BRAIN SPECIFIC PROTEINS AND NERVE FUNCTION

描述(改编自申请人的摘要)：质膜 CA2-ATPase(PMCA)在细胞调节中起主要作用 它们以高亲和力清除钙离子的能力。PMCA由一个 由差异剪接衍生的密切相关的亚型大家族 哺乳动物中至少四个不同基因的初级转录本。这个 到目前为止发现的亚型之间的结构差异只出现在 参与钙调蛋白、磷脂和钙调素活性调节的区域 蛋白激酶。它们在完整动物体内的表达受到严格调控 细胞既在基因水平上，也在RNA处理水平上。那个人造的 反义载体对PMCA1表达的抑制作用 PC-6细胞产生正常神经突起的能力 NGF最近被展示出来。PMCA1的丢失是伴随着丢失的 α1整合素的表达和伴随的黏附特性的丧失 以及离子霉素介导的钙离子通量的大幅下降和 糖皮质激素(地塞米松)依赖报告基因的增加 表情。相对分子质量相同的多肽迁移 由于SDS-PAGE上的PMCA在重量和意义上都是高度酪氨酸磷酸化的 转染PC-6细胞，但在反义转染体中不存在。 PMCA1/4的酪氨酸磷酸化在两项体外研究中都被证明 使用纯化的组分，并在人血小板中响应生理学 刺激。这些结果有力地证明了质膜Ca~(2+)-ATPase 在某些情况下受酪氨酸磷酸化调节，并可能发挥作用 在涉及酪氨酸激酶的信号转导中起直接作用。一个 生化、免疫学、药理学和重组的结合 DNA方法将用于阐明其性质、概括性和准确性 这种明显的酪氨酸磷酸化在调节血浆中的作用 膜钙泵在体外和体内的活性。研究对象： 稳定表达pp60src物种的Rat1细胞以及wt和 有缺陷的粘着斑激酶将检测这一潜在的作用 酪氨酸磷酸化调节PMCA的途径。 酵母双杂交选择程序将用于鉴定其他 PMCA调控酪氨酸激酶。由此产生的酪氨酸激酶(S)和 相应的纯化PMCA亚型，由经典的 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 小路。

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.