Identification of serum factor inducing ectodomain shedding of proHB-EGF and sStudies of noncleavable mutants of proHB-EGF.
The ectodomain of the transmembrane form of HB-EGF (proHB-EGF) is cleaved at the cell surface by proteases, yielding a soluble growth factor. A number of stimuli, including TPA, accelerate this cleavage. However, proHB-EGF is shed constitutively under normal culture conditions without any particular stimuli. We demonstrate here that constitutive cleavage resulted largely from factor(s) contained in supplemented FCS in a culture medium. Analysis of serum factors, including digestion with enzymes, separation by thin layer chromatography, and shedding assay with purified phospholipids, revealed that lysophosphatidic acid (LPA) is a major factor in FCS for stimulation of proHB-EGF shedding. We also studied here ectodomain shedding of two kinds of mutant form of proHB-EGF which have a single amino acid substitution around the putative cleavage sites. These mutant forms showed resistance to stimuli of both TPA and LPA, suggesting that proHB-EGF is cleaved at the similar site by stimulation with TPA and LPA.
Hirata
M
M
Department of Cell Biology, Osaka University, Osaka, Suita, 565-0871, Japan.
Umata
T
T
Takahashi
T
T
Ohnuma
M
M
Miura
Y
Y
Iwamoto
R
R
Mekada
E
E
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D003470:Culture Media; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000097:blood; D009154:Mutation; D013935:Thymic Factor, Circulating / Q000378:metabolism*; D014709:Vero Cells
D003470:Culture Media; D017931:DNA Primers; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D004815:Epidermal Growth Factor; D013935:Thymic Factor, Circulating