We report the identification of ligands for Tyro 3 (alternatively called Sky, rse, brt, or tif) and Axl (alternatively, Ark or UFO), members of a previously orphan family of receptor-like tyrosine kinases. These ligands correspond to protein S, a protease regulator that is a potent anticoagulant, and Gas6, a protein related to protein S but lacking any known function. Our results are reminiscent of recent findings that the procoagulant thrombin, a protease that drives clot formation by cleaving fibrinogen to form fibrin, also binds and activates intracellular signaling via a G protein-coupled cell surface receptor. Proteases and protease regulators that also activate specific cell surface receptors may serve to integrate coagulation with associated cellular responses required for tissue repair and growth, as well as to coordinate protease cascades and associated cellular responses in other systems, such as those involved in growth and remodeling of the nervous system.

Glioma-derived vascular endothelial cell growth factor (GD-VEGF) is a 46-kDa dimeric glycoprotein mitogen with apparently greater specificity for vascular endothelial cells than the well-characterized fibroblast growth factors. The GD-VEGF cDNA sequence encodes a 190-amino acid residue subunit that is converted, by removal of an aminoterminal hydrophobic secretory leader sequence, to the mature 164-residue subunit characterized by direct amino acid sequencing. The GD-VEGF homodimeric subunit is homologous to the platelet-derived growth factor A and B chains and its oncogene homologue v-sis. We report here the complete cDNAt and amino acid sequence of rat GD-VEGF. The sequence confirms its identification as a secretory homodimeric glycoprotein and reveals an unexpected homology to platelet-derived growth factor (PDGF), a mitogen for connective tissues cells but not vascular endothelial cells from large vessels. GD-VEGF as described (5). Purity was confirmed on all samples by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Aliquots (1-2 ,.g) of the purified protein, quantitated by using an extinction coefficient based on amino acid analysis (5), were reduced and carboxymethylated with iodo[2-14C]acetic acid as described (8). The 14C-carboxymethylated GD-VEGF product was repurified on a 4.6 mm x 5 cm Vydac C4 reversed-phase HPLC column by elution at 20'C with a linear gradient of 0-67% acetonitrile in 0.1% trifluoroacetic acid over 30 min at a flow rate of 0.75 ml/min.Enzymatic Digestion and Polypeptide Purification. Reduced and carboxymethylated GD-VEGF (725 ng) was digested on the carboxyl-terminal side of most lysine and arginine residues with 30 ng of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated bovine pancreatic trypsin (Worthington) in 200 ,ul of 0.1 M ammonium bicarbonate (pH 8.3) for 6 hr at 37°C. The polypeptide digestion mixture was loaded directly on a 4.6 mm x 25 cm Vydac C18 reversed-phase column and fractionated by elution at 20°C with a 0-67% (vol/vol) linear gradient of acetonitrile in 0.1% trifluoroacetic acid over 2 hr at a flow rate of 0.75 ml/min. Polypeptide peaks were identified by monitoring A210 and individually collected.A similar digest was performed on 925 ng of carboxymethylated GD-VEGF by using 50 ng of Lys-C endoproteinase (Lys-C implies lysine specific) (Boehringer Mannheim) in 50 ,ul of 0.1 M Tris, pH 8.5/1 mM EDTA at 37°C for 8 hr. Polypeptide products, the result of cleavage on the carboxylterminal side of lysine residues, were purified as described for the tryptic digest.A final enzymatic digestion was done on 1.1 ,ug of carboxymethylated GD-VEGF by using 65 ng ofStaphylococcus aureus V8 protease (Miles). The substrate was dissolved in 5 ,ul of6 M guanidinium chloride/0.1% EDTA buffered with 0.7 M Tris to pH 7.8. The protease was added in 65 ,ul of 0.1% EDTA buffered to pH 8.0 with 0.1 M ammonium bicarbonate. The digest was incubated at 37°C for 48 hr, and the polypeptides, generated primarily by cleavage on the carboxylterminal side ofglutamic ac...

