Salman B Hosawi1,2, Jonathan D Humphries1, Richard J Coward3, David Knight4, Martin J Humphries1, Rachel Lennon1,5. 1. Wellcome Centre for Cell-Matrix Research, University of Manchester, Manchester, M13 9PT, UK. 2. Department of Biochemistry, King AbdulAziz University, Jeddah, Saudi Arabia. 3. Academic Renal Unit, University of Bristol, Bristol, UK. 4. Biomolecular Analysis Core Facility, University of Manchester, Manchester, M13 9PT, UK. 5. Department of Paediatric Nephrology, Royal Manchester Children's Hospital, Manchester, UK.
Abstract
Background: Insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival. Insulin resistance describes the inability of cells to activate the insulin signalling pathway effectively; leading to pathological effects in multiple organ systems including the kidney. In diabetic kidney disease, there is progressive glomerular dysfunction and recent studies have demonstrated that the kidney podocyte is a direct target for insulin action. In this study we defined the literature-based insulin receptor (INSR) interactome and utilised an unbiased proteomic approach to examine INSR interactors in podocytes. Methods: Human podocytes expressing the INSR were characterised under basal and insulin resistant conditions. The INSR was isolated by whole cell immunoprecipitation following a time course stimulation of 2, 7, and 15 minutes with of 100nM insulin. The resulting INSR complexes were analysed by label-free mass spectrometry (MS) to detect protein interactors. Results: We identified 27 known, direct INSR interactors in addition to novel interactors including doublecortin domain-containing protein 2 (DCDC2). The interaction of DCDC2 with the INSR was confirmed by immunoprecipitation and immunofluorescence, and under insulin resistant conditions, DCDC2 had increased association with the INSR. siRNA knockdown of DCDC2 in podocytes resulted in cell morphological change and altered INSR localisation. Conclusion: This study provides insight into the complexity of INSR interactors in podocytes and highlights DCDC2 as a novel INSR binding protein. Involvement of this novel interactor in insulin signalling and podocyte biology may explain how insulin resistance alters morphology and integrity of the glomerular filtration barrier. Copyright:
Background: Insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival. Insulin resistance describes the inability of cells to activate the insulin signalling pathway effectively; leading to pathological effects in multiple organ systems including the kidney. In diabetic kidney disease, there is progressive glomerular dysfunction and recent studies have demonstrated that the kidney podocyte is a direct target for insulin action. In this study we defined the literature-based insulin receptor (INSR) interactome and utilised an unbiased proteomic approach to examine INSR interactors in podocytes. Methods: Human podocytes expressing the INSR were characterised under basal and insulin resistant conditions. The INSR was isolated by whole cell immunoprecipitation following a time course stimulation of 2, 7, and 15 minutes with of 100nM insulin. The resulting INSR complexes were analysed by label-free mass spectrometry (MS) to detect protein interactors. Results: We identified 27 known, direct INSR interactors in addition to novel interactors including doublecortin domain-containing protein 2 (DCDC2). The interaction of DCDC2 with the INSR was confirmed by immunoprecipitation and immunofluorescence, and under insulin resistant conditions, DCDC2 had increased association with the INSR. siRNA knockdown of DCDC2 in podocytes resulted in cell morphological change and altered INSR localisation. Conclusion: This study provides insight into the complexity of INSR interactors in podocytes and highlights DCDC2 as a novel INSR binding protein. Involvement of this novel interactor in insulin signalling and podocyte biology may explain how insulin resistance alters morphology and integrity of the glomerular filtration barrier. Copyright:
Authors: Markus Schueler; Daniela A Braun; Gayathri Chandrasekar; Heon Yung Gee; Timothy D Klasson; Jan Halbritter; Andrea Bieder; Jonathan D Porath; Rannar Airik; Weibin Zhou; Joseph J LoTurco; Alicia Che; Edgar A Otto; Detlef Böckenhauer; Neil J Sebire; Tomas Honzik; Peter C Harris; Sarah J Koon; Meral Gunay-Aygun; Sophie Saunier; Klaus Zerres; Nadina Ortiz Bruechle; Joost P H Drenth; Laurence Pelletier; Isabel Tapia-Páez; Richard P Lifton; Rachel H Giles; Juha Kere; Friedhelm Hildebrandt Journal: Am J Hum Genet Date: 2014-12-31 Impact factor: 11.025
Authors: Beatriz Santamaria; Eva Marquez; Abigail Lay; RoseaMarie M Carew; Águeda González-Rodríguez; Gavin I Welsh; Lan Ni; Lorna J Hale; Alberto Ortiz; Moin A Saleem; Derek P Brazil; Richard J Coward; Ángela M Valverde Journal: Biochim Biophys Acta Date: 2015-09-16
Authors: Dattatreya Mellacheruvu; Zachary Wright; Amber L Couzens; Jean-Philippe Lambert; Nicole A St-Denis; Tuo Li; Yana V Miteva; Simon Hauri; Mihaela E Sardiu; Teck Yew Low; Vincentius A Halim; Richard D Bagshaw; Nina C Hubner; Abdallah Al-Hakim; Annie Bouchard; Denis Faubert; Damian Fermin; Wade H Dunham; Marilyn Goudreault; Zhen-Yuan Lin; Beatriz Gonzalez Badillo; Tony Pawson; Daniel Durocher; Benoit Coulombe; Ruedi Aebersold; Giulio Superti-Furga; Jacques Colinge; Albert J R Heck; Hyungwon Choi; Matthias Gstaiger; Shabaz Mohammed; Ileana M Cristea; Keiryn L Bennett; Mike P Washburn; Brian Raught; Rob M Ewing; Anne-Claude Gingras; Alexey I Nesvizhskii Journal: Nat Methods Date: 2013-07-07 Impact factor: 28.547
Authors: Abigail C Lay; Jenny A Hurcombe; Virginie M S Betin; Fern Barrington; Ruth Rollason; Lan Ni; Lawrence Gillam; Grace M E Pearson; Mette V Østergaard; Hellyeh Hamidi; Rachel Lennon; Gavin I Welsh; Richard J M Coward Journal: Diabetologia Date: 2017-08-29 Impact factor: 10.122