2004 Fiscal Year Final Research Report Summary
Regulatory mechanism of urate transport function by urate transporter binding protein PDZK1
| Project/Area Number |
15590233
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
General pharmacology
|
|---|
| Research Institution | Kyorin University |
|---|
Principal Investigator |
ANZAI Naohiko Kyorin Univ., Sch. of Med., Dept. of Pharmacol. and Toxicol., Assistant Professor, 医学部, 助手 (70276054)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KANAI Yoshikatsu Kyorin Univ., Sch.of Med., Dept.of Pharmacol. and Toxicol., Professor, 医学部, 教授 (60204533)
IRIBE Yuji Kyorin Univ., Sch.of Med., Dept.of Pharmacol.and Toxicol., Assistant Professor, 医学部, 助手 (20348618)
HIRATA Taku Kyorin Univ., Sch.of Med., Dept of Pharmacol. and Toxicol., Assistant Professor, 医学部, 助手 (60372918)
YOKOYAMA Hirokazu Kyorin Univ., Sch.of Med., Dept. of Pharmacol.and Toxicol., Assistant Professor, 医学部, 助手 (60360103)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Keywords | Yeast Two-hybrid assay / Urate transporter / PDZ proteins / Co-immunoprecipitation / GST pull dow assay / Surface plasmon resonance / Surface biotinylation assay |
|---|
| Research Abstract |
Based on our preliminary results that the identification of intracellular binding protein PDZK1 for renal urate transporter URAT1 by yeast two-hybrid (Y2H) screening, we were planning to extend this research to clarify the functional regulatory mechanism of urate transport via urate transporter URAT1 by its binding protein PDZK1.
In 2003, we clarified these three points. (1)We carried out the in vitro biochemical binding experiments such as co-immunoprecipitation assay and GST-pull down assay to confirm the URAT1-PDZK1 interaction identified by Y2H experiments. Wild type URAT1 proteins could bind with PDZK1, but URAT1 lacking the last three amino acid residues including PDZ motif (T-Q-F) failed to bind with PDZK1. (2)We established URAT1-expressing HEK293 cells (HEK-URAT1) by lipofection method and performed urate transport kinetic study with or without PDZK1 cotransfection. PDZK1 transfection into HEK-URAT1 cell increased Vmax without changing Km. (3) We detected the colocalization of
… More
both URAT1 and PDZK1 at the apical membrane of renal proximal tubules by immunohistochemical analysis using human kidney serial sections. In addition, coimmunoprecipitation experiments using human kidney total protein demonstrated the binding between URAT1 and PDZK1. These two findings showed the physiological importance of this interaction.
In 2004, we further examined this URAT1-PDZK1 interaction. (1)We prepared the GST fused URAT1 C-terminal and MBP fused individual PDZ domains of PDZK1 and performed Surface Plasmon Resonance assay (Biacore) to check their binding affinities. The bindings were confirmed in PDZK1 PDZ domain 1, 2 and 4 by SPR. Their dissociation constant (K_D) is 1.9 - 516 nM. These values were compatible as PDZ interactions. (2)By the cell surface biotinylation experiments, we recognize that, at least, the enhancement of urate transport activity after the transfection of PDZK1 is due to the increased expression of URAT1 proteins at the plasma membrane of HEK293 cells, may be through the stabilization of URAT1 proteins by PDZK1 interaction. (3)By the database searches, we have already found two SNPs of human PDZK1. Y145H is located in the middle of the second PDZ domain of PDZK1 and V372E is located at the C-terminal side in the linker between PDZ domain 3 and 4. In the Y2H assay, we could not find the difference among three clones (wild type, Y145H, and V372E), whereas SPR experiments clearly exhibited the reduced affinity for Y145H mutants. These two PDZK1 SNP mutants did not affect the urate transport activity via URAT1. Less
|
-
-
-
-
[Journal Article] Immunocytochemical characterization of the incubated rat renal cortical slices.2005
Author(s)
Crljen V, Saboiic I, Susac J, Appenroth D, Herak-Kramberger CM, Ljubojevic M, Anzai N, Antolovic R, Burckhardt G, Fleck C, Sabolic I.
