Organic Anion Transporter OAT1 Undergoes Constitutive and Protein Kinase C-regulated Trafficking through a Dynamin- and Clathrin-dependent Pathway

Abstract

Organic anion transporter 1 (OAT1) mediates the body disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We previously demonstrate that OAT1 activity was down-regulated by activation of protein kinase C (PKC), kinetically revealed as a decrease in the maximum transport velocity Vmax without significant change in the substrate affinity Km of the transporter. In the current study, we showed that OAT1 constitutively internalized from and recycled back to the plasma membrane, and PKC activation accelerated OAT1 internalization without affecting OAT1 recycling. We further showed that treatment of OAT1-expressing cells with concanavalin A, depletion of K+ from the cells, or transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block the clathrin-dependent endocytotic pathway, significantly blocked constitutive and PKC-regulated OAT1 internalization. We finally showed that OAT1 colocalized with transferrin, a marker for clathrin-dependent endocytosis, at the cell surface and in the EEA1-positive early endosomes. Together, our findings demonstrated for the first time that (i) OAT1 constitutively traffics between plasma membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 trafficking, and (iii) OAT1 internalization occurs partly through a dynamin- and clathrin-dependent pathway. Organic anion transporter 1 (OAT1) mediates the body disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We previously demonstrate that OAT1 activity was down-regulated by activation of protein kinase C (PKC), kinetically revealed as a decrease in the maximum transport velocity Vmax without significant change in the substrate affinity Km of the transporter. In the current study, we showed that OAT1 constitutively internalized from and recycled back to the plasma membrane, and PKC activation accelerated OAT1 internalization without affecting OAT1 recycling. We further showed that treatment of OAT1-expressing cells with concanavalin A, depletion of K+ from the cells, or transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block the clathrin-dependent endocytotic pathway, significantly blocked constitutive and PKC-regulated OAT1 internalization. We finally showed that OAT1 colocalized with transferrin, a marker for clathrin-dependent endocytosis, at the cell surface and in the EEA1-positive early endosomes. Together, our findings demonstrated for the first time that (i) OAT1 constitutively traffics between plasma membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 trafficking, and (iii) OAT1 internalization occurs partly through a dynamin- and clathrin-dependent pathway. The organic anion transporter (OAT) 2The abbreviations used are: OAT, organic anion transporter; HIV, human immunodeficiency virus; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAH, p-[3H]aminohippuric acid; PBS, phosphate-buffered saline; ConA, concanavalin A; BUO, bilateral ureteral obstruction; MesNa, sodium 2-mercaptoethanesulfonate. 2The abbreviations used are: OAT, organic anion transporter; HIV, human immunodeficiency virus; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAH, p-[3H]aminohippuric acid; PBS, phosphate-buffered saline; ConA, concanavalin A; BUO, bilateral ureteral obstruction; MesNa, sodium 2-mercaptoethanesulfonate. family mediates the body disposition of a diverse array of environmental toxins, and clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories (1You G. Med. Res. Rev. 2002; 22: 602-616Crossref PubMed Scopus (90) Google Scholar, 2You G. Curr. Drug Metab. 2004; 5: 55-62Crossref PubMed Scopus (52) Google Scholar, 3You G. Med. Res. Rev. 2004; 24: 762-774Crossref PubMed Scopus (36) Google Scholar). Therefore, understanding the regulation of these transporters has profound clinical significance. Seven OATs (OAT1–7) have been cloned, and their expressions were identified in distinct tissues and cell membranes (4Sweet D.H. Wolff N.A. Pritchard J.B. J. Biol. Chem. 1997; 272: 30088-30095Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 5Sekine T. Watanabe N. Hosoyamada M. Kanai Y. Endou H. J. Biol. Chem. 1997; 272: 18526-18529Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 6Lopez-Nieto C.E. You G. Bush K.T. Barros E.J. Beier D.R. Nigam S.K. J. Biol. Chem. 1997; 272: 6471-6478Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 7Wolff N.A. Werner A. Burkhardt S. Burckhardt G. FEBS Lett. 1997; 417: 287-291Crossref PubMed Scopus (139) Google Scholar, 8Sekine T. Cha S.H. Tsuda M. Apiwattanakul N. Nakajima N. Kanai Y. Endou H. FEBS Lett. 1998; 429: 179-182Crossref PubMed Scopus (309) Google Scholar, 9Cihlar T. Lin D.C. Pritchard J.B. Fuller M.D. Mendel D.B. Sweet D.H. Mol. Pharmacol. 