The PDZ domain protein PDZK1 interacts with human peptide transporter PEPT2 and enhances its transport activity
The PDZ domain protein PDZK1 interacts with human peptide transporter PEPT2 and enhances its transport activity
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DOI：
10.1038/sj.ki.5001522
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发表时间：
2006-07-01
影响因子：
19.6
通讯作者：
Endou, H.
中科院分区：
文献类型：
--
作者：
Noshiro, R.;Anzai, N.;Endou, H.
质子偶联肽转运蛋白PEPT 2（SLC 15 A2）介导小肽以及各种口服肽类药物在肾脏中的高亲和力低容量转运。与其充分表征的转运特性相反，关于其调节机制的信息较少，尽管已经初步报道了PEPT 2与PDZ（PSD-95、DglA和ZO-1）结构域蛋白PDZK 1的相互作用。为了检测PDZK 1是否是肾脏中PEPT 2的生理伴侣，我们从以PEPT 2的C-末端（PEPT 2 C-末端（PEPT 2-CT））作为诱饵的人肾脏cDNA文库的酵母双杂交筛选开始。PDZK 1为阳性克隆之一。这种相互作用需要通过酵母双杂交试验、体外结合试验和免疫共沉淀法检测PEPT 2-CT的PDZ基序。通过表面等离子体共振测量PDZK 1的第二和第三PDZ结构域与PEPT 2-CT的结合亲和力。使用人肾膜组分的免疫共沉淀和PEPT 2在肾顶端近端小管中的定位揭示了这种相互作用在肾脏中的生理意义。此外，我们阐明了PDZK 1共表达后PEPT 2表达的HEK 293细胞中甘氨酰肌氨酸（Gly-Sar）转运活性增强的机制。这种增强伴随着经由PEPT 2的Gly-Sar转运的V-max的显著增加，并且其还与PEPT 2的表面表达水平增加相关。这些结果表明，PEPT 2-PDZK 1相互作用因此在人肾脏中的寡肽处理以及肽样药物转运中起着生理学上重要的作用。
The proton-coupled peptide transporter PEPT2 ( SLC15A2) mediates the high-affinity low-capacity transport of small peptides as well as various oral peptide-like drugs in the kidney. In contrast to its well-characterized transport properties, there is less information available on its regulatory mechanism, although the interaction of PEPT2 to the PDZ ( PSD-95, DglA, and ZO-1)-domain protein PDZK1 has been preliminarily reported. To examine whether PDZK1 is a physiological partner of PEPT2 in kidneys, we started from a yeast two-hybrid screen of a human kidney cDNA library with the C-terminus of PEPT2 ( PEPT2 C-terminus ( PEPT2-CT)) as bait. We could identify PDZK1 as one of the positive clones. This interaction requires the PDZ motif of PEPT2-CT detected by a yeast two-hybrid assay, in vitro binding assay and co-immunoprecipitation. The binding affinities of second and third PDZ domains of PDZK1 to PEPT2-CT were measured by surface plasmon resonance. Co-immunoprecipitation using human kidney membrane fraction and localization of PEPT2 in renal apical proximal tubules revealed the physiological meaning of this interaction in kidneys. Furthermore, we clarified the mechanism of enhanced glycylsarcosine (Gly-Sar) transport activity in PEPT2-expressing HEK293 cells after the PDZK1 coexpression. This augmentation was accompanied by a significant increase in the V-max of Gly-Sar transport via PEPT2 and it was also associated with the increased surface expression level of PEPT2. These results indicate that the PEPT2 - PDZK1 interaction thus plays a physiologically important role in both oligopeptide handling as well as peptide-like drug transport in the human kidney.