title: Analysis of the site of interaction of CD28 with its counter-receptors CD80 and CD86 and correlation with function.
abstract: CD28 and CTLA-4 are homologue members of the Ig superfamily of molecules, containing a single V-like domain, transmembrane, and cytoplasmic regions. Both receptors associate with the counter-receptors CD80 and CD86, but the avidity of interaction for CD28 is about 20-fold lower than for CTLA-4. The interaction between CD28 and its cognate receptors provides a costimulatory signal for optimal T cell activation. Our previous mutational analysis of CD28 defined the highly conserved "MYPPPY" motif in the CDR3-region of the V-like domain as a key site of common and selective recognition. We have extended our analysis to cover all residues in the membrane distal loops of the V region, examining their effect on association with CD80/CD86 in cell adhesion and novel protein-based binding assays, and determining correlation between binding and functional response. Conservative F substitutions at either Y residue in the MYPPPY motif selectively reduced binding to CD86, but mutation of the three amino acids immediately C-terminal to Y 104 equivalently reduced binding to both co-receptors. The conservative F substitution of Y 26 in the CDR1-like region also reduced binding to CD80 and CD86. Other substitutions in the CDR1 loop and mutations spanning the CDR2 and DE loops had no effect. We conclude that the CDR1 and CDR3 regions contribute to a common binding site for CD80/CD86, and that the CDR3 region also carries determinants for selective recognition of these counter-receptors within the MYPPPY motif. Furthermore, for CD28, the strength of functional response, as measured by IL-2 production, directly correlates with binding avidity.
authors.affiliation: Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
authors.forename: I
authors.initials: I
authors.lastname: Kariv
authors.forename: A
authors.initials: A
authors.lastname: Truneh
authors.forename: R W
authors.initials: RW
authors.lastname: Sweet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism* / Q000502:physiology; D018122:B7-1 Antigen / Q000378:metabolism* / Q000502:physiology; D051940:B7-2 Antigen; D001483:Base Sequence; D018106:CD28 Antigens / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007739:L Cells; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000276:immunology; D016415:Sequence Alignment
chemical_list: D015703:Antigens, CD; D018122:B7-1 Antigen; D051940:B7-2 Antigen; D018106:CD28 Antigens; C497964:CD86 protein, human; C497965:Cd86 protein, mouse; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins