1Institute of Microbiology, 3Institute of Molecular Genetics, 4Institute of Physiology, Academy of Sciences of Czech Republic, 14220 Praha 4, 2Department of Biochemistry, Faculty of Science, Charles University Prague, 12840 Praha 2.
Natural killer (NK) cells are cytotoxic effector lymphocytes which do not rearrange or express antigen-specific surface receptors . Although many NK cell receptors that transduce signals leading to activation or inhibition of natural killing have been identified, the details of their membrane topology and physiology remain poorly understood. In T-lymphocytes, many components of the activation receptor complexes are concentrated in glycolipid-enriched membrane rafts, which play important role in the membrane activation synapse . However, although morphological studies revealed movement of plasma membrane rafts into the area of contacts between NK cells and their sensitive targets , no detailed characterization of the membrane receptor complexes has been provided in NK cells.
We have used fresh sorted rat NK cells, and variants of the rat NK leukemic cell line RNK-16 as model systems to study the membrane environment of NKR-P1 receptor, a key activation antigen of rat NK cells . Studies with the bacterially expressed monomeric NKR-P1 receptor isoforms revealed important differences in the recognition of simple and complex carbohydrate ligands. The ability of carbohydrate dendrimers to precipitate recombinant dimeric forms of NKR-P1 receptors opened the way for studies of the native forms of these receptors isolated from their membrane microdomains. Carbohydrate dendrimers interact specifically with NK cells mainly through NKR-P1A isoform. They proved to be specific activators of NK cells mediating the increase in intracellular calcium, generation of inositol phosphates, and natural killing . Blue native electrophoresis revealed the native form of NKR-P1A receptor as a part of large 220 kDa protein complex. Moreover, a shotgun strategy based on microcapillary HPLC in combination with tandem mass spectrometry allowed us to study the complete protein profile of the NKR-P1+ membrane microdomain. We identified large sets of proteins associated with these microdomains but their detailed composition depended on the chemical environment used during the isolation. These proteins included additional NK cell receptors (CD2, CD18, CD44, CD45, membrane adaptors and signaling enzymes (LAT, N-TAL, lck, ras, rab), effector enzyme complexes (ATP synthase, VDAC proteins, annexins) as well as the proteins of the cytoskeleton scaffold (actin, clathrin, cofillin, ezrin, flotillin) .
Functional and structural characteristics of the individual protein-lipid complexes defining the molecular architecture of the entire microdomain are currently under active investigation in our laboratory.
We thank to Marek Cebecauer from the Ludwig Institute for Cancer Research, Epalinges, Switzerland, for stimulating discussions. Supported by Ministry of Education (MSM 113100001), by the Institutional Research Concept AVOZ5020903, and by Volkswagen Foundation.
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