1TK TYROSINE KINASE--NORMAL AND PATHOLOGIC FUNTION

受体型蛋白酪氨酸激酶（PTKs）是细胞表面 结合生长因子配体并启动多效性的分子 细胞内信号级联。 此外，大多数受体PTK是 原癌基因，有些与人类恶性肿瘤有关。 因此，对受体酪氨酸激酶的研究增加了我们的研究。 了解肿瘤，细胞生长因子和分子 细胞内信号传导的动力学。 我最近克隆、测序了 并鉴定了人ltk基因的全长cDNA， 受体PTK基因，其编码3.1 kB mRNA和100 kD蛋白， 显示酪氨酸激酶活性。 初步证据显示 LTK表达仅限于造血和神经嵴 衍生的细胞，表明其活性是相对组织特异性的。 然而，它的生理功能，假定的配体和致癌性， 潜力都是未知的。 这项建议的主要目的是界定 ltk基因产物的正常和病理功能， 重点是研究其作为细胞受体的可能作用， 生长因子及其作为原癌基因的可能作用。 具体地说， 提出了四组实验来实现这一目标。 第一、 将完成LTK基因和蛋白质的表征， 集中于LTK蛋白的体内生物合成和克隆 基因组序列和上游启动子区域的序列。 在第二盘 实验中，提出了两种方法来识别组织（S） ltk基因在生理上起作用的位置：表达调查和基因 “淘汰”实验 ltk的时空表达 小鼠胚胎及其在成年组织中的表达将通过 原位杂交、RNA酶保护分析和免疫荧光。 为了鉴定ltk发挥功能的组织， 将进行小鼠胚胎干细胞中的ltk基因，以 产生在LTK基因座处同源破坏的小鼠。 等 突变小鼠有望提供一种强大的LTK遗传模型 功能 在第三个目标中，ltk基因的致癌潜力将 通过确定是否有各种LTK衍生物， 在转化其他PTK的等位基因后， 成纤维细胞或淋巴样细胞，并通过筛选人肿瘤中的 存在激活性改变。 最终目标是确定 LTK在人类恶性肿瘤中的作用。最后提出了两种策略 用于分离LTK配体。 一个人利用一些人的能力 受体-配体对通过自分泌环机制转化细胞， 另一个是筛选已知的生长因子， 诱导ltk受体自身磷酸化作为间接测定 受体结合 ltk配体的鉴定将提供 对生长控制机制的进一步了解， 一种具有治疗价值的新型生长因子。

The receptor-type protein tyrosine kinases (PTKs) are cell surface molecules which bind growth factor ligands, and initiate pleotropic intracellular signaling cascades. In addition, most receptor PTKs are protooncogenes, and some have been implicated in human malignancies. Thus, the study of receptor tyrosine kinases has increased our understanding of neoplasia, cellular growth factors and the molecular dynamics of intracellular signaling. I have recently cloned, sequenced and characterized the full length cDNA of the human ltk gene, a new receptor PTK gene which encodes a 3.1 kB mRNA and a 100 kD protein with demonstrated tyrosine kinase activity. Preliminary evidence indicates that ltk expression is restricted to hematopoietic and neural crest derived cells, suggesting its activity is relatively tissue specific. However, its physiological function, putative ligand and oncogenic potential are all unknown. The broad aim of this proposal is to define the normal and pathologic function of the ltk gene product, with an emphasis on investigating its probable role as a receptor for a cellular growth factor and its possible role as a protooncogene. Specifically, four sets of experiments are proposed to achieve this objective. First, the characterization of the ltk gene and protein will be completed, focusing on the in vivo biosynthesis of the ltk protein and the cloning of the genomic sequences and upstream promoter region. In the second set of experiments, two approaches are proposed to identify the tissue(s) where the ltk gene acts physiologically: an expression survey and gene "knock out" experiments. The temporal and spatial expression of ltk in mouse embryos and its expression in adult tissues will be determined by in situ hybridization, RNAase protection analysis and immunofluorescence. To identify tissue(s) where ltk functions, targeted gene disruption of the ltk gene in mouse embryonic stem cells will be carried out, to produce mice which are homozygously disrupted at the ltk locus. Such mutant mice are expected to provide a powerful genetic model of ltk function. In the third aim, the oncogenic potential of the ltk gene will be investigated by determining whether various ltk derivatives, patterned after the transforming alleles of other PTKs, are able to transform fibroblasts or lymphoid cells, and by screening human tumors for the presence of activating alterations. The ultimate goal is to determine the role of ltk in human malignancy. Finally, two strategies are proposed for isolating the ltk ligand. One exploits the ability of some receptor-ligand pairs to transform cells via an autocrine loop mechanism, and the other involves screening known growth factors for their ability to induce ltk receptor autophosphorylation as an indirect assay of receptor binding. Identification of the ltk ligand would provide additional insights into the mechanism of growth control and may identify a novel growth factor with therapeutic value.