title: A novel system for convenient detection of low-affinity receptor-ligand interactions: chelator-lipid liposomes engrafted with recombinant CD4 bind to cells expressing MHC class II.
abstract: The interactions of cell surface receptors with their ligands, crucial for initiating many immunological responses, are often stabilized by receptor dimerization/oligomerization, and by multimeric interactions between receptors on one cell with their ligands or cognate receptors on the apposing cell. Current techniques for studying receptor-ligand interactions, however, do not always allow receptors to move laterally to enable dimerization/ oligomerization, or to interact multimerically with ligands on cell surfaces. For these reasons detection of low- affinity receptor-ligand interactions has been difficult. Utilizing a novel chelator-lipid, nitrilotriacetic acid di-tetradecylamine (NTA-DTDA), we have developed a convenient liposome system for directly detecting low-affinity receptor-ligand interactions. Our studies using recombinant soluble forms of murine CD40 and B7.1, and murine and human CD4, each possessing a hexhistidine tag, showed that these proteins can be anchored or 'engrafted' directly onto fluorescently labelled liposomes via a metal-chelating linkage with NTA-DTDA, permitting them to undergo dimerization/oligomerization and multimeric binding with ligands on cells. Fluorescence- activated cell sorter (FACS) analyses demonstrated that while there is little if any binding of soluble forms of murine CD40 and B7.1, and murine and human CD4 to cells, engrafted liposomes bind specifically to cells expressing the appropriate cognate receptor, often giving a fluorescence 4-6-fold above control cells. Such liposomes could detect directly the low-affinity interaction of murine CD40 and B7.1 with CD154- and CD28-expressing cells, respectively, and the interaction of CD4 with MHC Class II, which has hitherto defied direct detection except through mutational analysis and mAb blocking studies.
authors.affiliation: School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, Australian Capital Territory, Australia.
authors.forename: C L
authors.initials: CL
authors.lastname: Van Broekhoven
authors.forename: J G
authors.initials: JG
authors.lastname: Altin
mesh_terms: D000588:Amines / Q000737:chemistry; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D002614:Chelating Agents / Q000737:chemistry; D005434:Flow Cytometry; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D000588:Amines; D015704:CD4 Antigens; D002614:Chelating Agents; D000949:Histocompatibility Antigens Class II; D008024:Ligands; D008081:Liposomes; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C405382:nitrilotriacetic acid ditetradecylamine; D009571:Nitrilotriacetic Acid