The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.

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