-
Journal Title
Pflugers Arch. (in press)
Description
「研究成果報告書概要(欧文)」より
-
-
-
-
-
-
-
[Journal Article] The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal orate-anion exchanger URAT1 via its C terminus.2004
Author(s)
Anzai N, Miyazaki H, Noshiro R, Khamdang S, Chairoungdua A, Shin HJ, Enomoto A, Sakamoto S, Hirata T, Tomita K, Kanai Y, Endou H.
-
Journal Title
J Biol Chem. 279(44)
Pages: 45942-45950
Description
「研究成果報告書概要(欧文)」より
-
[Journal Article] Mutations in SLC6A19, encoding B^0AT 1, cause Hartnup disorders.2004
Author(s)
Kleta R, Romeo E, Ristic Z, Ohura T, Stuart C, Arcos-Burgos M, Dave MH, Wagner CA, Camargo SR, Inoue S, Matsuura N, Helip-Wooley A, Bockenhauer D, Warth R, Bernardini I, Visser G, Eggermann T, Lee P, Chairoungdua A, Jutabha P, Babu E, Nilwarangkoon S, Anzai N, Kanai Y, Verrey F, Gahl WA, Koizumi A.
-
Journal Title
Nat Genet. 36(9)
Pages: 999-1002
Description
「研究成果報告書概要(欧文)」より
-
-
-
-
[Journal Article] Interactions of human organic anion transporters with diuretics.2004
Author(s)
Hasannejad H, Takeda M, Taki K, Jung SH, Babu E, Jutabha P, Khamdang S, Aleboyeh M, Onodera ML, Tojo A, Enomoto A, Anzai N, Narikawa S, Huang XL, Niwa T, Endou H.
-
Journal Title
J Pharmacol Exp Ther. 308(3)
Pages: 1021-1029
Description
「研究成果報告書概要(欧文)」より
-
[Journal Article] Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters.2003
Author(s)
Babu E, Kanai Y, Chairoungdua A, Kim do K, Iribe Y, Tangtrongsup S, Jutabha P, Li Y, Ahmed N, Sakamoto S, Anzai N, Nagamori S, Endou H.
-
Journal Title
J Biol Chem. 278(44)
Pages: 43838-43845
Description
「研究成果報告書概要(欧文)」より
-
[Journal Article] Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule.2003
Author(s)
Jutabha P, Kanai Y, Hosoyamada M, Chairoungdua A, Kim do K, Iribe Y, Babu E, Kim JY, Anzai N, Chatsudthipong V, Endou H.
-
Journal Title
J Biol Chem. 278(30)
Pages: 27930-27938
Description
「研究成果報告書概要(欧文)」より
-
[Journal Article] Interaction of human and rat organic anion transporter 2 with various cephalosporin antibiotics.2003
Author(s)
Khamdang S, Takeda M, Babu E, Noshiro R, Onozato ML, Tojo A, Enomoto A, Huang XL, Narikawa S, Anzai N, Piyachaturawat P, Endou H.
-
Journal Title
Eur J Pharmacol. 465(1-2)
Pages: 1-7
Description
「研究成果報告書概要(欧文)」より
-
[Journal Article] Expression of human organic anion transporters in the choroid plexus and their interactions with neurotransmitter metabolites.2003
Author(s)
Alebouyeh M, Takeda M, Onozato ML, Tojo A, Noshiro R, Hasannejad H, Inatomi J, Narikawa S, Huang XL, Khamdang S, Anzai N, Endou H.
-
Journal Title
J Pharmacol Sci. 93(4)
Pages: 430-436
Description
「研究成果報告書概要(欧文)」より
-
