title: Recycling of Raft-associated prohormone sorting receptor carboxypeptidase E requires interaction with ARF6.
abstract: Little is known about the molecular mechanism of recycling of intracellular receptors and lipid raft-associated proteins. Here, we have investigated the recycling pathway and internalization mechanism of a transmembrane, lipid raft-associated intracellular prohormone sorting receptor, carboxypeptidase E (CPE). CPE is found in the trans-Golgi network (TGN) and secretory granules of (neuro)endocrine cells. An extracellular domain of the IL2 receptor alpha-subunit (Tac) fused to the transmembrane domain and cytoplasmic tail of CPE (Tac-CPE25) was used as a marker to track recycling of CPE. We show in (neuro)endocrine cells, that upon stimulated secretory granule exocytosis, raft-associated Tac-CPE25 was rapidly internalized from the plasma membrane in a clathrin-independent manner into early endosomes and then transported through the endocytic recycling compartment to the TGN. A yeast two-hybrid screen and in vitro binding assay identified the CPE cytoplasmic tail sequence S472ETLNF477 as an interactor with active small GTPase ADP-ribosylation factor (ARF) 6, but not ARF1. Expression of a dominant negative, inactive ARF6 mutant blocked this recycling. Mutation of residues S472 or E473 to A in the cytoplasmic tail of CPE obliterated its binding to ARF6, and internalization from the plasma membrane of Tac-CPE25 mutated at S472 or E473 was significantly reduced. Thus, CPE recycles back to the TGN by a novel mechanism requiring ARF6 interaction and activity.
authors.affiliation: Section of Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Arnaoutova
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Jackson
authors.forename: Omayma S
authors.initials: OS
authors.lastname: Al-Awar
authors.forename: Julie G
authors.initials: JG
authors.lastname: Donaldson
authors.forename: Y Peng
authors.initials: YP
authors.lastname: Loh
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D043423:Carboxypeptidase H / Q000378:metabolism*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005089:Exocytosis; D006056:Golgi Apparatus / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D009154:Mutation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D020798:Two-Hybrid System Techniques
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D011993:Recombinant Fusion Proteins; D043423:Carboxypeptidase H; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human