Noshiro R, Anzai N, Sakata T, Miyazaki H, Terada T, Shin H J, He X, Miura D, Inui K, Kanai Y, Endou H
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo, Japan.
Kidney Int. 2006 Jul;70(2):275-82. doi: 10.1038/sj.ki.5001522. Epub 2006 May 31.
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.
质子偶联肽转运体PEPT2（SLC15A2）介导肾脏中小肽以及各种口服类肽药物的高亲和力低容量转运。与其已明确的转运特性相比，尽管已经初步报道了PEPT2与PDZ（PSD-95、DglA和ZO-1）结构域蛋白PDZK1的相互作用，但其调节机制的相关信息较少。为了研究PDZK1是否是肾脏中PEPT2的生理伴侣，我们以PEPT2的C末端（PEPT2 C末端（PEPT2-CT））为诱饵，对人肾脏cDNA文库进行酵母双杂交筛选。我们能够将PDZK1鉴定为阳性克隆之一。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检测到的PEPT2-CT的PDZ基序。通过表面等离子体共振测量PDZK1的第二和第三PDZ结构域与PEPT2-CT的结合亲和力。使用人肾脏膜组分进行的免疫共沉淀以及PEPT2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，我们阐明了在共表达PDZK1后，表达PEPT2的HEK293细胞中甘氨酰肌氨酸（Gly-Sar）转运活性增强的机制。这种增强伴随着通过PEPT2的Gly-Sar转运的V(max)显著增加，并且还与PEPT2表面表达水平的增加相关。这些结果表明，PEPT2-PDZK1相互作用在人肾脏的寡肽处理以及类肽药物转运中均发挥着重要的生理作用。
