The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach.
A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser(108/121), HB-EGF-Cys/Ser(116/132), and HB-EGF-Cys/Ser(134/143)) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M(r) of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser(108/121) and HB-EGF-Cys/Ser(116/132) stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF(134/143) proteins competed with 125I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser(108/121) and HB-EGF-Cys/Ser(116/132) failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser(134/143) antagonizes EGFRs.
Hoskins
J T
JT
Department of Zoology, Miami University, Oxford, OH 45056, USA.
Zhou
Z
Z
Harding
P A
PA
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D005347:Fibroblasts / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D004220:Disulfides; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D014443:Tyrosine; D012694:Serine; D008668:Metallothionein; D066246:ErbB Receptors; D003545:Cysteine