BRAIN SPECIFIC PROTEINS AND NERVE FUNCTION

大脑特异性蛋白质和神经功能

• 批准号: 2883621
• 负责人: THOMAS C VANAMAN
• 金额: $ 23.47万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2883621
• 关键词: biological signal transduction; calcium; calcium flux; calcium transporting ATPase; calmodulin; cell adhesion; cell growth regulation; cell membrane; enzyme linked immunosorbent assay; enzyme mechanism; human tissue; integrins; isozymes; laboratory rat; neurogenesis; neurotrophic factors; phosphorylation; posttranslational modifications; protein structure function; protein tyrosine kinase; recombinant DNA; tissue /cell culture; transfection; yeast two hybrid system

DESCRIPTION (Adapted from applicant's abstract): The plasma membrane Ca2+-ATPases (PMCA) play a primary role in cellular regulation owing to their ability to remove Ca2+ with high affinity. PMCAs are composed of a large family of closely related isoforms derived by differential splicing of primary transcripts of at least four distinct genes in mammals. The structural differences thus far identified between isoforms occur only in regions involved in regulation of activity by calmodulin, phospholipids and protein kinases. Their expression is tightly regulated in intact animal cells both at the level of the gene and RNA processing. That artificial inhibition of PMCA1 expression by antisense vector transfection inhibits the ability of PC-6 cells to produce normal neuritic processes in response to NGF was recently shown. Loss of PMCA1 is accompanied by loss of alpha1-integrin expression and concomitant loss of adherence properties as well as substantial decrease in ionomycin-mediated calcium fluxes and increase in glucocorticoid (dexamethasone) dependent reporter gene expression. Polypeptides migrating with the same relative molecular weight as PMCAs on SDS-PAGE are heavily tyrosine phosphorylated in wt and sense transfected PC-6 cells, but are absent in the antisense transfectants. Tyrosine phosphorylation of PMCA1/4 has been shown both in in vitro studies with purified components and in human platelets in response to physiological stimulation. These results strongly that the plasma membrane Ca2+-ATPase is regulated by tyrosine phosphorylation in some settings, and that it may play a direct role in signal transduction involving tyrosine kinases. A combination of biochemical, immunological, pharmacological and recombinant DNA approaches will be used to elucidate the nature, generality and exact function of this apparent tyrosine phosphorylation in regulating plasma membrane calcium pump activity both in vitro and in vivo. Studies with stably transfected Rat1 cells expressing pp60src species, as well as, wt and defective focal adhesion kinase will examine the potential role of this pathway in mediating regulation of PMCA through tyrosine phosphorylation. Yeast two hybrid selection procedures will be used to identify additional PMCA directed tyrosine kinases. The resulting tyrosine kinase(s) and corresponding purified PMCA isoforms, prepared by a combination of classical and recombinant DNA approaches, will be used in detailed analyses of PMCA regulation through tyrosine phosphorylation and its role in cell signaling pathways.

描述（改编自申请人摘要）：质膜 Ca 2 +-ATP酶（PMCA）在细胞调节中起主要作用， 它们以高亲和力去除Ca 2+的能力。 PMCA由一个 一个大家族，通过不同的剪接产生密切相关的同种型， 哺乳动物中至少四种不同基因的初级转录本。 的 到目前为止，异构体之间的结构差异仅发生在 参与钙调蛋白、磷脂和 蛋白激酶 它们的表达在完整的动物中受到严格的调控 细胞在基因和RNA加工水平上。 人工 通过反义载体转染抑制PMCA 1表达抑制了PMCA 1的表达。 PC-6细胞产生正常神经炎过程的能力， NGF最近被证实。 PMCA 1的缺失伴随着 α 1-整联蛋白表达和伴随的粘附特性的丧失， 以及离子霉素介导的钙通量的显著降低， 糖皮质激素（地塞米松）依赖性报告基因增加 表情 相对分子质量相同的多肽迁移 因为SDS-PAGE上的PMCA在wt和正义中严重酪氨酸磷酸化， 转染的PC-6细胞，但在反义转染子中不存在。 在体外研究中，PMCA 1/4的酪氨酸磷酸化 与纯化的成分，并在人血小板中响应生理 刺激. 这些结果有力地证明了质膜Ca ~（2+）-ATP酶是一种 在某些情况下受酪氨酸磷酸化调节， 在涉及酪氨酸激酶的信号转导中的直接作用。 一 生物化学、免疫学、药理学和重组的组合 DNA方法将被用来阐明的性质，一般性和准确性， 这种明显的酪氨酸磷酸化在调节血浆 膜钙泵活性在体外和体内。 外贸 稳定转染的Rat 1细胞表达pp 60 SRC种类以及wt和 有缺陷的粘着斑激酶将研究这种蛋白的潜在作用。 在通过酪氨酸磷酸化介导PMCA的调节途径。 酵母双杂交选择程序将用于确定额外的 PMCA指导的酪氨酸激酶。 得到的酪氨酸激酶和 相应的纯化的PMCA同种型，通过经典的 和重组DNA方法，将用于PMCA的详细分析 酪氨酸磷酸化调控及其在细胞信号转导中的作用 途径。