The goal is to study the structure-function relationship for both the chemotactic peptide C5a and two major human leukocyte receptors: the C5a-receptor and the formyl peptide receptor. This collaborative study between cellular and molecular biologists will be performed by in vitro mutagenesis and subsequent expression of cloned DNA sequences in procaryotic and eucaryotic cells. The human and rat C5a peptides will be produced in E. coli, crystallized and their three dimensional structure determined by X-ray diffraction. C5a mutant peptides will be created by oligonucleotide directed mutagenesis, including C3a/C5a hybrid peptides, in order to define the receptor binding sites of C5a. Binding properties of wild-type and mutated peptides will be measured and their conformation in liquid will be studied by high resolution nuclear magnetic resonance. cDNA libraries will be prepared from peripheral blood neutrophil mRNA and mRNA from phorbol-ester stimulated U937 cells and screened with oligonucleotide probes derived from amino acid sequences of both receptors. Expression cDNA libraries will be constructed and screened with anti-receptor sera. In parallel, subtracted cDNA probes will be prepared from mouse L-cell lines transfected with human DNA, which express these receptors. These probes will be used to screen the cDNA libraries. The amino acid sequences of both receptors will be derived from cDNA. Genomic formyl peptide receptor clones will be isolated and the exon/intron structure of the gene will be determined in order to predict the location of functional domains in the peptide. The 5' flanking sequence of the gene including the transcription start region will be determined. Suitable cellular system for transfection with cloned cDNA will be examined, including mouse L-cells, cos-monkey cells and frog oocytes. mRNA prepared in vitro by copying of cDNA with SP6 polymerase will be used for microinjection into frog oocytes. An analysis of the ligand binding domain of the receptor will be initiated by a deletion scanning procedure and by expression of mutated receptor cDNA in a cellular expression system.
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