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 [1]. 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 [2]. However,
although morphological studies revealed movement of plasma membrane rafts into
the area of contacts between NK cells and their sensitive targets [3], 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 [4]. 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 [5]. 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) [6].
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.
[1] L.L. Lanier, Annu.Rev.Immunol., 16 (1998) 359-396. [2]
K. Simons & D. Toomre, Nat.Rev.Mol.Cell.Biol.,
1 (2000) 31-41. [3] M.S.Fassett, D.M. Davis, M.M. Valter, G.B. Cohen, J.L.
Strominger, Proc.Natl.Acad.Sci.USA 98
(2001) 14547-52. [4] K. Bezouška, J.Biotechnol,
90 (2002) 269-290. [5] O. Plíhal, J. Sklenář, et al., unpublished. [6] P.
Man, P.
Novák, M. Cebecauer, A. Fišerová, K. Bezouška, manuscript in preparation.