The NK Cell MHC Class I Receptor Ly49A Detects Mutations on H-2Dd Inside and Outside of the Peptide Binding Groove

Abstract

N atural killer cells are a group of lymphocytes that spontaneously lyse certain tumor targets (1). The finding that cell lines defective for MHC class I expression were efficiently lysed by NK cells led to the missing self hypothesis: NK cells kill cells that lack normal expression of MHC class I molecules (2). Recent studies have established that NK cells express inhibitory receptors for MHC class I molecules (3, 4). The receptors can be classified into two groups by their structural characteristics. One group that includes human killer cell Ig-like receptors is characterized by two or three Ig-like domains in the extracellular region and configuration of type I transmembrane proteins (5, 6). NK cell MHC class I receptors of Ig-type have thus far been found only in primates, but not in rodents. gp49B1, which belongs to the Ig superfamily, is expressed on mouse NK cells (7,8); however, the ligand for gp49B1 may not be MHC class I. Another group of NK cell MHC class I receptors is characterized by disulfide-linked dimer of type II transmembrane protein with an extracellular region homologous to carbohydrate recognition domain of C-type lectin. This group includes the Ly49 family in mice (9 -12) and rats (13) and CD94/NKG2 heterodimer found in humans (14) and rodents (15, 16).The inhibitory receptors of both groups have one or two immune receptor tyrosine-based inhibitory motifs (ITIM) 3 characterized by an IXYXXL sequence in the cytoplasmic region (17). Upon ligand recognition, the Tyr residue in the ITIM is phosphorylated and recruits the Src homology domain-containing protein tyrosine phosphatase 1 with Src homology 2 domains. The recruited Src homology domain-containing protein tyrosine phosphatase 1 is believed to dephosphorylate unknown critical substrates to shut down positive signaling. There are activating type of MHC class I receptors of C-type lectin and Ig-type with extracellular domains similar to those of the inhibitory receptors. Instead of cytoplasmic ITIM, the activating receptors have characteristic charged amino acid residues in the transmembrane region, and by that associate with DAP12, an adapter component with cytoplasmic immune receptor tyrosine-based activating motif (18). Engagement of such receptors initiates an activation signaling cascade analogous to T or B cell receptor signaling.Mouse NK cells express the Ly49 family of receptors, which includes Ͼ10 members (9 -12, 19). Several lines of evidence demonstrate that Ly49A, a primary member of Ly49 family, is an inhibitory receptor that specifically recognizes a conformational epitope on H- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Distinctive Interactions at Multiple Site 2 Subsites by Allele-Specific Rat and Mouse Ly49 Determine Functional Binding and Class I MHC Specificity

“…The unique orientation of Ly49C on H-2K b seemed to be due to a different mode of dimerization for Ly49C compared with Ly49A. In the Ly49C cocrystal, the Ly49C dimer was more open, allowing one Ly49C receptor to symmetrically bind two H-2K b molecules simultaneously, whereas in the Ly49A cocrystal, Ly49A dimerization was more closed, resulting in an asymmetrical binding orientation on a single H-2D d molecule (25,26). Ly49A has since been seen to assume a similar open dimerization state, according to nuclear magnetic resonance solution studies, capable of binding two H-2D d molecules simultaneously (44), making variable Ly49 dimerization states unlikely mediators of allele specificity.…”

“…Through mutagenesis studies and examination of KIR/MHC I cocrystal structures, it has been demonstrated that KIR bind MHC I on the top surfaces of the ␣1 and ␣2 helices of MHC I, and this principally involves interaction of polymorphic KIR residues with HLA-C residues 77 and 80 (21,22). The molecular interaction between mouse Ly49 and MHC I has also been studied by mutagenesis (23)(24)(25)(26)(27)(28)(29) and examination of cocrystal structures of Ly49A/H-2D d and Ly49C/H-2K b (30,31), and occurs primarily at a large interface known as site 2 that encompasses MHC I residues below the peptide-binding groove, within the ␣3 domain, and on the ␤ 2 -microglobulin (␤ 2 m). What has remained poorly understood is the mechanism by which this site 2 interface, which is highly conserved both within and between rat and mouse MHC I alleles, can engender the observed allele specificity of Ly49 receptors.…”

Natural killer cell licensing in mice with inducible expression of MHC class I

“…Crystallographic studies revealed two potential interaction sites on MHC class I at which interactions with Ly49 receptors might take place: Site 1 is on the left side of the peptide-binding cleft (when viewed from above with the α1 helix shown on top); site 2 is below the peptide-binding cleft (14). Mutational studies demonstrated that the Ly49 receptors engage site 2 in trans to inhibit effector functions, because point mutations at site 2 abrogated the capacity of transfected MHC class I molecules to inhibit NK cells in an Ly49-dependent manner (15)(16)(17). Furthermore, we recently showed that these same residues are involved in conferring Ly49-dependent licensing effects because transgenic (Tg) expression of site 2-mutant MHC class I molecules failed to induce licensed NK cells, whereas wild-type MHC class I alleles allowed licensing (18).…”

NK Cell Inhibitory Receptor Ly-49C Residues Involved in MHC Class I Binding

“…Three of the peptides used in this context (Refs. 25 d reduce binding to Ly-49A (57), whereas single mutations of residues 104 and 107 did not affect the interaction with Ly-49A (33,58). The Ly-49C mutation K225N/ I226T that showed reduced MHC class I binding is close to this loop when Ly-49C is modeled onto the Ly-49A-H-2D d co-crystal.…”