PHORBOL ESTER INDUCES THE RAPID PROCESSING OF CELL-SURFACE HEPARIN-BINDING EGF-LIKE GROWTH-FACTOR - CONVERSION FROM JUXTACRINE TO PARACRINEGROWTH-FACTOR ACTIVITY

Vero cell heparin-binding epidermal growth factor-like growth factor ( HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF pre cursor (proHB-EGF). Localization and processing of proHB-EGF, both con stitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, wa s examined in Vero cells overexpressing recombinant HB-EGF (Vero H cel ls). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the in terface of cell to cell contact. Cell surface biotinylation and immuno precipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species . Vero H cell surface proHB-EGF turned over constitutively with a half -life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proH B-EGF was processed and a 14-kDa species of bioactive HB-EGF was relea sed slowly, but most of the proHB-EGF was internalized, displaying a d iffuse immunofluorescent staining pattern and accumulation of proHB-EG F in endosomes. Addition of TPA induced a rapid processing of proHB-EG F at a pro(148)-Val(149) site with a half-life of 7 min. The TPA effec t was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is max imal at 30 min after addition of TPA and that proHB-EGF is resynthesiz ed and the initial cell surface levels are regained within 12-24 h. Lo ss of cell surface proHB-EGF was concomitant with appearance of 14- an d 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-ass ociated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in c oculture. Processing of proHB-EGF resulted in loss of juxtacrine activ ity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivit y by converting it from a cell-surface juxtacrine growth factor to a p rocessed, released soluble paracrine growth factor.