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.
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.
Hinkle
C Leann
CL
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Sunnarborg
Susan W
SW
Loiselle
David
D
Parker
Carol E
CE
Stevenson
Mary
M
Russell
William E
WE
Lee
David C
DC
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
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