IMPAIRED MHC CLASS-I (H-2D(D))-MEDIATED PROTECTION AGAINST LY-49A(-ACID SUBSTITUTIONS IN THE ANTIGEN-BINDING CLEFT() NK CELLS AFTER AMINO)

The MHC class I molecule H-2D(d) (D-d) acts as a ligand for the inhibi tory NK cell receptor Ly-49A. We have constructed altered D-d molecule s by site-directed mutagenesis, replacing residues with the correspond ing amino acids from the D-b molecule, which fails to inhibit via Ly-4 9A. Mutations at positions 73 and 156 (D(d)S73WD156Y) impaired the pro tective effect of the D-d molecule, as evaluated by testing lymphoma c ells transfected with the mutant gene for sensitivity to killing by Ly -49A(+) NK cells in vitro and rejection by NK cells in vivo. The alter ed residues form a hydrophobic ridge across the floor of the antigen b inding cleft. A mutation in the alpha helix of the alpha 2 domain, fac ing the solvent and without direct contact with the peptide (D(d)A150S ) had no effect. D-d recognition by Ly-49A(+) NK cells is considered t o be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational chang es in the D-d molecule that make the trimolecular complex less permiss ive for inhibition of Ly-49A(+) NK cells.