FUNCTIONAL DISSOCIATION OF CD8-ALPHA-S IG HOMOLOG AND CONNECTING PEPTIDE DOMAINS

The contribution of the CD8 alpha . IgV homologue domain to class I MH C binding was evaluated using a series of chimeric human CD8 alpha:Fs polypeptides incorporating alternative CD8 alpha extracellular domain components, Using a nonisotopic cellfree physical binding assay, those Fe chimeras encompassing the CD8 alpha:IgV homologue domain only (dis sociated from the 48-amino acid CD8 alpha connecting peptide) were sho wn to retain the capacity of the complete CD8 alpha extracellular doma in to bind to a recombinant soluble class I MHC alpha(3) domain unit o r to intact class I MHC. The specificity of the CD8 alpha:class I MHC alpha(3) domain interaction was verified by mAb and soluble polypeptid e blocking experiments, Furthermore, co-precipitation of an Fc chimera incorporating only the CD8 alpha . IgV homologue domain and a recombi nant soluble class I MHC alpha(3) domain unit was accomplished, In add ition, a glycosylphosphatidylinositol (GPI)-modified variant of the CD 8 alpha . IgV homologue domain was generated via chimerization with th e GPI signal sequence from decay-accelerating factor, Gal anchorage fo r this truncated CD8 alpha polypeptide was verified, and its capacity to promote intercellular adhesion through class I MHC binding was show n in a cell:cell binding assay, The findings indicate that the CD8 alp ha . IgV homologue domain acts as an independent structural unit when dissociated from the CD8 alpha connecting peptide, and in so doing ret ains class I MHC binding capacity. This further establishes the princi ple that Ig superfamily domains from receptor:counter-receptor pairs c an interact with each other as isolated units, providing an experiment al path for tailoring therapeutically useful IgSF protein derivatives.