Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria 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 HB-EGF, proHB-EGF, is biologically active, providing mitogenic stimulation to neighboring cells in a juxtacrine mode. ProHB-EGF forms a complex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a tetra membrane-spanning protein that upregulates the juxtacrine mitogenic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/CD9 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 overexpress HB-EGF, we also demonstrated the association of proHB-EGF with DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contact sites was observed by double-immunofluorescence staining. At cell-cell contact sites, DRAP27/CD9 was highly coincident with alpha-catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alpha 3 beta 1 are colocalized with adherence junction-locating proteins. These results indicate that direct interaction of growth factors and cell adhesion molecules may control cell proliferation during the cell-cell adhesion process.
Nakamura
K
K
Institute of Life Science, Kurume University, Fukuoka, Japan.
Iwamoto
R
R
Mekada
E
E
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006493:Heparin / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D039201:Integrin alpha3beta1; D016023:Integrins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007365:Intercellular Junctions / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D018613:Microscopy, Confocal; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D060245:Tetraspanin 29; D014162:Transfection; D014709:Vero Cells
D015703:Antigens, CD; C554869:CD9 protein, human; D003432:Cross-Linking Reagents; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D039201:Integrin alpha3beta1; D016023:Integrins; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D060245:Tetraspanin 29; D004815:Epidermal Growth Factor; D006493:Heparin