1999; 56: 570-580Crossref PubMed Scopus (306) Google Scholar, 10Lu R. Chan B.S. Schuster V.L. Am. J. Physiol. 1999; 276: F295-F303PubMed Google Scholar, 11Kusuhara H. Sekine T. Utsunomiya-Tata N. Tsuda M. Kojima R. Cha S.H. Sugiyama Y. Kanai Y. Endou H. J. Biol. Chem. 1999; 274: 13675-13680Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 12Cha S.H. Sekine T. Kusuhara H. Yu E. Kim J.Y. Kim D.K. Sugiyama Y. Kanai Y. Endou H. J. Biol. Chem. 2000; 275: 4507-4512Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 13Youngblood G.L. Sweet D.H. Am. J. Physiol. 2004; 287: F236-F244Crossref PubMed Scopus (117) Google Scholar, 14Monte J.C. Nagle M.A. Eraly S.A. Nigam S.K. Biochem. Biophys. Res. Commun. 2004; 323: 429-436Crossref PubMed Scopus (92) Google Scholar, 15Ekaratanawong S. Anzai N. Jutabha P. Miyazaki H. Noshiro R. Takeda M. Kanai Y. Sophasan S. Endou H. J. Pharmacol. Sci. 2004; 94: 297-304Crossref PubMed Scopus (177) Google Scholar, 16Shin H.J. Anzai N. Enomoto A. He X. Kim do K. Endou H. Kanai Y. Hepatology. 2007; 45: 1046-1055Crossref PubMed Scopus (94) Google Scholar). OAT1 and OAT3 are predominantly expressed at the basolateral membrane of kidney proximal tubule cells and the apical membrane of brain choroid plexus. OAT4 is expressed at the apical membrane of kidney proximal tubule cells and the basolateral membrane of placental trophoblast. OAT2 is expressed at the basolateral membrane of hepatocytes and is expressed in the kidney. The cellular localization of OAT2 in the kidney is still controversial. OAT5 is expressed only in the kidney. OAT6 is expressed in the olfactory mucosa, and OAT7 was identified in the liver. The cellular localization of OAT5–7 has not been defined. In the kidney, OAT1 and OAT3 utilize a tertiary transport mechanism to move organic anions across the basolateral membrane into the proximal tubule cells for subsequent exit across the apical membrane into the urine for elimination. Through this tertiary transport mechanism, Na+/K+-ATPase maintains an inwardly directed (blood-to-cell) Na+ gradient. The Na+ gradient then drives a sodium dicarboxylate cotransporter, sustaining an outwardly directed dicarboxylate gradient that is utilized by a dicarboxylate/organic anion exchanger, namely OAT, to move the organic anion substrate into the cell. This cascade of events indirectly links organic anion transport to metabolic and the Na+ the of a substrate gradient and the of the cell. of the OATs (4Sweet D.H. Wolff N.A. Pritchard J.B. J. Biol. Chem. 1997; 272: 30088-30095Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 5Sekine T. Watanabe N. Hosoyamada M. Kanai Y. Endou H. J. Biol. Chem. 1997; 272: 18526-18529Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 16Shin H.J. Anzai N. Enomoto A. He X. Kim do K. Endou H. Kanai Y. Hepatology. 2007; 45: 1046-1055Crossref PubMed Scopus (94) Google including by and C in the first between 1 and and in the between and and in the C from our the of OATs revealed that is for the of these transporters to the plasma membrane K. You G. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. You G. Mol. Pharmacol. PubMed Scopus Google Scholar). from our and from have that PKC activation in a of organic anion transport in kidney and in cells with and OAT4 Am. J. Physiol. 1998; 274: Google Scholar, Am. J. Physiol. Google Scholar, M. S. S.H. Am. J. Physiol. 1999; Google Scholar, A. S.H. Am. J. Physiol. 2000; PubMed Google Scholar, N.A. K. N. G. Burckhardt G. J. Am. PubMed Scopus Google Scholar, R. Chan B.S. Schuster V.L. Am. J. Physiol. 1999; 276: F295-F303PubMed Google Scholar, M. J. M. K. Am. J. Physiol. PubMed Google Scholar, R. M. A. K. M. Am. J. Physiol. Google Scholar, You G. J. Sci. PubMed Scopus Google Scholar, You G. Am. J. Physiol. 2007; PubMed Scopus Google Scholar, G. K. