Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor

Ward, R. J., Pediani, J. D. , Harikumar, K. G., Miller, L. J. and Milligan, G. (2017) Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor. Biochemical Journal, 474(11), pp. 1879-1895. (doi: 10.1042/BCJ20170184) (PMID:28424368) (PMCID:PMC5442643)

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Abstract

Previous studies have indicated that the G protein-coupled secretin receptor is present as a homo-dimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated, is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and Spatial Intensity Distribution Analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed demonstration that the Epidermal Growth Factor receptor is predominantly monomeric in the absence of ligand and whilst wild type receptor was rapidly converted to a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that at moderate expression levels the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. By contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein-protein interactions between copies of the receptor polypeptide, whilst short term treatment with secretin had no effect on organization of the wild type receptor but increased the dimeric proportion of the mutated receptor variant.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Pediani, Dr John and Ward, Dr Richard and Milligan, Professor Graeme
Authors: Ward, R. J., Pediani, J. D., Harikumar, K. G., Miller, L. J., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Biochemical Journal
Publisher:Portland Press
ISSN:0264-6021
ISSN (Online):1470-8728
Published Online:19 April 2017
Copyright Holders:Copyright © 2017 The Authors
First Published:First published in Biochemical Journal 474(11):1879-1895
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
656901The organisational structure of class A GPCRs: Implications for pharmacology, function and therapeutic regulationGraeme MilliganMedical Research Council (MRC)MR/L023806/1RI MOLECULAR CELL & SYSTEMS BIOLOGY