Mitchell Goldfarb

Requirement of FGF-4 for postimplantation mouse development

Fibroblast growth factors (FGFs) are thought to influence many processes in vertebrate development because of their diverse sites of expression and wide range of biological activities in in vitro culture systems. As a means of elucidating embryonic functions of FGF-4, gene targeting was used to generate mice harboring a disrupted Fgf4 gene. Embryos homozygous for the null allele underwent uterine implantation and induced uterine decidualization but did not develop substantially thereafter. As was consistent with their behavior in vivo, Fgf4 null embryos cultured in vitro displayed severely impaired proliferation of the inner cell mass, whereas growth and differentiation of the inner cell mass were rescued when null embryos were cultured in the presence of FGF-4 protein.

The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases

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.

An Orphan Receptor Tyrosine Kinase Family Whose Members Serve as Nonintegrin Collagen Receptors

Mammalian cells constantly monitor and respond to a myriad of extracellular signals, often by using cell surface receptors. Two important classes of cell surface receptors include the receptor tyrosine kinases, which recognize peptide growth factors such as insulin, and the integrins, which most often mediate binding to components of the extracellular matrix. We report that the collagens serve as ligands for the previously orphan family of discoidin domain-containing receptor-like tyrosine kinases. The unexpected realization that an extracellular matrix molecule can directly serve as a ligand for receptor tyrosine kinases provides an example of ligands shared by integrins and receptor tyrosine kinases, and this finding seems likely to change prevailing views about the mechanisms by which cells perceive and respond to the extracellular matrix.

Structure and activation of the human N-ras gene

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.

Human-tumor-derived cell lines contain common and different transforming genes

We have screened different cultured cell lines established from human tumors for the ability of their DNAs to induce transformed foci in NIH/3T3 cells. Based on restriction endonuclease digestions and the presence of human sequences in mouse transformants, we conclude that five of these human tumor cell lines contain a gene or genes capable of transforming mouse cells and that at least three different transforming genes are present in these five lines. Three cell lines, two derived from lung carcinomas and one derived from a colon carcinoma, transfer the same or closely related human genes. If these transforming genes are mediating the tumorigenic state of the human cells, then our results indicate that overlapping pathways leading to tumorigenesis may arise independently.

The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors.

We previously described the isolation of a human oncogene which had acquired transforming potential by a DNA rearrangement accompanying transfection of NIH 3T3 cells with human tumor DNA (X. Zhan, A. Culpepper, M. Reddy, J. Loveless, and M. Goldfarb, Oncogene 1:369-376, 1987). We now term this oncogene the FGF-5 gene, since it specifies the fifth documented protein related to fibroblast growth factors (FGFs. Two regions of the FGF-5 sequence, containing 122 of its 267 amino acid residues, were 40 to 50% homologous to the sequences of acidic and basic FGFs as well as to the sequences of the FGF-related oncoproteins int-2 and hst/KS3. The FGF-5 gene bears the three exon structures typical for members of this family. FGF-5 was found to be expressed in the neonatal brain and in 3 of the 13 human tumor cell lines examined. Several experiments strongly suggested that FGF-5 is a growth factor with properties common to those of acidic and basic FGFs. The rearrangement which activated the FGF-5 gene during DNA transfection had juxtaposed a retrovirus transcriptional enhancer just upstream from the native promoter of the gene.

The low affinity NGF receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins

NGF and the other neurotrophins all bind to the low affinity NGF receptor (LNGFR). Although early studies suggested that the LNGFR was absolutely required for the formation of a functional neurotrophin receptor, current evidence indicates that the Trk family of receptor tyrosine kinases, in the absence of the LNGFR, can directly bind to and mediate responses to the neurotrophins. Here we describe a functional approach, in fibroblasts, designed to assay for the ability of the LNGFR to potentiate Trk-mediated responses to the neurotrophins. We report that although collaboration between the LNGFR and the Trks could be detected in this system, a truncated form of the LNGFR displayed a much more dramatic ability to interact functionally with each of the Trks, potentiating masked autocrine loops as well as responses to limiting amounts of exogenously provided neurotrophins.

Genes in S. cerevisiae Encoding Proteins with Domains Homologous to the Mammalian ras Proteins

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.

Trkl3 mediates BDNF/NT-3-dependent survival and proliferation in fibroblasts lacking the low affinity NGF receptor

The neurotrophins (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], neurotrophin-3 [NT-3], and neurotrophin-4 [NT-4] ) have been defined by their ability to support neuronal survival. These factors utilize the Trk family of receptor tyrosine kinases, perhaps in conjunction with a second component known as the low affinity NGF receptor (LNGFR). Here we demonstrate that TrkB mediates survival and proliferation in response to both BDNF and NT-3 when expressed in a particular strain of NIH 3T3 fibroblasts, with BDNF the more potent ligand. Furthermore, the BDNF dose dependency displayed by these TrkB-expressing fibroblasts is similar to that required to support the survival of primary neurons. The LNGFR is not expressed in our fibroblast system, implying that this receptor is not essential for responses to physiological concentrations of the neurotrophins. We discuss our findings in the context of neurotrophin signaling pathways and mechanisms of neuronal cell death.

Functions of Fibroblast Growth Factors in Vertebrate Development

Fibroblast growth factors (FGFs) are a class of secreted polypeptide ligands which mediate diverse cellular responses during embryonic, fetal, and postnatal vertebrate development. The purposes of this review are to provide a condensed overview of FGFs and their receptors, to catalog and categorize the functions of FGFs in vertebrate development, to present recent discoveries relating to the interplay of FGFs with other secreted ligands in the control of tissue growth and patterning, and to discuss several potential directions for future research in the field.