This project involves the study of a rapidly emerging group of immune receptors. Many families of inhibitory immune receptors have recently been identified in both mice and humans. Interestingly, within each of these inhibitory families of receptors, there are proteins that have lost the inhibitory domains. Instead these receptors have gained a positively charged acid within their transmembrane domain, suggesting that they may interact with signal transduction chains and transmit positive signals. In this project, we study the signal transduction and biochemistry of both the positive and negative regulators of immune cell function. Through the study of the positive receptors, we and others demonstrated the association of some of these receptors with the novel signal transduction chain DAP12. Since then we have been characterizing the biochemistry of the the DAP12 signal transduction pathway. This work has included demonstration of the kinases involved in the early signaling of DAP12, delineation of the adaptors involved, and study of the regulation of these pathways. To facilitate these studies, we have developed and are utilizing, a reconstitution system that involves transfection of the receptor, DAP12, a kinase, and a reporter plasmid. This system allows for the analysis of various components of the pathway via co-transfection. In addition, biochemical analysis of the pathway has defined several substrates of DAP12-activated kinases. The exact roles of these substrates in mediating specific DAP12-driven responses is under investigation. In addition, to DAP12, we are beginning the study of DAP10, a second chain known to associate with receptors within NK cells and monocytes that is located just 130 bp from DAP12 on Chromosome 19. We are pursuing studies of the promotors of DAP12 and DAP10 and plan to tag the proteins in mice. These studies should help us to fully understand not only the genetic components that control the cell type specific expression of these chains within NK cells, monocytes and dendritic cells, but any role that their subcellular localization may play in DAP12 and DAP10 biology as well. Our studies of the negative receptors are centered on the aggressive study of two members of the sialic acid binding lectin (Siglec) family of adhesion molecules, CD33 and Siglec 7. CD33 is expressed only on myeloid cells and leukemias of myeloid origin. We have recently demonstrated the ability of CD33 to function as an inhibitory receptor in monocytes through the recruitment of the SHP-1 and SHP-2 protein tyrosine phosphatases via its various cytoplasmic tyrosine motifs. Siglec 7 is an inhibitory receptor expressed on monocytes, macrophages, dendritic cells, and NK cells. We have identified a murine cDNA that apparently encodes the mouse homologue of Siglec 7. We have cloned the gene, characterized its ability to interact with phosphatases, and determined its genomic structure and mRNA splicing. We are currently preparing constructs to target this gene via homologous recombination in mice. These mice, together with our continued study of the biochemistry of CD33 and Siglec 7, should shed light on the biology and immunology of this rapidly growing family of inhibitory receptors.
