Matrix metalloproteinase-3 releases active heparin-binding EGF-like growth factor by cleavage at a specific juxtamembrane site.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored precursor that is cleaved to release the soluble mature growth factor. The two forms are active as juxtacrine and paracrine/autocrine growth factors, respectively. The enzymes that process the HB-EGF transmembrane form are unknown. Accordingly, an in vitro assay was established using a fusion protein in which alkaline phosphatase (AP) replaced the transmembrane and cytoplasmic domains of HB-EGF (HB-EGF JM-AP). The fusion protein was anchored to agarose beads coated with anti-AP antibodies. Several matrix metalloproteinases (MMPs) were tested for the ability to release soluble HB-EGF in the in vitro system. MMP-3 released soluble 12-kDa immunoreactive and mitogenic HB-EGF within 30 min. On the other hand neither MMP-2 nor MMP-9 had any cleavage activities. A non-cleavable mutant was prepared by replacing the juxtamembrane (JM) region of HB-EGF with the JM region of CD4. The mutant HB-EGF, which in its full-length form was as active a juxtacrine growth factor as was the wild type HB-EGF in vivo, was not cleaved by MMP-3 in the in vitro assay. The C-terminal portion of the cleaved HB-EGF JM-AP that remained attached to the anti-AP beads was N-terminally sequenced and the MMP-3 cleavage site was determined to be Glu151-Asn152, a site within the JM domain. MMP-3 treatment also released soluble HB-EGF in vivo from MC2 cells expressing transmembrane HB-EGF precursor, at a level of about 2-fold above control. It was concluded that MMP-3 cleaves HB-EGF at a specific site in the JM domain and that this enzyme might regulate the conversion of HB-EGF from being a juxtacrine to a paracrine/autocrine growth factor.
Suzuki
M
M
Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Raab
G
G
Moses
M A
MA
Fernandez
C A
CA
Klagsbrun
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data
C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D004815:Epidermal Growth Factor; D006493:Heparin; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9