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). You G. J. Sci. PubMed Scopus Google Scholar, G. K. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google revealed that the transport activity of OAT1 and OAT4 by activation of PKC from a transport velocity without significant change in the substrate affinity Km of the transporter. are by which PKC has been for membrane S. E. Y. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, G. Y. Am. J. Physiol. 1998; 275: PubMed Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). We PKC activation activity through of the transporter. showed G. K. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google that and PKC to the of OAT1 This that is to the for of OAT1 This was further by Wolff N.A. K. N. G. Burckhardt G. J. Am. PubMed Scopus Google that of OAT1 activity is of the PKC in PKC activation an internalization of membrane transporters the of transporters into plasma membrane plasma membrane are in the cell surface only in to cellular Pharmacol. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, P. T. M. Pharmacol. Sci. 22: Full Text Full Text PDF PubMed Scopus Google Scholar). In the transporter of and cells, constitutively between the cell surface and with the the the of in a a of at the plasma This occurs through the of recycling without significant of internalization A. PubMed Scopus Google Scholar). PKC internalization and recycling has been for brain transporter and constitutively are by internalization and recycling in to PKC activation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. M. S. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the we (i) OAT1 constitutive between cell surface and (ii) of OAT1 activity occurs by altering the of this and (iii) the through which OAT1 was from and were from cells and were from Eps15 was by from of was from of was from and were from and all were from of cells were in with and in a at were at of human with at C M. T. K. M. You G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (36) Google was into cells a the of in were to for and was The of phosphate-buffered and 1 and the time in the the was by the and the cells with The cells were then in in and for The was by the of protein in surface of OAT1 was the were in of cells was with 1 of in in with The was for was with of then with the for to of the The cells were then for 1 in of 1 with The cell were by at at of was then to the to cell membrane OAT1 was in the of surface by and the by J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and X. J. S.A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). OAT1-expressing cells with at of cells was with and at to the surface OAT1 and cells in the were with 1 or and with the for and at cell surface was by cells for with in 1 was for cells in at were in with were from by from of cellular and were by and OAT1 internalized was as of the cell surface OAT1 the by J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and X. J. S.A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). OAT1-expressing cells with at for to cell surface of of cells was at in the were to and at the time in the was and OAT1 was by and as OAT1 recycled was as the between OAT1 at and OAT1 at and were and to The were blocked for 1 with in and for 1 at with by The were by protein were by with the cells were for in PBS, and with 1 in at for 1 to the of to by at to the internalization of The cells were then with for at with for and with at for 1 the cells were with to and with at The were then with or at for the were for and were with a was a of The was from was of was of OAT1 in the regulation of we cells The transport of PAH, a organic across the cell membrane was the Km for was and Vmax was the that OAT1 activity was down-regulated by activation of PKC in cells and in kidney proximal Am. J. Physiol. 1998; 274: Google Scholar, Am. J. Physiol. Google Scholar, M. S. S.H. Am. J. Physiol. 1999; Google Scholar, A. S.H. Am. J. Physiol. 2000; PubMed Google Scholar, N.A. K. N. G. Burckhardt G. J. Am. PubMed Scopus Google Scholar, R. Chan B.S. Schuster V.L. Am. J. Physiol. 1999; 276: F295-F303PubMed Google Scholar, M. J. M. K. Am. J. Physiol. PubMed Google Scholar, R. M. A. K. M. Am. J. Physiol. Google Scholar, You G. J. Sci. PubMed Scopus Google Scholar, You G. Am. J. Physiol. 