Vascular endothelial growth factor (VEGF) is a potent and selective mitogen for endothelial cells that is angiogenic in vivo and induced by hypoxia. A homologous protein, placenta growth factor (PlGF), is also reported to be mitogenic for endothelial cells in culture. The rat GS-9L glioma cell line produces not only VEGF homodimers but also PlGF homodimers and a novel heterodimer composed of VEGF and PlGF subunits. All three dimeric forms were purified to apparent homogeneity, and their structures and mitogenic activities were compared. VEGF.PlGF heterodimers are vascular endothelial cell mitogens nearly as potent as VEGF homodimers. Therefore, some of the biological activities attributed to VEGF homodimers might be mediated by VEGF.PlGF heterodimers. In contrast, pure PlGF homodimers are mitogenic for endothelial cells only at high, possibly non-physiologic concentrations; thus the biological relevance of their mitogenic activity for these cells is not obvious. However, the existence of not only homodimers but also heterodimers clearly extends the similarity between the VEGF/PlGF and the homologous platelet-derived growth factor systems.

A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of~'1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line. The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction.This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts, properties that further distinguish it from other well-characterized vascular endothelial cell mitogens. In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor, which have no secretory leader sequences and might only be released by leakage from damaged cells, the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway. This secretable growth factor could, therefore, be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with bloodvessel growth and maintenance. genes (1-7). All of these FGFs, with the exception of the int-2 and FGF-6 gene products, which have not been isolated, have been shown to be potent vascular endothelial cell mitogens in vitro. Furthermore, exogenously applied aFGF (8) and bFGF (9) induce angiogenesis in vivo. An angiogenic platelet-derived endothelial cell growth factor (PD-ECGF), a monomeric 45-kDa protein, has also been purified and structurally characterized. This protein, like aFGF and bFGF, lacks a secretory leader sequence and so is presumably released by lysis of platelets (10,11 We have identified and purified a vascular endothelial cell mitogen that is not only produced but also apparently secreted by cultured cells derived from a chemically induced rat glioma (13). This glioma-derived vascular endothelial cell growth factor (GD-VEGF) is structurally unrelated to the previously well-characterized mitogens for large-vessel endothelium. Pure GD-VEGF approaches the specific mitogenic activity of FGFs for cultured human umbilical vein endothelial (HUVE) cells. MATERIALS AND METHODSMitogenic Assays. HUVE cells were isolated and maintained as described (14). The cells were plated in gelatincoated 48-well tissue culture dishes (Costar) at 6000 cells per well in 400 ,ul of medium 199 (GIBCO) containing 15 mM Hepes buffer and supplemented with 20% heat-inactivated fetal bovine serum (Hazelton Research Products, Reston, VA) and antibiotics (penicillin G, 100 units/ml; streptomycin sulfate, 100lg/ml; amphotericin B, 0.25 ,ug/ml; GIBCO). Samples to be assayed were added at the time of cell plating and the tissue culture dishes were incubated at 37°C under 5% CO2. After a 12-hr incubation, 0.64 ,Ci of [methyl-3H]-thymidine (20 Ci/mmol; New England Nuclear; 1 Ci = 37 GBq) was added per well and the dishes were incubated for an additional 60 hr. The cells were then washed with Hanks' ...

Hepatocyte growth factor (HGF) has been shown to function as a potent mitogen for a variety of cells, transducing its signal through the c-met tyrosine kinase receptor. Ciliary neurotrophic factor (CNTF) is a cytokine that has been shown to promote survival of motor neurons. We show here that c-met mRNA is present in the embryonic rat spinal cord. Peak expression of c-met (at E14) coincides with the period of naturally occurring cell death in motor neurons, suggesting a possible role of HGF in the regulation of this process. Utilizing a neuron-enriched culture system, we established that HGF, like CNTF, stimulates choline acetyltransferase (CAT) activity in motor neurons. When co-administered to motor neuron cultures, saturating concentrations of HGF and CNTF produced a synergistic increase in CAT levels. We show that this synergy reflects enhanced motor neuron survival. Exposure of motor neuron cultures to the cytostatic agent vincristine markedly decreased CAT levels; co-treatment with HGF and CNTF (but not either factor alone) restored CAT activity to control levels. Our findings indicate that HGF is a survival factor for motor neurons, that it acts synergistically with CNTF, and that HGF and CNTF can together be neuroprotective in the face of vincristine toxicity.