title: G protein coupling and second messenger generation are indispensable for metalloprotease-dependent, heparin-binding epidermal growth factor shedding through angiotensin II type-1 receptor.
abstract: A G protein-coupled receptor agonist, angiotensin II (AngII), induces epidermal growth factor (EGF) receptor (EGFR) transactivation possibly through metalloprotease-dependent, heparin-binding EGF (HB-EGF) shedding. Here, we have investigated signal transduction of this process by using COS7 cells expressing an AngII receptor, AT1. In these cells AngII-induced EGFR transactivation was completely inhibited by pretreatment with a selective HB-EGF inhibitor, or with a metalloprotease inhibitor. We also developed a COS7 cell line permanently expressing a HB-EGF construct tagged with alkaline phosphatase, which enabled us to measure HB-EGF shedding quantitatively. In the COS7 cell line AngII stimulated release of HB-EGF. This effect was mimicked by treatment either with a phospholipase C activator, a Ca2+ ionophore, a metalloprotease activator, or H2O2. Conversely, pretreatment with an intracellular Ca2+ antagonist or an antioxidant blocked AngII-induced HB-EGF shedding. Moreover, infection of an adenovirus encoding an inhibitor of G(q) markedly reduced EGFR transactivation and HB-EGF shedding through AT1. In this regard, AngII-stimulated HB-EGF shedding was abolished in an AT1 mutant that lacks G(q) protein coupling. However, in cells expressing AT1 mutants that retain G(q) protein coupling, AngII is still able to induce HB-EGF shedding. Finally, the AngII-induced EGFR transactivation was attenuated in COS7 cells overexpressing a catalytically inactive mutant of ADAM17. From these data we conclude that AngII stimulates a metalloprotease ADAM17-dependent HB-EGF shedding through AT1/G(q)/phospholipase C-mediated elevation of intracellular Ca2+ and reactive oxygen species production, representing a key mechanism indispensable for EGFR transactivation.
authors.affiliation: Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: Mizuo
authors.initials: M
authors.lastname: Mifune
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Ohtsu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Hidekatsu
authors.initials: H
authors.lastname: Nakashima
authors.forename: Eugen
authors.initials: E
authors.lastname: Brailoiu
authors.forename: Nae J
authors.initials: NJ
authors.lastname: Dun
authors.forename: Gerald D
authors.initials: GD
authors.lastname: Frank
authors.forename: Tadashi
authors.initials: T
authors.lastname: Inagami
authors.forename: Shigeki
authors.initials: S
authors.lastname: Higashiyama
authors.forename: Walter G
authors.initials: WG
authors.lastname: Thomas
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Eckhart
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Dempsey
authors.forename: Satoru
authors.initials: S
authors.lastname: Eguchi
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000256:Adenoviridae / Q000235:genetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry*; D066257:Heparin-binding EGF-like Growth Factor; D006861:Hydrogen Peroxide / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D007763:Lac Operon; D008024:Ligands; D008666:Metalloendopeptidases / Q000378:metabolism; D045726:Metalloproteases / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D051381:Rats; D017382:Reactive Oxygen Species; D044139:Receptor, Angiotensin, Type 2 / Q000737:chemistry*; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007476:Ionophores; D008024:Ligands; D017382:Reactive Oxygen Species; D044139:Receptor, Angiotensin, Type 2; D004815:Epidermal Growth Factor; D006493:Heparin; D006861:Hydrogen Peroxide; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D045726:Metalloproteases; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606464:Adam17 protein, rat; D019204:GTP-Binding Proteins; D002118:Calcium