2007; PubMed Scopus Google Scholar, G. K. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google we OAT1 by PKC activation in treatment with phorbol 12-myristate a PKC at 1 for we a significant decrease in transport The of OAT1 activity by was to a decrease in the maximum transport velocity Vmax without significant change in the substrate affinity Km demonstrate that OAT1 in cells all the of OAT1 as in cell Am. J. Physiol. 1998; 274: Google Scholar, Am. J. Physiol. Google Scholar, M. S. S.H. Am. J. Physiol. 1999; Google Scholar, A. S.H. Am. J. Physiol. 2000; PubMed Google Scholar, N.A. K. N. G. Burckhardt G. J. Am. PubMed Scopus Google Scholar, R. Chan B.S. Schuster V.L. Am. J. Physiol. 1999; 276: F295-F303PubMed Google Scholar, M. J. M. K. Am. J. Physiol. PubMed Google Scholar, R. M. A. K. M. Am. J. Physiol. Google Scholar, You G. J. Sci. PubMed Scopus Google Scholar, You G. Am. J. Physiol. 2007; PubMed Scopus Google Scholar, G. K. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google and that cells are for the regulation of this transporter. of decrease in the Vmax of transport in to PKC activation by a decrease in transporter at the cell surface or a decrease in transport between these we used cell surface to the cellular of OAT1 in cells with or without We showed that treatment of cells with in a decrease of OAT1 at the cell surface with a of OAT1 in the OAT1 were not significantly treatment that in cell surface OAT1 is not by Therefore, activation of PKC in a of OAT1 from the cell surface to The of OAT1 from the cell surface is for the decrease in Vmax OAT1 OAT1 that OAT1 to constitutive internalization from and recycling back to the cell this we a We first OAT1 constitutively OAT1-expressing cells were with The cells were then back to to internalization to time of from the surface was by treatment with MesNa, a that and from at the cell The of to treatment was as of protein a and showed that of was OAT1 or of or of OAT1 was in the Therefore, OAT1 constitutive internalization in were in OAT1-expressing cells a and that OAT1 internalization is not cell is a of this of constitutive OAT1 internalization in OAT1 internalization was as and by as a for of OAT1 in cell was in to in a by of from a as as from OAT1 was expressed as of cell surface OAT1 are We OAT1 constitutively back to the cell surface OAT1-expressing cells first with at for to the cell surface of of cells was at in the were to and at J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, X. J. S.A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The was that OAT1 constitutively traffics between the cell surface and the then significantly the of OAT1 as with The were first with which is to in membrane recycling and the of the which is a negative for recycling J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, X. J. S.A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). in the of the of recycling was that the of recycling the of Na+/K+-ATPase was the and the of recycling the we further showed that the of OAT1 at was that at and demonstrated OAT1 constitutively back to the cell the of OAT1 to the cell surface we and surface OAT1 protein was blocked with treatment a time of significant OAT1 surface or OAT1 not Therefore, the of OAT1 to the cell surface is not significant in the time in which our were OAT1 of OAT1 from cell surface to from an in OAT1 a decrease in OAT1 or a of the as we used to constitutive OAT1 trafficking, we activation of PKC OAT1 We a and that the of OAT1 internalized in the of was that in the of PMA, that activation of PKC by accelerated OAT1 internalization into the treatment significant OAT1 recycling back to the plasma membrane of OAT1 recycling. OAT1 recycling and was as and in the and the of by as a for of OAT1 in cell was in to in the by of from as as from OAT1 was expressed as of OAT1 at are OAT1 for transporter internalization is their with the cellular internalization internalization have been for (i) (ii) and (iii) and internalization. internalization occurs at membrane a a is to surface as transporters for internalization S.A. T. R. Biochem. J. 2004; PubMed Scopus Google Scholar). from the membrane and move as internalization into the an in this have been is expressed in dynamin-2 is and is expressed in and to a in and internalization is an to internalization A. 2002; PubMed Scopus Google Scholar, 2004; 5: PubMed Scopus Google Scholar). are of plasma membrane of cell in and as as a protein from the membrane and move as internalization into the OAT1 clathrin-dependent pathway, the constitutive and OAT1 internalization were the that block clathrin-dependent internalization H. S. M. A. Y. Takeda K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Y. R. M. Am. J. Physiol. 