Molekularbiologische und funktionelle Charakterisierung neuer "Chicken Ig-like" Rezeptoren
Some recently described receptor families on myeloid and lymphoid cells in man and mouse have both inhibitory and activating receptors with important functions in the regulation of the immune system. Due to the lack of information regarding these receptors in other vertebrates, in the present thesis seven new members of this receptor family were identified in the chicken. All receptors identified were located on one single clone of a chicken BAC library, which indicates that they are tightly clustered on the chromosome. This localisation and the high amino acid homology suggests, that they belong to a multigene-family. Analysis of the genomic organisation showed, that the signalpeptide was split into two exons, each Ig-domain and the transmembrane region were encoded by separate exons and the long cytoplasmic tail (in case of inhibitory receptors) was encoded by two exons, the last exon containing the ITIMs. The exon/intron boundaries conformed to the gt-ag rule and the exon phases were highly conserved. In conclusion, the genomic organisation showed conserved features of mammalian Ig-like receptor genes. The novel identified CHIR were grouped into three different types of receptors. The activating CHIR-A had two extracytoplasmic Ig-domains and a short cytoplasmic tail, but a positively charged amino acid in the transmembrane region, which could associate to an ITAM-containing adaptor molecule. CHIR-A2 had a relative molecular weight of ~ 42 kDa and was detected in the mRNA of bursa, spleen, PBL, NK-cells and different macrophage- and B-cell lines. The inhibitory CHIR-B consisted of two Ig-domains and a long cytoplasmic tail with two ITIMs in it. CHIR-B2 had a relative molecular weight of ~ 46 kDa and was only detectable on B-cells by a newly produced specific monoclonal antibody (mAb). Upon Pervanadate treatment the ITIMs in the cytoplasmic tail were phosphorylated and recruited the protein-tyrosine- phosphatases SHP-1 and -2. Additionally ligation of CHIR-B2 had an inhibitory effect on the cell proliferation of a B-cell line. The potentially inhibitory and activating CHIR-AB had one Ig-domain, a long cytoplasmic tail with one ITIM in it and a positively charged amino acid in the trans-membrane region, which could associate with an ITAM-containing adaptor molecule. The only CHIR-AB homologue in the literature is KIR2DL4, that represents a highly conserved Ig-like Receptor expressed on all primate NK-cells with both activating and inhibitory function. CHIR-AB1 had a molecular weight of ~ 40 kDa and its expression on B-cells, on the B-cell line DT-40, on primary macrophages, different macrophage cell lines and on NK-cells in the gut was detected by a specific mAb.
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