Regulation of Human Organic Anion Transporter 4 by Protein Kinase C and NHERF-1: Altering the Endocytosis of the Transporter

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. 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. 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. 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.