2002; PubMed Scopus Google Scholar, S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google (i) treatment of the cells with concanavalin and (ii) depletion of K+ from the cells and and (iii) transfection of dominant negative of dynamin-2 into the cells an showed that the of OAT1 internalized in the and in the of was significantly these as with that of the The of OAT1 internalization OAT1 cellular and in cells with dominant negative of dynamin-2 was further OAT1 internalization in an in cell surface of OAT1 with decrease in without significant change in a OAT1 transport activity was depletion blocked constitutive and OAT1 internalization. cells were at for in and in and then and with K+ depletion 1 1 for S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). OAT1 internalization was then as and in the and the of 1 by as a for the of OAT1 in cell was in to in the by of from as as from OAT1 was expressed as of cell surface OAT1 are negative of blocked constitutive and OAT1 internalization. cells were with dominant negative of dynamin-2 OAT1 internalization was as and in the and the of 1 by as a for the of OAT1 in cell was in to in the by of from as as from OAT1 was expressed as of cell surface OAT1 are the of the of OAT1 at cell in as as in cell into cells with activity was expressed as a of the in are the of dynamin-2 OAT1 internalization was in cells with dominant negative of of A. M. N. A. J. Sci. 1999; PubMed Google OAT1 activity was of cellular of OAT1 was by is to cells, to Mol. Biol. 2002; PubMed Scopus Google Scholar). is then to EEA1-positive early and recycled back to the cell surface Mol. Biol. 2002; PubMed Scopus Google Scholar). In this of was first with cells at for 1 to the of by at to the internalization of into the The were at time showed that OAT1 colocalized with at the cell surface as and in the showed that OAT1 colocalized with in EEA1-positive to significant of OAT1 with marker The organic anion transporter (OAT) family mediates the body disposition of a diverse array of environmental toxins, and clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and Therefore, understanding the regulation of these transporters has profound clinical significance. We previously showed G. K. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google that OAT1 activity is down-regulated by activation of PKC, kinetically revealed as a decrease in the maximum transport velocity Vmax without significant change in the substrate affinity Km of the and that of OAT1 activity is not to of the transporter. that of OAT1 activity from internalization of the transporter the of the transporter into membrane PKC a protein that is with OAT1 in the our has of these is a substrate for is In the current study, we in cells cells for of the organic anion transporter. (i) cells were from the kidney and have been in understanding transport and cellular including organic transport X. S.H. Am. J. Physiol. 2002; Scopus Google Scholar, K. N. M. T. A. T. M. M. E. H. S. Biochem. Biophys. PubMed Scopus Google Scholar). (ii) This cell not transporter. Therefore, of OAT1 in cells to the transport of OAT1 in a without the of organic anion (iii) PKC and and a for the transport S. Biochem. Biophys. PubMed Scopus Google Scholar, J. H. T. E.J. Am. J. Physiol. 2002; PubMed Google Scholar). The transport of OAT1 in cells and by PKC activation were in a with that in Am. J. Physiol. 1998; 274: Google Scholar, Am. J. Physiol. Google Scholar, M. S. S.H. Am. J. Physiol. 1999; Google Scholar, A. S.H. Am. J. Physiol. 2000; PubMed Google Scholar, N.A. K. N. G. Burckhardt G. J. Am. PubMed Scopus Google Scholar, R. Chan B.S. Schuster V.L. Am. J. Physiol. 1999; 276: F295-F303PubMed Google Scholar, M. J. M. K. Am. J. Physiol. PubMed Google Scholar, R. M. A. K. M. Am. J. Physiol. Google Scholar, You G. J. Sci. PubMed Scopus Google Scholar, You G. Am. J. Physiol. 