A brighter method to illuminate the dark side of adhesion G protein-coupled receptors: detection of the ADGRG1 dimers by Bioluminescence Resonance Energy Transfer (BRET)

Murat, Merve
G protein-coupled receptors (GPCRs) form oligomeric complexes in living cells, and these complex structures affect the receptor maturation, trafficking, and signaling processes. Adhesion G protein-coupled receptors (aGPCRs), the second-largest sub-family of GPCRs, interact with extracellular matrix and ligands on the adjacent cell surface to modulate tissue and organ development. However, it is still unclear whether aGPCRs interact with each other. In this study, ADGRG1 was used as a model aGPCR to investigate the aGPCR dimerization in living cells using Bioluminescence Resonance Energy Transfer (BRET). BRET donor (NLuc) or acceptor (EGFP) tagged ADGRG1 receptors at various positions at C-terminus were expressed in HEK 293 cells. The cellular localizations of tagged receptors verified by laser scanning confocal imaging. The expression of C-terminally tagged constructs were determined using Western blot technique and their function was assessed with a bioluminesence based Serum Response Element (SRE) assay. The EGFP-tagged ADGRG1 constructs were all functional with elevated SRE activity as in the case for WT, and N-terminally truncated receptors. Since both NanoLuc (NLuc) and Renilla luciferase (RLuc) oxidize coelenterazine, NLuc-tagged ADGRG1 receptors could not be tested for the function using this method. However, as the tagging position was same and the tag NLuc was smaller in size these constructs were assumed to be functional. Various constructs of ADGRG1 tagged with NLuc were co-transfected with ADGRG1 EGFP constructs to determine the in vivo dimerization of ADGRG1. BRET resulting from the interacting receptor dimers was measured. Observed BRET increased in constructs tagged after 667th amino acid at C-terminus compared to the constructs with NLuc and EGFP appended at the end of C-terminus. The results given herein, suggest for the first time the homodimerization and may be oligomerization of ADGRG1, an aGPCR.


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Citation Formats
M. Murat, “A brighter method to illuminate the dark side of adhesion G protein-coupled receptors: detection of the ADGRG1 dimers by Bioluminescence Resonance Energy Transfer (BRET),” M.S. - Master of Science, Middle East Technical University, 2021.