Guofeng You

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 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.

Regulation of human organic anion transporter 1 by ANG II: involvement of protein kinase Cα

Human organic anion transporter 1 (hOAT1) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT1 is abundantly expressed in the kidney. In the current study, we examined the regulation of hOAT1 by ANG II in kidney COS-7 cells. ANG II induced a concentration- and time-dependent inhibition of hOAT1 transport activity. Such inhibition mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased maximal velocity without significant change in Michaelis constant. ANG II-induced inhibition of hOAT1 activity could be prevented by treating hOAT1-expressing cells with staurosporine, a general protein kinase C (PKC) inhibitor. To obtain further information on which PKC isoform mediates ANG II regulation of hOAT1 activity, cellular distribution of various PKC isoforms was examined in cells treated with or without ANG II. We showed that ANG II treatment resulted in a significant translocation of PKCalpha from cytosol to membrane, and such translocation was blocked by treating hOAT1-expressing cells with Gö-6976, a PKCalpha-specific inhibitor. We further showed that ANG II-induced inhibition of hOAT1 activity and retrieval of hOAT1 from the cell surface could also be prevented by treating hOAT1-expressing cells with Gö-6976. We concluded that ANG II inhibited hOAT1 activity through activation of PKCalpha, which led to the redistribution of the transporter from the cell surface to the intracellular compartments.

The Putative Transmembrane Segment 7 of Human Organic Anion Transporter hOAT1 Dictates Transporter Substrate Binding and Stability

Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs. We examined the role of the putative transmembrane segment (TM) 7 in the function of hOAT1. Each residue within putative TM7 was replaced by alanine, and the uptake of para-aminohippurate was studied in cells expressing the mutants. We discovered four critical amino acid residues: Trp-346, Thr-349, Tyr-353, and Tyr-354. Substitution of Tyr-353 and Tyr-354 with alanine led to the loss of transport activity without affecting the surface expression of the transporter, whereas substitution of Trp-346 and Thr-349 with alanine lead to the loss of the total expression of the transporter. The effect of side chains of Tyr-353 and Tyr-354 on transporter functions were further evaluated by replacing these residues with Phe or Trp. Among all the mutants studied (Y353W, Y353F, Y354W, and Y354F), only mutant Y353F regained 30% transport activity, which was lost from replacement of Tyr-353 with alanine, suggesting that both the -OH group and the size of the side chain at positions 353 and 354 are critical for maintaining the full transport activity. To investigate the mechanisms underlying the loss of total protein expression when Trp-346 and Thr-349 were replaced with alanine, mutant-expressing cells were treated with lysosomal or proteasomal inhibitors. Our results showed that only proteasomal inhibitors resulted in the accumulation of mutant proteins, indicating that proteasome is involved in the degradation of the mutant transporters. Therefore, Trp-346 and Thr-349 are critically involved in the stability of the transporter.

Functional role of the C terminus of human organic anion transporter hOAT1.

Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536–550) or the last 30 amino acids (residues 521–550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506–550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506–520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.

An Essential Role of Nedd4-2 in the Ubiquitination, Expression, and Function of Organic Anion Transporter-3.

Organic anion transporter-3 (OAT3) is a member of the organic anion transporter family that mediates the body disposition of a diverse array of clinically important drugs. We previously demonstrated that activation of protein kinase C (PKC) inhibits OAT3 transport activity by accelerating OAT3 internalization from cell surface into intracellular compartments. In the current study, we established that PKC-induced inhibition of OAT3 transport activity occurred through an enhanced OAT3 ubiquitination, a process catalyzed by an E3 ubiquitin-protein ligase Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2). Overexpression of Nedd4-2 enhanced OAT3 ubiquitination, decreased OAT3 expression at the cell surface, and inhibited OAT3 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-2/C821A or siRNA knockdown of endogenous Nedd4-2 had opposite effects on OAT3. Furthermore, immunoprecipitation experiments conducted both in culture cells and with rat kidney slices showed that there was a physical interaction between OAT3 and Nedd4-2. In conclusion, our results provided the first evidence that Nedd4-2 is an important regulator for OAT3 ubiquitination, expression, and transport activity.

Nedd4-2 but not Nedd4-1 is Critical for Protein Kinase C-Regulated Ubiquitination, Expression and Transport Activity of Human Organic Anion Transporter 1

Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.

Transmembrane Peptide as Potent Inhibitor of Oligomerization and Function of Human Organic Anion Transporter 1

Human organic anion transporter 1 (hOAT1) plays a critical role in the body disposition of environmental toxins and clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We have demonstrated previously that hOAT1 forms homo-oligomers in cultured cells and in rat kidney. However, the functional consequence of such oligomerization has never been elucidated. In the current study, we used a novel approach by examining the effects of short hydrophobic peptides corresponding to transmembrane domains (TMDs) 1 to 12 of hOAT1 on the oligomerization and function of the transporter. We constructed expression vectors encoding short fusion peptides corresponding to TMDs 1 to 12 of hOAT1. These peptides were transfected into hOAT1-expressing COS-7 cells. Our results showed that among all 12 peptides examined, only the peptide corresponding to TMD 6 of hOAT1 significantly disrupted hOAT1 oligomerization demonstrated by cross-linking and coimmunoprecipitation experiments. The same peptide also caused a reduced expression of hOAT1 at the cell surface. As a result, hOAT1-mediated transport activity was compromised. Our data suggest that the peptide corresponding to TMD 6 of hOAT1 is a potent inhibitor of hOAT1 oligomerization and that oligomerization of hOAT1 is critical for the expression of the transporter at the cell surface and consequently for the proper function of the transporter.

Putative Transmembrane Domain 12 of the Human Organic Anion Transporter hOAT1 Determines Transporter Stability and Maturation Efficiency

Human organic anion transporter hOAT1 plays a critical role in the body disposition of clinically important drugs. In transmembrane segment (TM) 12, residues Tyr-490 and dileucine Leu-503/Leu-504 were identified to be critical for hOAT1 function. Substitution of Tyr-490 with alanine led to a dramatic reduction in protein expression of hOAT1 and its transport activity. The contribution of the side chain of Tyr-490 to transport activity was then evaluated by replacing this residue with Trp or Phe. Substitution of Tyr-490 with Trp or Phe partially or fully recovered the protein expression of hOAT1 and its transport activity, respectively, that were lost by substitution of Tyr-490 with alanine, suggesting that the aromatic ring and the size of the side chain of Tyr-490 are critical for hOAT1 expression and function. Studies with protease inhibitors and pulse-chase labeling further showed that the loss of expression of hOAT1 and its transport activity by replacing Tyr-490 with alanine resulted from accelerated degradation of the transporter, whereas its maturation efficiency was not affected. In contrast to Tyr-490, substitution of Leu-503/Leu-504 with alanine also resulted in complete loss of protein expression of hOAT1 and its transport activity. However, such loss of protein expression could not be prevented by treating mutant-expressing cells with protease inhibitors. Pulse-chase experiments showed that the mutant transporter (L503/L504A) was trapped in the endoplasmic reticulum without conversion into mature form of the transporter. Our results are the first to highlight the central role of TM 12 in maintaining the stability and in promoting the maturation efficiency of hOAT1.

Serum- and glucocorticoid-inducible kinase SGK2 regulates human organic anion transporters 4 via ubiquitin ligase Nedd4-2.

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, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT4 is abundantly expressed in the kidney and placenta. In the current study, we examined the regulation of hOAT4 by serum- and glucocorticoid-inducible kinase 2 (sgk2) in the kidney COS-7 cells. We showed that sgk2 stimulated hOAT4 transport activity. Such stimulation mainly resulted from an increased cell surface expression of the transporter, kinetically revealed as an increased maximal transport velocity Vmax without significant change in substrate-binding affinity Km. We further showed that regulation of hOAT4 activity by sgk2 was mediated by ubiquitin ligase Nedd4-2. Overexpression of Nedd4-2 enhanced hOAT4 ubiquitination, and inhibited hOAT4 transport activity, whereas overexpression of ubiquitin ligase-dead mutant Nedd4-2/C821A or siRNA knockdown of endogenous Nedd4-2 had opposite effects on hOAT4. Our co-immunoprecipitation experiment revealed that sgk2 weakened the association between hOAT4 and Nedd4-2. In conclusion, our study demonstrated for the first time that sgk2 stimulated hOAT4 transport activity by abrogating the inhibitory effect of Nedd4-2 on the transporter.

REGULATION OF HUMAN ORGANIC ANION TRANSPORTER 3 BY PEPTIDE HORMONE BRADYKININ

Human organic anion transporter (hOAT) 3 belongs to a family of organic anion transporters that play critical roles in the body disposition of numerous clinically important drugs. In the current study, we examined the regulation of hOAT3 by peptide hormone bradykinin (BK) in COS-7 cells. BK (≤500 nM) induced a concentration- and time-dependent stimulation of hOAT3 activity, kinetically revealed as an increased Vmax. Such an increase in Vmax resulted from an increased cell surface expression without a change in total cell expression of the transporter. BK-induced stimulation of hOAT3 activity could be prevented by treating hOAT3-expressing cells with staurosporine, a general inhibitor for protein kinase C (PKC). To obtain further information on which PKC isoform mediates BK regulation of hOAT3 activity, cellular distribution of various PKC isoforms was examined in cells treated with BK. We showed that BK treatment resulted in a significant translocation of PKCδ, PKCε, and PKCζ from cytosol to membrane. We further showed that BK treatment enhanced association of hOAT3 with PKCδ, PKCε, and PKCζ and that isoform-specific inhibitor for PKCδ, PKCε, and PKCζ reversed BK effect on hOAT3 activity. We therefore concluded that BK stimulated hOAT3 activity through activation of PKCδ, PKCε, and PKCζ, which then led to the redistribution of hOAT3 from the intracellular compartments to the cell surface and to the up-regulation of hOAT3 activity.