Screening of the interaction between xenobiotic transporters and PDZ proteins
Screening of the interaction between xenobiotic transporters and PDZ proteins
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DOI：
10.1023/b:pham.0000045244.83999.43
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发表时间：
2004-10-01
影响因子：
3.7
通讯作者：
Tsuji, A
中科院分区：
文献类型：
--
作者：
Kato, Y;Yoshida, K;Tsuji, A
目的。异种转运体已被提出参与各种治疗剂的膜渗透。由于关于其功能调控的分子机制的信息很少，我们试图澄清这类转运蛋白的蛋白质-蛋白质相互作用，作为鉴定其调控因子的第一步。通过酵母双杂交筛选，研究了多种外源转运体羧基末端与含PDZ （PSD95、Dlg和ZO1）结构域蛋白之间的相互作用。在拉下、免疫沉淀和转运研究中也评估了相互作用和功能调节。几种外源性转运蛋白，包括PEPT1、PEPT2、OCT3、OCTN1、OCTN2、OAT4、OATP-A、OATP-D和OATP-F，与PDZ蛋白有特异性相互作用。在PEPT2和PDZK1之间观察到强有力的相互作用，而PEPT2 c端最后四个氨基酸的缺失几乎完全消除了这种相互作用。重组PEPT2 c端融合蛋白可以与纯化的His(6)标记的PDZK1结合，证实PDZK1中四个PDZ结构域中的两个参与了相互作用。PEPT2的丙氨酸扫描突变揭示了一个负责PDZK1相互作用的共识序列（-T-X-L）的存在。转染PDZK1增加了PEPT2对甘氨酸肌氨酸的摄取，而在最后四个氨基酸缺失的PEPT2中没有观察到这种刺激。这些结果首次确定了PDZ蛋白与各种外源转运体的胞质尾部之间的相互作用。PDZK1直接与PEPT2相互作用，对其转运活性进行功能调控。目前的研究结果表明，PEPT2定位于由PDZK1和其他转运蛋白构建的蛋白质网络中。
Purpose. Xenobiotic transporters have been proposed to be involved in membrane penetration of various therapeutic agents. As little information is available on molecular mechanism of their functional regulation, we have attempted to clarify the protein-protein interactions of such transporters as a first step to identify their regulators.Methods. Yeast two-hybrid screening was performed to examine the interaction between carboxylic terminus of various xenobiotic transporters and PDZ (PSD95, Dlg and ZO1) domain-containing proteins. The interaction and functional regulation were also evaluated in pull-down, immunoprecipitation and transport studies.Results. Specific interaction with PDZ proteins was identified for several xenobiotic transporters including PEPT1, PEPT2, OCT3, OCTN1, OCTN2, OAT4, OATP-A, OATP-D, and OATP-F. The potent interaction was observed between PEPT2 and PDZK1, and deletion of the last four amino acids of the PEPT2 C-terminus almost completely abrogated such interaction. Recombinant PEPT2 C-terminus fusion protein can bind to purified His(6)-tagged PDZK1, confirming the involvement of two of four PDZ domains within PDZK1 in the interaction. Alanine-scanning mutation in PEPT2 revealed the presence of a consensus sequence (-T-X-L) that is responsible for the PDZK1 interaction. Transfection of PDZK1 increased the uptake of glycylsarcosine by PEPT2, whereas such stimulation was not observed for PEPT2 with the last four amino acids deleted.Conclusions. These results first identified the interaction between PDZ proteins and the cytosolic tail of various xenobiotic transporters. PDZK1 directly interacts with PEPT2, exerting functional regulation of its transporting activity. The current findings imply the localization of PEPT2 within a protein network constructed from PDZK1 and other transporter proteins.