The proposed research will describe the structure, expression and function of a set of related murine proteins whose common feature is the binding of fragments of C3. These proteins, known as the complement receptors, have been implicated in a number of roles of the immune response including the control of the activation of the complement cascade enhancing the mammalian humoral response including the control of the activation of the complement cascade enhancing the mammalian humoral response and priming the proliferation of subsets of cells within the immune response. The focus of this application is to continue the analysis of the genes and gene products of the murine complement receptors and to extend this investigation to include their roles in the biology of the cells which express them. Specifically, this will include the tissue specific expression of the Crry gene (which is the murine homologue to human CR1) and the analysis of the two variant proteins produced form Crry transcripts. Using transfection analyses, the domains of the Crry protein important in this presumed roles of the control of complement activation and phagocytosis of immune complexes will localized and scrutinized via mutations introduced into the normal gene. In addition, a genomic sequence which contains sequences homologous to the Crry gene will be isolated and examined for coding sequences similar to those of Crry. our analysis of the murine CR will also include the Cr2 gene which appears to produce a wider variety of gene products than the human homologue, CR2. The mechanisms of providing for this diversity (tissue specific transcription and alternative splicing) will be analyzed as will the functions of the gene products via transfection analyses. The role of these proteins in the biology of the animal will be further investigated by creating mutant strains of mice which no longer express the Crry or Cr2 genes into the Crry or Cr2 genes of an ES cell, which is capable of developing into a mouse. Any phenotypic change which can be detected in these mice can be used to screen the human population to look for similar defects in the expression of the human complement receptors. GRANT=R01AI23483 Many of the hematopoietic cells that participate in immune and inflammatory responses [including mast cells, basophils, natural killer cells, cytotoxic T lymphocytes, eosinophils, neutrophils, macrophages, and platelets] store a unique family of proteoglycans in an intracellular secretory granule compartment. Because these proteoglycans are present in such a wide range of effector cells of the immune response and because the gene that encodes the peptide core of these proteoglycans is expressed very early in the differentiation of hematopoietic cells, it is now clear that the regulation of this particular gene is extremely important for the development and function of these hematopoietic cells. The objectives of the research proposed in this application are to determine the primary sequence of the secretory granule proteoglycan peptide core gene in two evolutionarily distant species, and then to understand how this gene is regulated in hematopoietic cells. Genomic fragments of ~18 kilobases in size have been isolated that contain the gene that encodes the peptide core of this novel family of proteoglycans. Double stranded nucleotide sequencing using chemically synthesized oligonucleotide primers will be used to determine the complete nucleotide sequence of the human and mouse gene. Data have already been obtained that the nucleotide sequences that encode the N- terminus of the peptide core are highly conserved through evolution, as are specific nucleotide sequences within the 5' and 3' untranslated regions of their mRNA transcripts. Likewise, when the 5' flanking region of the human gene was compared to the corresponding 5' flanking region of the analogous mouse gene, a region that immediately precedes the transcription-initiation site was found to be nearly identical. The observation that this latter sequence is even more highly conserved than any corresponding region of the gene that is translated into protein implies that this 5' flanking region contains cis acting regulatory elements that are critical for expression of this gene in hematopoietic cells. Hematopoietic cells and non- hematopoietic cells will be transfected with chimeric constructs to elucidate the regions of the gene which are important for its transcriptional regulation. Antibodies will be raised that recognize the peptide core of the mouse proteoglycan to study the translation and glycosaminoglycan modification of this proteoglycan in transfected cells.
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