MEMBRANE-ANCHORED HEPARIN-BINDING EGF-LIKE GROWTH-FACTOR (HB-EGF) ANDDIPHTHERIA-TOXIN RECEPTOR-ASSOCIATED PROTEIN (DRAP27) CD9 FORM A COMPLEX WITH INTEGRIN ALPHA-3-BETA-1 AT CELL-CELL CONTACT SITES/

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF receptor to exert mitogenic activity. The membrane-anchored form of H B-EGF, proHB-EGF, is biologically active, providing mitogenic stimulat ion to neighboring cells in a juxtacrine mode. ProHB-EGF forms a compl ex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a t etra membrane-spanning protein that upregulates the juxtacrine mitogen ic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/C D9 resulted in preferential coprecipitation of integrin alpha 3 beta 1 from Vero cell, A431 cell and MG63 cell lysates. Anti-integrin alpha 3 or anti-integrin beta 1 coprecipitated DRAP27/CD9 from the same cell lysates. Chemical cross-linking confirmed the physical association of DRAP27/CD9 and integrin alpha 3 beta 1. Using Vero-H cells, which ove rexpress HB-EGF, we also demonstrated the association of proHB-EGF wit h DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contac t sites was observed by double-immunofluorescence staining. At cell-ce ll contact sites, DRAP27/CD9 was highly coincident with alpha-catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alph a 3 beta 1 are colocalized with adherence junction-locating proteins. These results indicate that direct interaction of growth factors and c ell adhesion molecules may control cell proliferation during the cell- cell adhesion process.