HUMAN LEUKOCYTE RECEPTORS FOR CHEMOTACTIC PEPTIDES

我们的目标是研究两者的结构-功能关系 趋化肽C5a和两种主要的人类白细胞受体 C5a受体和甲酰肽受体。这项合作研究 细胞生物学家和分子生物学家之间的关系将由体外进行 克隆DNA序列的诱变及后续表达 原核细胞和真核细胞。人和大鼠的C5a多肽将是 在大肠杆菌中产生、结晶和它们的三维结构 用X射线衍射法测定。C5a突变多肽将由 寡核苷酸诱导的突变，包括C3a/C5a杂交肽，在 以确定C5a的受体结合部位。的绑定属性 野生型和突变型多肽将被测量，并在 液体将通过高分辨率核磁共振进行研究。 将从外周血中性粒细胞mRNA和 佛波酯刺激U937细胞的mRNA及其筛选 从两者的氨基酸序列衍生的寡核苷酸探针 感受器。将构建表达cDNA文库，并用 抗受体血清。同时，将制备消减的cdna探针。 从转染人DNA的小鼠L细胞系中，表达这些 感受器。这些探针将用于筛选cDNA文库。这个 这两种受体的氨基酸序列都将从cDNA中推导出来。 将分离基因组甲酰肽受体克隆和外显子/内含子 基因的结构将被确定，以便预测位置 多肽中的功能结构域。该基因的5‘侧翼序列 包括转录起始区的序列将被确定。适切 将对克隆的cdna的细胞系统进行检测， 包括小鼠L细胞、同系猴细胞和青蛙卵母细胞。已准备好的mRNA 在体外，通过用SP6聚合酶复制cDNA将用于 显微注射入青蛙卵母细胞。配基结合域的分析 将通过删除扫描程序和通过 突变受体基因在细胞表达系统中的表达

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.