title: Selective roles for tumor necrosis factor alpha-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family: the juxtamembrane stalk determines cleavage efficiency.
abstract: Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
authors.forename: C Leann
authors.initials: CL
authors.lastname: Hinkle
authors.forename: Susan W
authors.initials: SW
authors.lastname: Sunnarborg
authors.forename: David
authors.initials: D
authors.lastname: Loiselle
authors.forename: Carol E
authors.initials: CE
authors.lastname: Parker
authors.forename: Mary
authors.initials: M
authors.lastname: Stevenson
authors.forename: William E
authors.initials: WE
authors.lastname: Russell
authors.forename: David C
authors.initials: DC
authors.lastname: Lee
mesh_terms: D051722:ADAM Proteins; D000072197:ADAM10 Protein; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D066258:Amphiregulin; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D066259:Betacellulin; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019483:Disintegrins / Q000378:metabolism; D066255:EGF Family of Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D066260:Epiregulin; D000939:Epitopes; D066246:ErbB Receptors / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014162:Transfection
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; C587849:Areg protein, mouse; C587851:BTC protein, human; D066259:Betacellulin; C587852:Btc protein, mouse; D018076:DNA, Complementary; D019483:Disintegrins; D066255:EGF Family of Proteins; C587880:EREG protein, human; D066260:Epiregulin; D000939:Epitopes; C587881:Ereg protein, mouse; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D004815:Epidermal Growth Factor; D006493:Heparin; D066246:ErbB Receptors; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; C498316:Adam9 protein, mouse; D008666:Metalloendopeptidases; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; C491234:Adam10 protein, mouse; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse