MAJOR HISTOCOMPATIBILITY ANTIGENS & MEMBRANE RECEPTOR

我们的目标是显示MHC抗原影响配体的程度。 结合肽激素受体，并反过来被修饰为 配体与特定受体结合的结果。 我们已经证明， HLA（基因型和表型）与 B淋巴母细胞的胰岛素受体。 我们现在将进一步分析 HLA与胰岛素受体亲和力和延伸的遗传关联 分析了抗原与抗原之间的物理关系， 受体分子 基因分析将通过检查 突变的淋巴母细胞，缺失了一些HLA抗原的表达， 已经被缺失抗原的基因所转染 我们还将 试图拯救Daudi细胞中HLA抗原的表达，以检查 这种拯救对Daudi结合胰岛素的影响。 物理分析 将采取两种方法：1. 一种尝试，根据先例， 文献中，共沉淀胰岛素受体，通过其 内源性酪氨酸激酶活性，与I类MHC抗原，使用 特异性抗受体和抗I类单克隆抗体和2. 确定I类MHC抗原和胰岛素的接近度 受体在完整的细胞，使用共振能量转移之间 内源性（β-2-m）标记的I类抗原和胰岛素受体 我们已经证明它可以被生物素胰岛素特异性标记， 抗生物素蛋白 能量传递将在流动中测量 在供体猝灭敏化受体发射方面， 我们计划将我们的工作扩展到表皮生长的代谢效应 因子结合的野生型的展示和磷酸化状态， 突变I类MHC抗原，使用流式细胞术定量展示 以及标准免疫化学和生物化学方法来确定 抗原的磷酸化状态与细胞内 显示.

Our goal is to show the extent to which MHC antigens influence ligand binding by peptide hormone receptors and in turn are modified as a consequence of ligand binding to specific receptors. We have shown as association between HLA (genotype and phenotype) and the affinity of insulin receptors on B lymphoblasts. We will now further analyze the genetic associations between HLA and insulin receptor affinity and extend the analysis to the physical relationships between the antigens and receptor molecules. The genetic analysis will proceed through examination of mutant lymphoblasts, deleted for expression of some HLA antigens, which have been transfected with genes for the missing antigens. We will also attempt to rescue expression of HLA antigens in Daudi cells, to examine the effects of such rescue on insulin binding by Daudi. The physical analysis will take two approaches: 1. an attempt, based on precedents in the literature, to co-precipitate insulin receptors, detected by their endogenous tyrosine kinase activity, with class I MHC antigens, using specific anti-receptor and anti-class I monoclonal antibodies and 2. determination of the proximity of class I MHC antigens and insulin receptors in intact cells, using resonance energy transfer between endogenously (Beta-2-m) labeled class I antigens, and insulin receptors which we have shown can be specifically labeled with biotinyl insulin and avidin phycobilliproteins. Energy transfer will be measured in the flow cytometer in terms of donor quenching sensitized emission of acceptor. We plan to extend our work on the metabolic effects of epidermal growth factor binding on the display and phosphorylation state of wild-type and mutant class I MHC antigens, using flow cytometry to quantitate the display and standard immunochemical and biochemical approaches to determine the phosphorylation state of the antigens associated with changes in the display.