title: The cytoplasmic domain of a disintegrin and metalloproteinase 10 (ADAM10) regulates its constitutive activity but is dispensable for stimulated ADAM10-dependent shedding.
abstract: The membrane-anchored metalloproteinase a disintegrin and metalloprotease 10 (ADAM10) is required for shedding of membrane proteins such as EGF, betacellulin, the amyloid precursor protein, and CD23 from cells. ADAM10 is constitutively active and can be rapidly and post-translationally enhanced by several stimuli, yet little is known about the underlying mechanism. Here, we use ADAM10-deficient cells transfected with wild type or mutant ADAM10 to address the role of its cytoplasmic and transmembrane domain in regulating ADAM10-dependent protein ectodomain shedding. We report that the cytoplasmic domain of ADAM10 negatively regulates its constitutive activity through an ER retention motif but is dispensable for its stimulated activity. However, chimeras with the extracellular domain of ADAM10 and the transmembrane domain of ADAM17 with or without the cytoplasmic domain of ADAM17 show reduced stimulated shedding of the ADAM10 substrate betacellulin, whereas the ionomycin-stimulated shedding of the ADAM17 substrates CD62-L and TGFα is not affected. Moreover, we show that influx of extracellular calcium activates ADAM10 but is not essential for its activation by APMA and BzATP. Finally, the rapid stimulation of ADAM10 is not significantly affected by incubation with proprotein convertase inhibitors for up to 8 h, arguing against a major role of increased prodomain removal in the rapid stimulation of ADAM10. Thus, the cytoplasmic domain of ADAM10 negatively influences constitutive shedding through an ER retention motif, whereas the cytoplasmic domain and prodomain processing are not required for the rapid activation of ADAM10-dependent shedding events.
authors.affiliation: From the Arthritis and Tissue Degeneration Program, Hospital for Special Surgery and.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Maretzky
authors.affiliation: From the Arthritis and Tissue Degeneration Program, Hospital for Special Surgery and the Department of Dermatology Venerology and Allergology, University Hospital Schleswig Holstein, Campus Kiel, Kiel D-24105, Germany, and.
authors.forename: Astrid
authors.initials: A
authors.lastname: Evers
authors.affiliation: From the Arthritis and Tissue Degeneration Program, Hospital for Special Surgery and.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Le Gall
authors.affiliation: From the Arthritis and Tissue Degeneration Program, Hospital for Special Surgery and the Tri-Institutional M.D./Ph.D. Program, Rockefeller University/Memorial Sloan-Kettering Cancer Center/Weill Cornell Medical College, New York, New York 10021.
authors.forename: Rolake O
authors.initials: RO
authors.lastname: Alabi
authors.affiliation: the Department of Dermatology Venerology and Allergology, University Hospital Schleswig Holstein, Campus Kiel, Kiel D-24105, Germany, and.
authors.forename: Nancy
authors.initials: N
authors.lastname: Speck
authors.affiliation: the Department of Dermatology Venerology and Allergology, University Hospital Schleswig Holstein, Campus Kiel, Kiel D-24105, Germany, and.
authors.forename: Karina
authors.initials: K
authors.lastname: Reiss
authors.affiliation: From the Arthritis and Tissue Degeneration Program, Hospital for Special Surgery and the Departments of Medicine and of Physiology, Biophysics and Systems Biology, Weill Cornell Medical College, New York, New York 10021, blobelc@hss.edu.
authors.forename: Carl P
authors.initials: CP
authors.lastname: Blobel
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000072197:ADAM10 Protein; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology*; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D059748:Proteolysis; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491234:Adam10 protein, mouse
keywords: ADAM; Cell Surface Enzyme; Cytoplasmic Domain; Metalloprotease; Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9); Protein Ectodomain Shedding; Shedding