CHEMISTRY OF TUMORICIDAL MACROPHAGE SURFACE ANTIGENS

我们将描述配体特异性，配体结合位点， 调节对配体的亲和力，以及 白细胞粘附受体Mac-1和p150，95. Mac-L是两者都是 补体受体（CR 3）和细胞粘附受体; p150，95 是一种在骨髓细胞上表达的密切相关的粘附受体 而且大量存在于毛状白血病细胞上。 遗传缺陷 这些受体导致反复的细菌感染， 往往在儿童时期就致命。 这些受体的α亚单位 控制配体特异性，我们假设串联重复序列 含有假定的二价阳离子结合位点的配体 结合位点。 为了确定配体结合位点，我们将切割和 粘贴α亚基cDNA克隆以制备杂交Mac-1 × LFA-1， p150，95 x LFA-1 α亚基，其将使用 逆转录病毒载体与共同的β亚基上 T淋巴瘤细胞的表面。 单克隆抗体表位将被定位 并与MAb对功能的影响相关。 与iC 3b的结合 溶液或E上;以及与新的细胞配体结合， 溶液、人工平面膜和完整细胞上的细胞， 定义对不同配体特异性的结合位点， 这些位点对亲和力的贡献。 同样表示 截短的α亚基将被用来定义的重要性 跨膜和胞质结构域在调节 配体和细胞骨架结合。 我们假设细胞 动态调节粘附受体对配体的亲和力。 这 将检测中性粒细胞上的天然分子， T淋巴瘤表达的重组杂合和截短分子 通过单体iC 3b和细胞表面的Scatchard结合的细胞 配体，用fMLP或佛波醇酯刺激后。 将通过清洁剂平行测量细胞骨架结合 增溶作用 在上述研究的同时， 在中性粒细胞聚集中被Mac-l识别的分子， 对内皮细胞的粘附将用MAb定义。 配体 分子将被表征，并在 蛋白质和cDNA水平。 识别序列，假设 将被定义为RGD。 纯化天然和重组 分泌的分子将用于推配体结合 问题研究 fMLP可调控的Mac的细胞内储存位点， 还将对嗜中性粒细胞中Mae-1和鼠Mae-1 cDNA克隆进行 表征了

We will characterize the ligand specificity, ligand binding sites, regulation of affinity for ligand, and cytoskeletal association of the leukocyte adhesion receptors Mac-l and p150,95. Mac-l is both a complement receptor (CR3) and a cell adhesion receptor; p150,95 is a closely related adhesion receptor expressed on myeloid cells and abundantly on hairy leukemia cells. Genetic deficiency of these receptors results in recurring bacterial infections which are often fatal in childhood. The alpha subunits of these receptors control ligand specificity, and we hypothesize that tandem repeats which contain putative divalent cation binding sites are the ligand binding sites. To define ligand binding sites, we will cut and paste alpha subunit cDNA clones to prepare hybrid Mac-l x LFA-l and p150,95 x LFA-l alpha subunits which will be expressed using retroviral vectors in association with the common beta subunit on the surface of T lymphoma cells. MAb epitopes will be localized and correlated with MAb effect on function. Binding to iC3b in solution or on E; and binding to novel cellular ligands in solution, artificial planar membranes, and on intact cells will define the binding sites specific for different ligands, and the contribution of these sites to affinity. Similarly expressed truncated alpha subunits will be used to define the importance of transmembrane and cytoplasmic domains in regulating affinity for ligand and cytoskeletal association. We hypothesize that cells dynamically regulate adhesion receptor affinity for ligand. This will be tested for native molecules on neutrophils and for recombinant hybrid and truncated molecules expressed on T lymphoma cells by Scatchard binding of monomeric iC3b and cell surface ligands, after stimulation with fMLP or phorbol esters. Cytoskeletal association will be measured in parallel by detergent solubilization. In parallel with the above studies, novel ligand molecules recognized by Mac-l in neutrophil aggregation and adhesion to endothelial cells will be defined with MAb. Ligand molecules will be characterized and primary sequence defined at the protein and cDNA levels. Recognition sequences, hypothesized to be RGD-like, will be defined. Purified natural and recombinant secreted molecules will be used in the shove ligand binding studies. The intracellular storage site of fMLP-regulatable Mac- l in neutrophils and s murine Mae-l cDNA clone will also be characterized.