title: Spatiotemporal visualization of proHB-EGF ectodomain shedding in living cells.
abstract: Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family, each of which is produced as a type I transmembrane precursor. The juxtamembrane domain of proHB-EGF, a precursor of HB-EGF, is cleaved by a disintegrin and metalloproteases. HB-EGF is released into the extracellular space and strongly activates EGF receptor. The relevance of better understanding proHB-EGF shedding relates to the importance of the process in the proliferation, differentiation and survival of various types of cells. Shedding of proHB-EGF is normally evaluated using an alkaline phosphatase-tagged proHB-EGF assay or a western blotting assay that involves multiple cells, which makes it difficult to observe spatiotemporal differences in the activities of the individual cells. In this study, we developed a fluorescent proHB-EGF-based metalloprotease biosensor, named Fluhemb, to visualize spatiotemporal regulation of proHB-EGF shedding in individual cells using a simple method that measures changes in fluorescence ratios. Fluhemb might be very useful for detecting the activity of proHB-EGF shedding in various types of cells under different conditions in vitro and in vivo.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan. hinoue66@m.ehime-u.ac.jp
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Inoue
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Sakaue
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
authors.forename: Shigeki
authors.initials: S
authors.lastname: Higashiyama
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004800:Enzymes, Immobilized; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006493:Heparin / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D045726:Metalloproteases; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D018613:Microscopy, Confocal; D009361:Neoplasm Invasiveness; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019737:Transplants
chemical_list: D004800:Enzymes, Immobilized; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011498:Protein Precursors; D004815:Epidermal Growth Factor; D006493:Heparin; D045726:Metalloproteases
keywords: EGF family; HB-EGF; ectodomain shedding; fluorescence bioimaging; tumour