BRAIN SPECIFIC PROTEINS AND NERVE FUNCTION

大脑特异性蛋白质和神经功能

• 批准号: 2037173
• 负责人: THOMAS C VANAMAN
• 金额: $ 22.48万
• 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/2037173
• 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（改编自申请人的摘要）：质膜 Ca2+-ATPases（PMCA）在细胞调节中起主要作用 他们具有高亲和力去除CA2+的能力。 PMCA由 通过差剪接得出的密切相关同工型的大型家族 哺乳动物中至少四个不同基因的主要转录本。 这 到目前为止，同工型之间发现的结构差异仅发生在 通过钙调蛋白，磷脂和 蛋白激酶。 它们的表达在完整的动物中受到严格调节 细胞在基因和RNA加工水平上。 那个人造 通过反义载体转染抑制PMCA1表达抑制 PC-6细胞产生正常神经过程的能力。 最近显示了NGF。 PMCA1的损失伴随着损失 α1-整合蛋白的表达和依从性丧失作为 以及离子霉素介导的钙通量的大幅下降和 糖皮质激素（地塞米松）依赖性记者基因的增加 表达。 多肽以相同的相对分子量迁移 由于SDS-PAGE上的PMCA在WT和Sense中大量酪氨酸磷酸化 转染的PC-6细胞，但在反义转染剂中不存在。 PMCA1/4的酪氨酸磷酸化已在体外研究中显示 纯化的成分和人类血小板响应生理 刺激。 这些结果强烈认为质膜Ca2+-ATPase是 在某些情况下由酪氨酸磷酸化调节，并且可能会发挥作用 在涉及酪氨酸激酶的信号转导中的直接作用。 一个 生化，免疫学，药理和重组的组合 DNA方法将用于阐明性质，一般性和精确性 这种明显的酪氨酸磷酸化在调节血浆中的功能 体外和体内膜钙泵活性均可 研究 表达PP60SRC物种以及WT和WT和WT的稳定转染的Rat1细胞 有缺陷的局灶性粘附激酶将检查此的潜在作用 通过酪氨酸磷酸化介导PMCA调节的途径。 酵母将使用两个混合选择程序来识别其他 PMCA指挥酪氨酸激酶。 由此产生的酪氨酸激酶（S）和 相应的纯化PMCA同工型，通过经典的组合制备 和重组DNA方法将用于PMCA的详细分析 通过酪氨酸磷酸化调节及其在细胞信号中的作用 途径。