1TK TYROSINE KINASE--NORMAL AND PATHOLOGIC FUNTION

受体类蛋白酪氨酸激酶(PTKs)是细胞表面蛋白 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，其中一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 对肿瘤、细胞生长因子和分子生物学的认识 细胞内信号的动力学。我最近克隆并测序了 并鉴定了一种新的人类ltk基因的全长cdna。 受体PTK基因，编码3.1kB的mRNA和100kD的蛋白 证明了酪氨酸激酶的活性。初步证据表明 Ltk的表达仅限于造血峰和神经峰。 来源的细胞，表明其活性是相对组织特异性的。 然而，其生理功能、可能的配体和致癌作用 潜力都是未知的。这项提案的主要目的是界定 Ltk基因产物的正常和病理功能，以及 重点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。具体来说， 为了实现这一目标，提出了四组实验。第一, Ltk基因和蛋白的表征将完成， 关注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。在第二组中 在实验中，提出了两种方法来识别组织(S) Ltk基因在哪里起生理作用：表达调查和基因 “击倒”实验。Ltk的时间和空间表达 小鼠胚胎及其在成年组织中的表达将由以下因素决定 原位杂交、核糖核酸酶保护分析和免疫荧光。 为了确定组织(S)中ltk的功能，靶向的基因干扰 将在小鼠胚胎干细胞中进行ltk基因，以 产生在ltk基因上纯合子干扰的小鼠。是这样的 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 通过确定各种LTK衍生品是否图案化进行调查 在其他PTK的等位基因转化后，都能够转化 成纤维细胞或淋巴样细胞，并通过筛查人类肿瘤 活跃性变化的存在。最终目标是确定 Ltk在人类恶性肿瘤中的作用。最后提出了两个策略。 用于分离ltk配体。一个人利用了一些人的能力 受体-配体配对通过自分泌环路机制转化细胞， 另一项涉及筛选已知的生长因子，以了解它们的能力 诱导ltk受体自身磷酸化作为一种间接检测 受体结合。Ltk配体的鉴定将提供 对增长控制机制的其他见解，并可能确定 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.