The goal of this application is to define mechanisms of transmembrane signaling by Ly-49 receptors on mouse natural killer (NK) cells. Mouse Ly-49A (mLy49-A) binds to H-2Dd on target cells and prevents mLY-49A+ NK cells from lysing targets. To examine the pathways by which mLY-49A prevents natural killing, the applicants have developed a model in which mLY-49A is functionally expressed in the rat NK cell line, RNK-16. They have also defined 12-mer peptides, corresponding to residues 73-84 from the a1 helix of H-Dd, which initiate transmembrane signaling through mLy-49A. They have created a chimeric receptor, linking the extracellular domain of NK1.1 to the transmembrane and cytoplasmic domains of mLy-49A, and have used this model to demonstrate a role for the cytoplasmic domain of mLy-49A in transmembrane signals that inhibit NK cell cytotoxicity. Their objective now is to define the functional signals and the properties of mLy-49A that permit their activation. To this end, they have six specific aims: 1) Create truncation and site-specific mutations of mLY-49A expressed in RNK-16 to determine functional effects on cytotoxicity. 2) Compare the capacity of intact or mutated mLy-49A to generate transmembrane signals in response to a1 helix peptides and compare signaling to function (in Specific Aim 1). 3) As an additional and selective probe, express mLy-49A/NK1.1 chimeric receptors on RNK-16 cells. Correlate the generation of transmembrane signals with effect on NK cell activity. 4) Immunoprecipitate mLy-49A from stimulated or unstimulated RNK-16.mLy-49A cells and probe for associated molecules. 5) Use the yeast two hybrid system to clone, sequence, and express, cytoplasmic proteins from mouse IL-2-activated NK cells that bind to the cytoplasmic domain of mLY-49A. 6) Compare transmembrane signaling by Ly-49A with signaling by other members of the Ly-49 gene family.
