Screening of the interaction between xenobiotic transporters and PDZ proteins.
OBJECTIVE
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, D1g 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 His6-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.
Kato
Yukio
Y
Department of Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan.
Yoshida
Kazuhiro
K
Watanabe
Chizuru
C
Sai
Yoshimichi
Y
Tsuji
Akira
A
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins; D009154:Mutation / Q000502:physiology; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters / Q000378:metabolism; D015262:Xenobiotics / Q000737:chemistry / Q000378:metabolism*; D015003:Yeasts / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; C484659:PDZK1 protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins; D027981:Symporters; D015262:Xenobiotics; C093694:hydrogen-coupled oligopeptide transporter PepT2; D005978:Glutathione