The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions

Abstract

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (K a ) of 2-4 ؋ 10 8 M ؊1 , using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization K a , greater than 90% of the circulating SCF would be in the monomeric form. When 125 I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10 -100 ng/ml, consistent with the K a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer K a values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH 2 CONH 2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys 165 -Cys 165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.Stem cell factor is a cytokine that is active toward early hematopoietic cells and also plays roles in gametogenesis, melanogenesis, and mast cell function. Its biological and other properties have been extensively reviewed (1, 2). It is found in both membrane-bound and soluble forms, with the latter being derived from a membrane-bound form by proteolytic cleavage. The soluble SCF has 165 amino acids.Both soluble Escherichia coli-derived and CHO 1 cell-derived recombinant human SCF have been reported to be non-covalently associated dimers, as determined by sedimentation equilibrium and size exclusion chromatography at protein concentrations above 0.4 mg/ml (3). In a previous paper (4), we demonstrated that SCF dimer is dissociable under non-denaturing conditions and the dissociation rate constant (k d ) of E. coli-derived rhSCF dimer is approximately 1.35 ϫ 10 Ϫ4 s Ϫ1 at pH 4.8, 25°C. In the present work, we arrive at a value of 2-4 ϫ 10 8 M Ϫ1 for the dimer association constant (K a ) of E. coli-derived rhSCF, based on several approaches including ultracentrifugation and size exclusion chromatography at low SCF concentrations. Since the SCF concentration in human serum has been found previously to be a few nanograms/ml (5), the K a value suggests that the majority of SCF in serum may be monomeric. We use 125 I-SCF as a tracer added to serum to show that this does in fact appear to be the case.The binding of ligands to cell receptors, followed by receptor dimerization, is essential for signal transduction by the family of transmembrane receptor tyrosine kinases (6 -8). The receptor for SCF on target cells is Kit (see Refs....

Local presentation of Steel factor increases expression of c-kit immunoreactive interstitial cells of Cajal in culture

“…Both isoforms of SF initially produce membranebound SF, and, to different degrees, both produce soluble SF (11). To distinguish the effects of soluble from membrane-bound SF on ICC in culture, we used a genetically engineered form of SF known as membrane restricted.…”

“…Both soluble and membrane-bound SF can stimulate c-kit in vitro, but soluble SF does not support long-term hematopoietic stem cell growth (11). To determine whether soluble SF was sufficient to support ICC, freshly dispersed murine jejunal cells were cocultured with fibroblasts expressing soluble SF.…”

“…SF is present in low concentration in serum but is thought to act locally, close to the site of production where the concentration is likely much higher (2). Soluble SF circulates as a dimer and a monomer, but the dimeric form is more biologically active (11). Membrane-bound SF is a more effective agonist for the c-kit receptor compared with soluble SF (25).…”

Structure-Function Analysis of FLT3 Ligand-FLT3 Receptor Interactions Using a Rapid Functional Screen

“…Phe 67 in SCF altered monomer-dimer equilibrium toward monomer and reduced the biological activity of SCF (23). The observation that the L27P mutation mapped to the dimerization interface and appeared to alter monomer-dimer equilibrium helped validate M-CSF as a template for modeling FLT3L structure.…”

“…Other members of this family share little identity, yet have remarkable structural similarity (9). FLT3L (1), SCF (23), and M-CSF (21) are dimeric, but unlike M-CSF, FLT3L, and SCF they are non-disulfide-linked dimers. The M-CSF dimerization interface was used to model the dimerization interface of FLT3L.…”