Regulation of human organic anion transporter 4 by protein kinase C and NHERF-1: altering the endocytosis of the transporter.
OBJECTIVE
Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs. We have previously shown that the activity of hOAT4 was down-regulated by activation of PKC and up-regulated by PDZ protein NHERF-1. Here, we investigated the mechanisms underlying such regulations.


METHODS
COS-7 cells expressing hOAT4 were treated with PKC activator phorbol 12-myristate 13-acetate (PMA) or transfected with dominant negative mutants of dynamin-2 or Eps15 or transfected with NHERF-1. The internalization and the function of hOAT4 were then determined.


RESULTS
We showed that hOAT4 constitutively internalized from and recycled back to plasma membrane. Transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block clathrin-dependent endocytotic pathway, significantly blocked hOAT4 internalization. Treatment of cells with PMA accelerated hOAT4 internalization, whereas transfection of cells with NHERF-1 attenuated hOAT4 internalization.


CONCLUSIONS
Our studies demonstrated that i) hOAT4 undergoes constitutive trafficking between cell surface and intracellular compartments, ii) hOAT4 internalization partly occurs through clathrin-dependent pathway, iii) the down-regulation of hOAT4 activity by activation of PKC and the up-regulation of hOAT4 activity by NHERF-1 are mediated through alteration of hOAT4 internalization.
Zhang
Qiang
Q
Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854, USA.
Pan
Zui
Z
You
Guofeng
G
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D006801:Humans; D029362:Organic Anion Transporters, Sodium-Independent / Q000032:analysis / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000032:analysis
D002966:Clathrin; D029362:Organic Anion Transporters, Sodium-Independent; D010750:Phosphoproteins; C427207:SLC22A11 protein, human; D017923:Sodium-Hydrogen Exchangers; D033921:Vesicular Transport Proteins; C093376:sodium-hydrogen exchanger regulatory factor; D011493:Protein Kinase C