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.
- G-protein-coupled receptors
- Spatial Intensity Distribution Analysis
- quaternary structure
- ©2017 The Author(s)
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