ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx.
Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.
Sanderson
Michael P
MP
Pacific Northwest Research Institute, Seattle, Washington 98122, USA.
Erickson
Sarah N
SN
Gough
Peter J
PJ
Garton
Kyle J
KJ
Wille
Paul T
PT
Raines
Elaine W
EW
Dunbar
Andrew J
AJ
Dempsey
Peter J
PJ
D051722:ADAM Proteins; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases; D001483:Base Sequence; D066259:Betacellulin; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D017136:Ion Transport; D008565:Membrane Proteins / Q000502:physiology*; D008666:Metalloendopeptidases / Q000502:physiology*; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology*
C587851:BTC protein, human; D066259:Betacellulin; C587852:Btc protein, mouse; D017931:DNA Primers; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C010024:4-aminophenylmercuriacetate; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D010662:Phenylmercuric Acetate; D002118:Calcium