The majority of stem cell factor exists as monomer under physiological conditions

被引:60
作者
Hsu, YR
Wu, GM
Mendiaz, EA
Syed, R
Wypych, J
Toso, R
Mann, MB
Boone, TC
Narhi, LO
Lu, HS
Langley, KE
机构
[1] Amgen Inc., Amgen Center, Thousand Oaks
[2] Amgen Inc. 14-2-E, Amgen Center, Thousand Oaks, CA 91320-1789
关键词
D O I
10.1074/jbc.272.10.6406
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 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 K-a, greater than 90% of the circulating SCF would be in the monomeric form. When I-125-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-CH2CONH2 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.
引用
收藏
页码:6406 / 6415
页数:10
相关论文
共 33 条
[11]  
Hsu YR, 1996, PROTEIN SCI, V5, P1165
[12]   PLATELET-DERIVED GROWTH-FACTOR - IDENTIFICATION OF CONSTITUENT POLYPEPTIDE-CHAINS [J].
JOHNSSON, A ;
HELDIN, CH ;
WESTERMARK, B ;
WASTESON, A .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1982, 104 (01) :66-74
[13]   PROPERTIES OF VARIANT FORMS OF HUMAN STEM-CELL FACTOR RECOMBINANTLY EXPRESSED IN ESCHERICHIA-COLI [J].
LANGLEY, KE ;
MENDIAZ, EA ;
LIU, NL ;
NARHI, LO ;
ZENI, L ;
PARSEGHIAN, CM ;
CLOGSTON, CL ;
LESLIE, I ;
POPE, JA ;
LU, HS ;
ZSEBO, KM ;
BOONE, TC .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1994, 311 (01) :55-61
[14]   PURIFICATION AND CHARACTERIZATION OF SOLUBLE FORMS OF HUMAN AND RAT STEM-CELL FACTOR RECOMBINANTLY EXPRESSED BY ESCHERICHIA-COLI AND BY CHINESE-HAMSTER OVARY CELLS [J].
LANGLEY, KE ;
WYPYCH, J ;
MENDIAZ, EA ;
CLOGSTON, CL ;
PARKER, VP ;
FARRAR, DH ;
BROTHERS, MO ;
SATYGAL, VN ;
LESLIE, I ;
BIRKETT, NC ;
SMITH, KA ;
BALTERA, RF ;
LYONS, DE ;
HOGAN, JM ;
CRANDALL, C ;
BOONE, TC ;
POPE, JA ;
KARKARE, SB ;
ZSEBO, KM ;
SACHDEV, RK ;
LU, HS .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1992, 295 (01) :21-28
[15]  
LANGLEY KE, 1993, BLOOD, V81, P656
[16]   STEEL FACTOR AND C-KIT PROTOONCOGENE - GENETIC LESSONS IN SIGNAL-TRANSDUCTION [J].
LEV, S ;
BLECHMAN, JM ;
GIVOL, D ;
YARDEN, Y .
CRITICAL REVIEWS IN ONCOGENESIS, 1994, 5 (2-3) :141-168
[17]   INTERSPECIES MOLECULAR CHIMERAS OF KIT HELP DEFINE THE BINDING-SITE OF THE STEM-CELL FACTOR [J].
LEV, S ;
BLECHMAN, J ;
NISHIKAWA, SI ;
GIVOL, D ;
YARDEN, Y .
MOLECULAR AND CELLULAR BIOLOGY, 1993, 13 (04) :2224-2234
[18]  
LEV S, 1992, J BIOL CHEM, V267, P15970
[19]  
LEV S, 1992, J BIOL CHEM, V267, P10866
[20]   SPONTANEOUS DISSOCIATION ASSOCIATION OF MONOMERS OF THE HUMAN-STEM-CELL FACTOR DIMER [J].
LU, HS ;
CHANG, WC ;
MENDIAZ, EA ;
MANN, MB ;
LANGLEY, KE ;
HSU, YR .
BIOCHEMICAL JOURNAL, 1995, 305 :563-568