title: Efficient trafficking of MDR1/P-glycoprotein to apical canalicular plasma membranes in HepG2 cells requires PKA-RIIalpha 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.
authors.affiliation: Section of Membrane Cell Biology, Department of Cell Biology, University Medical Center Groningen, University of Groningen, 9713 AV Groningen, The Netherlands.
authors.forename: Kacper A
authors.initials: KA
authors.lastname: Wojtal
authors.forename: Erik
authors.initials: E
authors.lastname: de Vries
authors.forename: Dick
authors.initials: D
authors.lastname: Hoekstra
authors.forename: Sven C D
authors.initials: SC
authors.lastname: van Ijzendoorn
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001648:Bile Canaliculi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D016764:Cell Polarity / Q000187:drug effects; D002478:Cells, Cultured; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005963:Glucosylceramides / Q000096:biosynthesis / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D021381:Protein Transport / Q000187:drug effects
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D048868:Adaptor Proteins, Signal Transducing; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D005963:Glucosylceramides; C517819:PRKAR2A protein, human; D003994:Bucladesine; D017868:Cyclic AMP-Dependent Protein Kinases