Stewart D. Lyman

Identification of a ligand for the c-kit proto-oncogene

We report the purification and N-terminal amino acid sequence of a novel mast cell growth factor, termed MGF, from the supernatants of a murine stromal cell line. A panel of interleukin 3-dependent cell lines were screened for responsiveness to partially purified MGF in [3H]thymidine incorporation assays; proliferative stimulation of these cells in response to MGF correlated with expression of mRNA for the c-kit protooncogene. MGF was shown to be a ligand for c-kit by cross-linking 125I-labeled MGF to c-kit-expressing cells with subsequent immunoprecipitation of the complex with antiserum specific for the C-terminus of c-kit. This establishes MGF as a ligand for the c-kit protein.

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.

Molecular cloning of a ligand for the flt3 flk-2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles

Many spontaneous, chemical-induced, and radiation-induced dominant white spotting (W) and steel (Sl) mutations have been identified in the mouse. W and Sl mutations have similar phenotypic effects including deficiencies in pigment cells, germ cells, and blood cells, Numerous studies have suggested that W acts within the affected cell while Sl instead exerts its effects in the extracellular environment. Recent findings demonstrating that W encodes the c-kit proto-oncogene, a tyrosine kinase membrane receptor, have suggested that Sl encodes a ligand for c-kit. In the accompanying article we report the identification and purification of mast cell growth factor (MGF), a c-kit ligand. Here we describe the cloning of sequences encoding MGF. Furthermore, we show that Mgf maps near Sl in the distal region of mouse chromosome 10 and is deleted in a number of Sl alleles. These findings strongly support the notion that Sl encodes the mast cell growth factor.

A new cytokine receptor superfamily

The amino acid sequences of several, recently cloned cytokine receptors show significant homologies, primarily in their extracellular, ligand-binding domains. With one exception, their cognate cytokines mediate biological activities on a variety of hematopoietic cell types; thus we have designated the receptors as the hematopoietic receptor superfamily.

Embryonic RNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development.

Mutations at the dominant white spotting (W) and Steel (Sl) loci in mouse exert deleterious effects on three migratory cell lineages (primordial germ cells, melanocytes and hematopoietic stem cells) resulting in loss of pigmentation, reduced fertility and anemia. The W locus encodes the c‐kit protein tyrosine kinase (TK) receptor. More recently, the Sl locus has been shown to encode a ligand for c‐kit, which is variously known as mast cell growth factor (MGF), stem cell growth factor and c‐kit ligand. Here we report an in situ hybridization analysis comparing the expression profiles of MGF and c‐kit transcripts during mouse embryogenesis. The data are consistent with the c‐kit receptor‐ligand complex providing a homing mechanism during stem cell migration in early development and in stem cell proliferation, differentiation, or survival in late development. In the nervous system, an unexpected and complex pattern of expression is uncovered that suggests involvement of the W and Sl gene products in the organization of the neural tube and brain.

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c‐kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c‐kit gene products with cytoplasmic signalling proteins, using transient c‐kit expression assays in COS cells. In addition to the previously identified c‐kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in‐frame insertion, Gly‐Asn‐Asn‐Lys, within the extracellular domain, was detected in murine mast cell cultures and mid‐gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3′ kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP‐associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI‐3′ kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582‐‐‐‐Lys) or W41 (Val831‐‐‐‐Met) mutant alleles into c‐kit expression constructs abolished (W37) or reduced (W41) the Steel factor‐induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

The steel factor

Steel factor (SLF) is a recently identified growth factor which is the gene product of the murine Steel locus and a ligand for the c-kit tyrosine kinase receptor, the product of the dominant white spotting locus (W). Defects at these genetic loci result in aberrant melanocyte, germ cell, and hematopoietic development. Both the receptor (c-kit) and the ligand (SLF) have been shown to undergo tissue-specific mRNA splicing to produce distinct isoforms which have unique biological functions. As predicted by the phenotype of these mutations, SLF influences the growth and differentiation of melanocytes, primordial germ cells, and a broad spectrum of cell types in the hematopoietic progenitor and stem cell hierarchy. SLF has also been shown to have effects on hematopoietic lineages not predicted by defects seen in the Steel mouse.

Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor

Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

Steel Factor Is Required for Maintenance, But Not Differentiation, of Melanocyte Precursors in the Neural Crest

Skin melanocytes are derived from neural crest cells that migrate into the dermis during embryogenesis. Two mouse mutants, Steel and White dominant-spotting, which have defects in melanocyte production, have recently been shown to have deletions in the genes that code for a new growth factor, steel factor (SLF), and its receptor, respectively. Here, we have investigated the role that SLF plays in melanogenesis using cultures of mouse neural crest and found that its primary action is the maintenance of melanocyte precursors. It has no effect on the final stage of melanocyte differentiation, the production of melanin, which appears to require an additional factor whose action is mimicked by the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate).