PHOSPHORYLATION AND DRUG TRANSPORT BY P-GLYCOPROTEIN

P-糖蛋白（Pgp）是一种ATP酶， 从细胞中提取抗癌药物。 Pgp的过度表达似乎是 化疗失败的一个常见原因， 癌症的形式。 Pgp也在正常组织中表达，包括 肾上腺皮质、肾脏近端肾小管和内皮 细胞，在那里它似乎参与分泌醛固酮， 药物和代谢物的转运。 似乎 蛋白激酶A（PKA）和蛋白激酶C（PKC）磷酸化Pgp 激活药物转运，并且已经表明PKC活性是 在几种多药耐药细胞系中升高。 显然，PKA 和PKC磷酸化只有3个丝氨酸（两个重叠）， 位于两个同源半体之间的接头区 Pgp（minilinker domain）。 这项建议的主要目的是 了解PKA和PKC介导的 Pgp的磷酸化影响有机底物的转运， 明确Pgp活性调节的基本机制， PKC活性的变化。为了实现这些目标，我们将研究 PKA和PKC的Pgp磷酸化对药物的影响 运输和结合，以及ATP结合和水解。 我们将 在完整细胞上进行“体内”研究，并使用完整的 长度Pgp和含有N-末端ATP-和 药物结合结构域和小连接体结构域。在完整的细胞中， 我们将通过测定丝氨酸的氨基酸序列来鉴定功能相关的丝氨酸。 磷酸化对表达野生型和突变体的细胞的影响 Pgps。 在突变体Pgps中，小连接体结构域的丝氨酸将被 被丙氨酸单独或组合取代。 最后我们 将使用Pgp突变体，该突变体不对磷酸化反应， PKC，以评估激酶在Pgp刺激中的作用， 对暴露于细胞毒性药物、雌二醇的反应， 抗雌激素他莫昔芬。 对生物化学的分子基础的理解 PKA和PKC对Pgp介导的药物转运的影响将是 这对我们了解Pgp的调控有重要贡献 功能 本提案中的实验还将提供 旨在调节Pgp功能和多药治疗的研究框架 低毒性试剂的抗性（例如，内源激素）。

P-glycoprotein (Pgp) is an ATPase thought to extrude actively anticancer drugs from the cells. Overexpression of Pgp seems to be a frequent cause of failure of chemotherapy in the treatment of some forms of cancer. Pgp is also expressed in normal tissues including adrenal cortex, proximal renal tubules of the kidney and endothelial cells, where it appears to be involved in secretion of aldosterone and transport of drugs and metabolites, respectively. It seems that phosphorylation of Pgp by protein kinases A (PKA) and C (PKC) activates drug transport, and it has been shown that PKC activity is elevated in several multidrug-resistant cell lines. Apparently, PKA and PKC phosphorylate only 3 serines each (two overlapping), which are located in the linker region between the two homologous halves of Pgp (minilinker domain). The main objectives of this proposal are to understand the mechanisms by which PKA- and PKC-mediated phosphorylation of Pgp affect to transport of organic substrates, and to define the basic mechanisms of regulation of Pgp activity via changes in PKC activity. To accomplish these aims, we will study the effects of Pgp phosphorylation of PKA and PKC on drug transport and binding, and ATP binding and hydrolysis. We will carry out studies 'in vivo' on intact cells and 'In vitro' using full- length Pgp and Pgp fragments that contain the N-terminal ATP- and drug-binding domains and the minilinker domain. In the intact cells, we will identify the functionally relevant serines by determining the effects of phosphorylation on cells expressing wild-type and mutant Pgps. In the mutant Pgps, serines of the minilinker domain will be substituted by alanines, individually or in combinations. Finally, we will use a Pgp mutant that does not respond to phosphorylation by PKC to assess the role of the kinase in the stimulation of Pgp in response to exposure to cytotoxic agents, estradiol, and the antiestrogen tamoxifen. The understanding of the molecular bases of the effects of PKA and PKC on Pgp-mediated drug transport will be an important contribution to our knowledge of the regulation of Pgp function. The experiments in this proposal will also provide the framework for studies aimed to regulate Pgp function and multidrug resistance by low-toxicity agents (e.g., endogenous hormones).