-phosphorylated 89-kDa component after FGF-2 stimulation. Stimulation with other growth factors did not lead to tyrosine phosphorylation of p89. As shown by “far-Western” analysis, p89 bound directly to the Grb2 SH2 domain, and this interaction was inhibited by a peptide containing the Y(P)-X-N motif. Tyrosine-phosphorylated p89 was found exclusively in the membrane fraction, indicating its role in bringing Grb2, as well as Sos, to the plasma membrane. These data support the concept of growth factor-specific coupling of Grb2 to the Ras pathway.