M. Suzuki et al., MATRIX METALLOPROTEINASE-3 RELEASES ACTIVE HEPARIN-BINDING EGF-LIKE GROWTH-FACTOR BY CLEAVAGE AT A SPECIFIC JUXTAMEMBRANE SITE, The Journal of biological chemistry, 272(50), 1997, pp. 31730-31737
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is
synthesized as a membrane-anchored precursor that is cleaved to relea
se the soluble mature growth factor. The two forms are active as juxta
crine and paracrine/autocrine growth factors, respectively, The enzyme
s that process the HB EGF transmembrane form are unknown, Accordingly,
an in vitro assay was established using a fusion protein in which alk
aline phosphatase (AP) replaced the transmembrane and cytoplasmic doma
ins of HB-EGF (HB-EGF JM-AP). The fusion protein was anchored to agaro
se beads coated with anti-AP antibodies, Several matrix metalloprotein
ases (MMPs) were tested for the ability to release soluble HB-EGF in t
he in vitro system. MMP-3 released soluble 12-kDa immunoreactive and m
itogenic 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 n
ot cleaved by MMP-3 in the in vitro assay. The C-terminal portion of t
he cleaved HB-EGF JM-AP that remained attached to the anti-AP beads wa
s N-terminally sequenced and the MMP-3 cleavage site was determined to
be Glu(151)-Asn(152), a site within the JM domain. MMP-3 treatment al
so released soluble HB-EGF in vivo from MC2 cells expressing transmemb
rane HB-EGF precursor, at a level of about 2-fold above control. It wa
s concluded that MMP-3 cleaves HB-EGF at a specific site in the JM dom
ain and that this enzyme might regulate the conversion of HB-EGF from
being a juxtacrine to a paracrine/autocrine growth factor.