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Elucidating Interactions Between Human Mas‐related G‐protein Coupled Receptors
Author(s) -
Van Remoortel Samuel,
Arora Rohit,
Labro Alain J.,
Snyders Dirk J.,
Buckinx Roeland,
Timmermans JeanPierre
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1021.2
Subject(s) - g protein coupled receptor , receptor , förster resonance energy transfer , transfection , microbiology and biotechnology , hek 293 cells , fluorescence recovery after photobleaching , complementation , photobleaching , luciferase , bimolecular fluorescence complementation , biology , protein–protein interaction , chemistry , biophysics , biochemistry , gene , fluorescence , membrane , phenotype , physics , quantum mechanics
G‐protein coupled receptors (GPCR's) are important targets in drug discovery research because of their extensive regulatory effects on cellular functioning and their profound molecular diversity. A decade ago a new GPCR family, referred to as Mas‐related G‐protein coupled receptors (MRGPR), was discovered and since then shown to be involved in nociception and mast cell physiology. MRGPR are thus interesting targets in pain treatment and neuro‐immune communication. Previously, we were able to show the involvement of MRGPR in gastro‐intestinal neuro‐immune communication and observed a marked co‐expression of MRGPRD, MRGPRE and MRGPRF in the murine enteric nervous system (Avula et al. 2011; Avula et al. 2013). Given the well‐known concept of GPCR heteromerization, as well as its importance in functional modulation, we investigated heteromeric properties of the human orthologues of MRGPRD, MRGPRE and MRGPRF. Material and methods Heteromeric MRGPR interactions were efficiently screened using a novel luciferase complementation‐based platform. Further validation of interacting partners was performed with Fluorescence Resonance Energy Transfer (FRET). Cells were transiently transfected with eCFP‐ and/or eYFP‐coupled receptors, and FRET efficiency was determined with acceptor photobleaching. For MRGPRD plasmids, receptor functionality was tested according to the known MRGPRD‐mediated inhibition of KCNQ2‐potassium currents. Results Transient transfections with fluorescently tagged vector constructs confirmed membrane expression of the vector protein product. Ligand‐induced activation of tagged MRGPRD resulted in reversible inhibition of KCNQ2‐mediated potassium currents, indicating functionality of fluorescently tagged MRGPR. Interaction screening with luciferase complementation provided clear evidence for heteromeric interactions between MRGPRE and MRGPRF. FRET analysis on transiently co‐transfected cells confirmed this MRGPRE‐MRGPRF interaction. Conclusion This study provides novel insights on the interactions between human MRGPR. The observed MRGPRE‐MRGPRF interaction contributes to the further elucidation of the function of these orphan receptors, and strengthens the relevance of heteromeric interactions in their receptor biology. Furthermore, we provide a proof‐of‐concept for the applicability of a new complementation‐based screening assay as a high‐throughput screening tool for protein interactions. Support or Funding Information This study was financially supported by FWO grant G019314N.