title: The majority of stem cell factor exists as monomer under physiological conditions. Implications for dimerization mediating biological activity.
abstract: Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 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 Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-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 Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 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.
authors.affiliation: Amgen Inc., Amgen Center, Thousand Oaks, California 91320, USA.
authors.forename: Y R
authors.initials: YR
authors.lastname: Hsu
authors.forename: G M
authors.initials: GM
authors.lastname: Wu
authors.forename: E A
authors.initials: EA
authors.lastname: Mendiaz
authors.forename: R
authors.initials: R
authors.lastname: Syed
authors.forename: J
authors.initials: J
authors.lastname: Wypych
authors.forename: R
authors.initials: R
authors.lastname: Toso
authors.forename: M B
authors.initials: MB
authors.lastname: Mann
authors.forename: T C
authors.initials: TC
authors.lastname: Boone
authors.forename: L O
authors.initials: LO
authors.lastname: Narhi
authors.forename: H S
authors.initials: HS
authors.lastname: Lu
authors.forename: K E
authors.initials: KE
authors.lastname: Langley
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D012995:Solubility; D013050:Spectrometry, Fluorescence; D019089:Stem Cell Factor / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D011994:Recombinant Proteins; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit