Correlation between Apical Localization of Abcc2/Mrp2 and Phosphorylation Status of Ezrin in Rat Intestine

Abstract

ABSTRACT:The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 ؎ 2.1 nmol/cm to 18.1 ؎ 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.The small intestine is a highly differentiated organ with a barrier function against xenobiotics and a gateway function for nutrients. The ATP-binding cassette (ABC) transporter family, including P-glycoprotein/multidrug resistance protein 1 (P-gp/Mdr1/Abcb1), breast cancer resistant protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2), are well known efflux transporters that are located on the brush-border membrane (BBM) of small intestinal epithelia to limit the absorption of a broad range of compounds (Takano et al., 2006). The distinct but sometimes overlapping substrate specificities of these efflux transporters have been shown previously (Chan et al., 2004). Indeed, a significant increase in the oral absorption of their substrates, including drugs and carcinogens, was confirmed in animals genetically deficient in these transporters (Dietrich et al., 2001;Yamaguchi et al., 2002).Several reports indicated the disparity between mRNA and protein expression of Abcc2 in the intestine. Naud et al. (2007) reported that Abcc2 protein expression was reduced to approximately 40% of that of the control during chronic renal failure in rats, but its mRNA expression was unaffected. Dietrich et al. (2004) also reported ABCC2 protein expression was reduced to approximately 27% of that of the control patients in the human intestine du...

Changes in PtdIns(4,5)P2 Induced by Etoposide Treatment Modulates Small Intestinal P-Glycoprotein <i>via</i> Radixin

“…44) Accordingly, whether certain membrane proteins decide which to choose out of the ERM as each partner for their plasma membrane localization may be dependent on the kinds of tissues and/or membrane protein itself. 6,45,[48][49][50]52) It also remains to be determined what increases in the amount of both radixin and P-gp bound to PI(4,5) P2 means under our experimental condition. Because of increases in the both protein expressions of the input as shown in our previously study, 6) it may be merely due to that contact probability of P-gp and radixin could be simply elevated, which may in turn magnifies the number of molecular complex at the small intestinal membrane.…”

“…Several studies have clearly demonstrated that PtdIns(4,5) P2 is a critical factor for promoting the dissociation of ERM, 22,25,26) which enables ERM translocation to the plasma membrane where the ERM proteins can interact with various membrane proteins. [44][45][46][47][48][49] Moreover, we recently demonstrated the existence of molecular interactions between P-gp and each of the ERM proteins in the small intestinal membrane by immunoprecipitation analysis using an anti-P-gp antibody.…”

Radixin Influences the Changes in the Small Intestinal P-Glycoprotein by Etoposide Treatment

“…4,14,20) As is well documented using histological methods, ezrin or moesin are mainly localized on the epithelial cells of villi surfaces or the endothelial cells of the sub-epithelial capillaries, respectively. 28,29,40) In contrast, radixin has been shown to be widely distributed in the epithelial and/or endothelial cells of the small intestine as indicated by immunofluorescence analyses. 12,29) In recent years, some researchers have proposed that radixin functions as a scaffold protein for P-gp and other membrane transporters not only in the canalicular membrane of the hepatocytes, 14,20) but also in the small intestine, similarly to ezrin.…”

“…12) Ezrin is expressed in the epithelium, 12,18,29) and is the only ERM protein that has been proven to interact with P-gp by use of immunoprecipitation analysis. 28) On the other hand, radixin is expressed in the liver 14,17,20,31) and kidney, 29) as well as in the epithelium of the small intestine, 32) although not to the extent of ezrin. 18,29) Furthermore, radixin has been just recently proposed to play a crucial role in regulating the membrane localization and functional activity of P-gp in the small intestinal by using mice lacking radixin.…”

“…Perhaps for this reason, there have been no reports describing the protein-protein interactions between P-gp and each ERM member in the small intestine except for ezrin. 28) Although all ERM proteins are expressed in the small intestine, 12,18,29,30) each proteins show a different distribution in this tissue. Among the ERM proteins expressed in the small intestine, ezrin is believed to present most abundant.…”