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

Richard J. Ward, John D. Pediani, Kaleeckal G. Harikumar, Laurence J Miller, Graeme Milligan

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, 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 us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into 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. In 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, while 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.

Original languageEnglish (US)
Pages (from-to)1879-1895
Number of pages17
JournalBiochemical Journal
Volume474
Issue number11
DOIs
StatePublished - Jun 1 2017

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Secretin
Dimerization
Cell membranes
GTP-Binding Proteins
Receptor-Interacting Protein Serine-Threonine Kinases
Ligands
Facings
Butyric Acid
G-Protein-Coupled Receptors
Epidermal Growth Factor Receptor
Calibration
Luminance
Proteins
Up-Regulation
Down-Regulation
Cell Membrane
secretin receptor
Lipids
Peptides
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor. / Ward, Richard J.; Pediani, John D.; Harikumar, Kaleeckal G.; Miller, Laurence J; Milligan, Graeme.

In: Biochemical Journal, Vol. 474, No. 11, 01.06.2017, p. 1879-1895.

Research output: Contribution to journalArticle

Ward, Richard J. ; Pediani, John D. ; Harikumar, Kaleeckal G. ; Miller, Laurence J ; Milligan, Graeme. / Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor. In: Biochemical Journal. 2017 ; Vol. 474, No. 11. pp. 1879-1895.
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