PHOSPHORYLATION AND DRUG TRANSPORT BY P-GLYCOPROTEIN

P-糖蛋白(Pgp)是一种被认为是主动挤出的ATPase 从细胞中提取抗癌药物。Pgp的过度表达似乎是 化疗失败的常见原因在某些疾病的治疗中 各种形式的癌症。Pgp在正常组织中也有表达，包括 肾上腺皮质、近端肾小管和内皮细胞 细胞，在那里它似乎参与了醛固酮和 药物和代谢物的运输。似乎 蛋白激酶A(PKA)和C(PKC)对Pgp的磷酸化 激活药物转运，已有研究表明PKC活性是 在几个多药耐药细胞系中升高。显然，PKA 和PKC每个只磷酸化3个丝氨酸(两个重叠)，这 位于两个同源部分之间的连接区 PGP(微链接域)。这项建议的主要目标是 了解PKA和PKC介导的机制 Pgp的磷酸化影响有机底物的运输，以及 明确PGP活性调节的基本机制，通过 PKC活性的变化。为了达到这些目标，我们将研究 Pgp对PKA和PKC的磷酸化对药物的影响 转运和结合，以及三磷酸腺苷的结合和水解。我们会 在完整的细胞上进行体内研究，并使用完整的 含有N-末端ATP-和的PGP和PGP片段的长度 药物结合域和微链接域。在完好的细胞中， 我们将通过确定功能相关的丝氨酸 磷酸化对野生型和突变型细胞表达的影响 Pgps。在突变的Pgps中，微链结构域的丝氨酸将是 被丙氨酸单独或组合取代。最后，我们 将使用一种对磷酸化无反应的Pgp突变体 PKC在Pgp刺激中的作用 对暴露于细胞毒剂、雌二醇和 抗雌激素他莫昔芬。对分子基础的认识 PKA和PKC在PgP介导的药物转运中的作用 对我们了解PGP调控的重要贡献 功能。该提案中的实验还将提供 旨在调节Pgp功能和多药的研究框架 低毒药物(如内源性激素)的抗药性。

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).