2007; PubMed Scopus Google Scholar, G. K. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). these cells, we OAT1 was between cell surface and with the the of PKC by the cellular of OAT1 in a a of OAT1 in the Therefore, of OAT1 as a maximum transport velocity from a of OAT1 from cell surface to the OAT1 that OAT1 to constitutive between cell surface and transporters were to at the cell a body of demonstrated that transporters internalization from and recycling back to cell surface constitutively or in to transporter and are P. T. M. Pharmacol. Sci. 22: Full Text Full Text PDF PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Y. R. M. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). The constitutive OAT1 from our We showed OAT1 internalized from and recycled back to the cell surface to a cell surface of the transporter. The for internalization and recycling were to that for a protein that internalization and recycling M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The of OAT1 to the cell surface in the time in which our were treatment with an for protein a time of significant OAT1 surface or OAT1 further showed that PKC OAT1 activity by OAT1 internalization without significantly affecting OAT1 recycling PKC internalization and recycling have been for transporters as brain transporter J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and K. A. M. S. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are the cellular the constitutive and PKC-regulated OAT1 internalization have been for (i) clathrin-dependent (ii) and (iii) and internalization. from our that the constitutive and the OAT1 internalization partly through a dynamin- and clathrin-dependent pathway. treatment of OAT1-expressing cells with ConA, depletion of K+ from the cells, or transfection of dominant negative mutants of dynamin-2 into the cells, all of which blocked clathrin-dependent H. S. M. A. Y. Takeda K. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Y. R. M. Am. J. Physiol. 2002; PubMed Scopus Google Scholar, S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google significantly blocked OAT1 internalization OAT1 internalization was significantly in cells with dominant negative of of A. M. N. A. J. Sci. 1999; PubMed Google was to in S. Rev. Mol. Biol. 2007; PubMed Scopus Google that in as Therefore, the of Eps15 is a to these the internalization of which has been to through clathrin-dependent Mol. Biol. 2002; PubMed Scopus Google OAT1 internalization only blocked by these that internalization to OAT1 This is further OAT1 partly through a dynamin- and clathrin-dependent pathway, was further by our is to cells, to Mol. Biol. 2002; PubMed Scopus Google Scholar). is then to EEA1-positive early and recycled back to the cell surface Mol. Biol. 2002; PubMed Scopus Google Scholar). We showed that OAT1 colocalized with at the cell surface and in the EEA1-positive early endosomes, that OAT1 at in through the as that of In the of with and from has been that recycling are in their transport and K. G. K. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, that of in a of recycling from that in is to that is in In an is not to that not with and In in the current study, we demonstrated for the first time that (i) OAT1 constitutively traffics between cell membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 OAT1 internalization without significantly affecting OAT1 and (iii) OAT1 internalization occurs partly through a dynamin- and clathrin-dependent pathway. We have that of as OAT3 and OAT1 the kidney to the is that the transporter in a a is for the to is of and in body to environmental OAT1 to the in bilateral ureteral is a and clinical and an important of S. G. The and Scholar, A. Anzai N. Endou H. Full Text Full Text PDF PubMed Scopus Google Scholar). is A. Anzai N. Endou H. Full Text Full Text PDF PubMed Scopus Google that in of was partly to a of OAT1 from cell surface to In BUO, has an of S. G. The and Scholar, S. 1998; Google Scholar, S. J. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). was that through activation of PKC Biophys. 2002; PubMed Scopus Google Scholar). Therefore, OAT1 through a pathway. current important into the and clinical