Organic anion transporter OAT1 undergoes constitutive and protein kinase C-regulated trafficking 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 V(max) without significant change in the substrate affinity K(m) 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.
Zhang
Qiang
Q
Department of Pharmaceutics, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
Hong
Mei
M
Duan
Peng
P
Pan
Zui
Z
Ma
Jianjie
J
You
Guofeng
G
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019921:Biotinylation; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D027362:Organic Anion Transport Protein 1 / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002966:Clathrin; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D027362:Organic Anion Transport Protein 1; D010750:Phosphoproteins; D011493:Protein Kinase C; D034281:Dynamins