Identification of serum factor inducing ectodomain shedding of proHB-EGF and studies of noncleavable mutants of proHB-EGF

The ectodomain of the transmembrane form of HB-EGF (proHB-EGF) is cleaved a t 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 f rom factor(s) contained in supplemented FCS in a culture medium. Analysis o f serum factors, including digestion with enzymes, separation by thin layer chromatography, and shedding assay with purified phospholipids, revealed t hat 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 aro und the putative cleavage sites. These mutant forms showed resistance to st imuli of both TPA and LPA, suggesting that proHB-EGF is cleaved at the simi lar site by stimulation with TPA and LPA. (C) 2001 Academic Press.