TY - JOUR
T1 - Association of paracellin-1 with ZO-1 augments the reabsorption of divalent cations in renal epithelial cells
AU - Ikari, Akira
AU - Hirai, Naho
AU - Shiroma, Morihiko
AU - Harada, Hitoshi
AU - Sakai, Hideki
AU - Hayashi, Hisayoshi
AU - Suzuki, Yuichi
AU - Degawa, Masakuni
AU - Takagi, Kuniaki
PY - 2004/12/24
Y1 - 2004/12/24
N2 - Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether 45Ca2+ transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Nest, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (ΔTRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the ΔTRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of 45Ca2+ transport from apical to basal compartments, compared with those expressing the ΔTRV, ARV, and TRA mutants and the mock cells. 45Ca2+ transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced 45Ca2+ transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1·ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.
AB - Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether 45Ca2+ transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Nest, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (ΔTRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the ΔTRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of 45Ca2+ transport from apical to basal compartments, compared with those expressing the ΔTRV, ARV, and TRA mutants and the mock cells. 45Ca2+ transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced 45Ca2+ transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1·ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.
UR - http://www.scopus.com/inward/record.url?scp=11144243665&partnerID=8YFLogxK
U2 - 10.1074/jbc.M406331200
DO - 10.1074/jbc.M406331200
M3 - 学術論文
C2 - 15496416
AN - SCOPUS:11144243665
SN - 0021-9258
VL - 279
SP - 54826
EP - 54832
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 52
ER -