Efficient Trafficking of MDR1/P-Glycoprotein to Apical Canalicular Plasma Membranes in HepG2 Cells Requires PKA-RIIα Anchoring and Glucosylceramide

Abstract

In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIalpha from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5'NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5'NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIalpha displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIalpha anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1.

Anchoring of Protein Kinase A-Regulatory Subunit IIα to Subapically Positioned Centrosomes Mediates Apical Bile Canalicular Lumen Development in Response to Oncostatin M but Not cAMP

“…Bile canalicular lumens were first identified with phase contrast and scored as NBD positive or negative with epifluorescence. Figure 6, A-D, shows that the lipid analogue reached the apical surface domain in untreated or OSM-treated cells expressing the AKAP-IS peptide, similarly as previously shown in control HepG2 cells Hoekstra, 1999, 2000;van der Wouden et al, 2002) and consistent with previous results (Wojtal et al, 2006). Basolaterally derived NBD-sphingomyelin reached 74 and 89% of the bile canalicular lumens after a chase of 15 and 30 min, respectively, in parental cells.…”

“…The expression level of PKA-RII␣ in cells expressing the peptide was not changed compared with parental HepG2 cells (cf. Wojtal et al, 2006). Importantly, in contrast to parental HepG2 cells or HepG2 cells expressing the scrambled peptide, treatment of cells expressing the AKAP-IS peptide with OSM at 37°C for 4 h did not result in an increase in the percentage of centrosomes that contained PKA-RII␣ ( Figure 3A), confirming that OSM stimulated the association of PKA-RII␣ with centrosomes in parental HepG2 cells by promoting its AKAP-mediated anchoring.…”

“…Stable transfectants were selected, and they were shown to express AKAP-IS or the scrambled peptide and displace PKA-RII␣ from natural anchoring sites or not, respectively (cf. Wojtal et al, 2006). In cells stably expressing the AKAP-IS peptide, but not the scrambled peptide, the percentage of PKA-RII␣-positive centrosomes (identified by ␥-tubulin) was significantly reduced ( Figure 3A).…”

“…HepG2 cells stably expressing the GFP/V5/His-tagged AKAP-IS peptide or scrambled peptide (GFP-QDVEIQLKAAYNQKLIAI-V5/His) (provided by John Scott (Howard Hughes Medical Institute, Vollum Institute, Oregon Health & Science University, Portland, OR 97239) and described in Alto et al (2003)) were created as described previously (Wojtal et al, 2006). Stable transfectants were cultured in medium supplemented with 800 g/ml Geneticin (G-418; Invitrogen, Carlsbad, CA).…”

MDR1 in immunity: friend or foe?

“…4 Several studies have suggested the ability of PKC and PKA to increase the functionality of MDR1 or its membrane integration. 29,30 Other studies showed that the preferential localization of MDR1 in the lipid rafts and caveolae, characterized as detergent resistant membranes (DRM), is required for its functionality. 31,32 Indeed, cholesterol depletion that disrupts lipid rafts, abolishes membrane MDR1 localization and is associated with reduced efflux capacities.…”