Analysis of the tetraspanin CD9-integrin alphaIIbbeta3 (GPIIb-IIIa) complex in platelet membranes and transfected cells.
The platelet integrin, alphaIIbbeta3 (GPIIb-IIIa), and the tetraspanin, CD9, are integral membrane proteins that are abundant in platelet membranes. We have identified several proteins, including CD9, which were co-precipitated by anti-alphaIIbbeta3 antibody from untreated, resting platelets that were solubilized with the poly(oxyethylene) non-ionic detergent, Brij-35. Immunoblot and quantitative immunoprecipitation showed that the association of alphaIIbbeta3 with CD9 is specific and stoichiometric. The interaction between CD9 and alphaIIbbeta3 is probably hydrophobic, as Triton X-100 and hydrophobic detergents of the Brij series completely dissociated the CD9-alphaIIbbeta3 complex. Recombinant CD9 and alphaIIbbeta3 can associate after transfection into Chinese hamster ovary cells, as seen by co-immunoprecipitation and co-localization in the periphery of spreading cells and in the lamellipodia of cells plated on fibrinogen. This co-localization is absent from focal adhesions. Furthermore, anti-CD9-coated latex beads clustered alphaIIbbeta3 with CD9. This work indicates that the tetraspanin, CD9, is associated with beta3 integrins in resting platelets and transfected cells.
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Department of Vascular Biology, The Scripps Research Institute, 10666 N. Torrey Pines Rd, La Jolla, CA 92307, USA.
Diaz-Gonzalez
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Ginsberg
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D019921:Biotinylation; D001792:Blood Platelets / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003902:Detergents; D006801:Humans; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D060245:Tetraspanin 29; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554869:CD9 protein, human; D003902:Detergents; D008562:Membrane Glycoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D060245:Tetraspanin 29