The binding of heparin-binding EGF-like growth factor (HB-EGF) to the epidermal growth factor (EGF) receptor of human endometrial carcinoma cells was compared to that of EGF using an 125I-EGF radioreceptor assay. The inhibitory effect of HB-EGF on 125I-EGF binding was reversed either in the presence of heparin (but not by chondroitin sulfate) or by pre-treating the cells with heparinase. These treatments did not affect the binding of EGF to its receptor. To map potential regions in the HB-EGF molecule that mediate its heparin-dependent interaction with the EGF receptor, HB-EGF peptides were synthesized that were non-homologous to EGF. Accordingly residues 20-25 and 36-41, but not residues 8-19, of HB-EGF were found to be (i) heparin-binding and (ii) modulators of HB-EGF (but not of EGF) binding to the EGF receptor.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially synthesized as a membrane bound protein that is subsequently processed to yield an approximately 74 amino acid secreted product. To investigate the biological activities of HB-EGF and its role(s) in tumor formation, the full-length HB-EGF cDNA was cloned under the regulation of the mouse metallothionein promoter and stably expressed in HB-EGF deficient mouse L cells. HB-EGF immunoreactive proteins of 21 and 24 kDa were observed from transfected MLC lysates, and these lysates exhibited the ability to bind to the EGF receptor, stimulate 3H-thymidine uptake in BALB/c-3T3 cells, and induce anchorage independent growth (AIG) of normal rat kidney (NRK) cells. Furthermore, NRK cells treated with either E. coli-derived or vaccinia virus-derived HB-EGF, as well as NRK cells directly transfected with the HB-EGF construct, demonstrated AIG. We conclude that HB-EGF is a potent growth factor capable of stimulating altered cell growth and anchorage independence.
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