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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA069299-03
Application #
2733199
Study Section
Experimental Immunology Study Section (EI)
Program Officer
Finerty, John F
Project Start
1996-07-01
Project End
2000-06-30
Budget Start
1998-09-04
Budget End
2000-06-30
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Northern California Institute Research & Education
Department
Type
DUNS #
City
San Francisco
State
CA
Country
United States
Zip Code
94121
Nakamura, M C; Seaman, W E (2001) Ligand interactions by activating and inhibitory Ly-49 receptors. Immunol Rev 181:138-48
Daws, M R; Lanier, L L; Seaman, W E et al. (2001) Cloning and characterization of a novel mouse myeloid DAP12-associated receptor family. Eur J Immunol 31:783-91
Nakamura, M C; Hayashi, S; Niemi, E C et al. (2000) Activating Ly-49D and inhibitory Ly-49A natural killer cell receptors demonstrate distinct requirements for interaction with H2-D(d). J Exp Med 192:447-54
Seaman, W E (2000) Natural killer cells and natural killer T cells. Arthritis Rheum 43:1204-17
Nakamura, M C; Linnemeyer, P A; Niemi, E C et al. (1999) Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity. J Exp Med 189:493-500
Nakamura, M C; Naper, C; Niemi, E C et al. (1999) Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor. J Immunol 163:4694-700
Sundback, J; Nakamura, M C; Waldenstrom, M et al. (1998) The alpha2 domain of H-2Dd restricts the allelic specificity of the murine NK cell inhibitory receptor Ly-49A. J Immunol 160:5971-8
Nakamura, M C; Niemi, E C; Fisher, M J et al. (1997) Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. J Exp Med 185:673-84
Ryan, J C; Seaman, W E (1997) Divergent functions of lectin-like receptors on NK cells. Immunol Rev 155:79-89