Jo Suda1, Lixin Zhu, Curtis T Okamoto, Serhan Karvar. 1. Division of Gastrointestinal & Liver Diseases, University of Southern California Keck School of Medicine, Los Angeles, California 90033, USA.
Abstract
BACKGROUND & AIMS: Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation. METHODS: We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation. RESULTS: A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion. CONCLUSIONS: Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion.
BACKGROUND & AIMS: Rabs are monomeric guanosine triphosphatases that regulate membrane trafficking and acid secretion in gastric parietal cells. Using a proteomics approach, we identified a new Rab, Rab27b, in tubulovesicle membranes and determined its role in parietal cell activation. METHODS: We used mass spectrometry (MS) to identify Rab27b in purified tubulovesicular membrane fractions and used immunoblot and immunofluorescence analyses to study its expression. Wild-type, constitutively active (Rab27bQ78L), and dominant negative (Rab27bN133I) forms of Rab27b were tagged with yellow fluorescent protein (YFP) and expressed in parietal cells using adenoviral constructs to study localization and function. Localization was visualized by fluorescence microscopy in resting and stimulated cells. Acid secretion in primary cell cultures was measured by aminopyrine accumulation. RESULTS: A tandem MS peptide mass fingerprint was matched to 7 peptides of Rab27b. Rab27b localized to tubulovesicle membranes, based on immunoblot and immunocytochemical analyses. Endogenous Rab27b, YFP/wild-type Rab27b, Rab27bQ78L, and Rab27bN133I all distributed throughout the cytoplasm of resting parietal cells. After stimulation, wild-type Rab27b and YFP-Rab27bQ78L translocated to the apical membrane, but YFPR-ab27bN133I did not. Expression of wild-type YFP-Rab27b or YFP-Rab27bQ78L did not affect acid secretion, whereas expression of Rab27bN133I almost completely inhibited acid secretion. CONCLUSIONS: Rab27b is associated with tubulovesicle membranes in the parietal cell and Rab27b may play a role in stimulation-associated membrane recruitment and gastric acid secretion.
Authors: Lilian Chiang; Julie Ngo; Joel E Schechter; Serhan Karvar; Tanya Tolmachova; Miguel C Seabra; Alistair N Hume; Sarah F Hamm-Alvarez Journal: Am J Physiol Cell Physiol Date: 2011-04-27 Impact factor: 4.249
Authors: Janine F Felix; Jonathan P Bradfield; Claire Monnereau; Ralf J P van der Valk; Evie Stergiakouli; Alessandra Chesi; Romy Gaillard; Bjarke Feenstra; Elisabeth Thiering; Eskil Kreiner-Møller; Anubha Mahajan; Niina Pitkänen; Raimo Joro; Alana Cavadino; Ville Huikari; Steve Franks; Maria M Groen-Blokhuis; Diana L Cousminer; Julie A Marsh; Terho Lehtimäki; John A Curtin; Jesus Vioque; Tarunveer S Ahluwalia; Ronny Myhre; Thomas S Price; Natalia Vilor-Tejedor; Loïc Yengo; Niels Grarup; Ioanna Ntalla; Wei Ang; Mustafa Atalay; Hans Bisgaard; Alexandra I Blakemore; Amelie Bonnefond; Lisbeth Carstensen; Johan Eriksson; Claudia Flexeder; Lude Franke; Frank Geller; Mandy Geserick; Anna-Liisa Hartikainen; Claire M A Haworth; Joel N Hirschhorn; Albert Hofman; Jens-Christian Holm; Momoko Horikoshi; Jouke Jan Hottenga; Jinyan Huang; Haja N Kadarmideen; Mika Kähönen; Wieland Kiess; Hanna-Maaria Lakka; Timo A Lakka; Alexandra M Lewin; Liming Liang; Leo-Pekka Lyytikäinen; Baoshan Ma; Per Magnus; Shana E McCormack; George McMahon; Frank D Mentch; Christel M Middeldorp; Clare S Murray; Katja Pahkala; Tune H Pers; Roland Pfäffle; Dirkje S Postma; Christine Power; Angela Simpson; Verena Sengpiel; Carla M T Tiesler; Maties Torrent; André G Uitterlinden; Joyce B van Meurs; Rebecca Vinding; Johannes Waage; Jane Wardle; Eleftheria Zeggini; Babette S Zemel; George V Dedoussis; Oluf Pedersen; Philippe Froguel; Jordi Sunyer; Robert Plomin; Bo Jacobsson; Torben Hansen; Juan R Gonzalez; Adnan Custovic; Olli T Raitakari; Craig E Pennell; Elisabeth Widén; Dorret I Boomsma; Gerard H Koppelman; Sylvain Sebert; Marjo-Riitta Järvelin; Elina Hyppönen; Mark I McCarthy; Virpi Lindi; Niinikoski Harri; Antje Körner; Klaus Bønnelykke; Joachim Heinrich; Mads Melbye; Fernando Rivadeneira; Hakon Hakonarson; Susan M Ring; George Davey Smith; Thorkild I A Sørensen; Nicholas J Timpson; Struan F A Grant; Vincent W V Jaddoe Journal: Hum Mol Genet Date: 2015-11-24 Impact factor: 6.150