Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study

Abstract

TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers – likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase

“…4C), or as a function of the ratio [A]/[D] (Fig. 4F), using a custom calibration procedure (see supporting information), as shown by others (14,15). In all cases, the FRET efficiency increases with the absolute or relative amount of acceptor.…”

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy

“…to date, only a very few reports exist of membrane receptor proteins organized in chain-like arrangements, among them rhodopsin (24), TGR5, a bile acid-sensing G-protein coupled receptor (25), and EGFR (26); interestingly, all of these receptors function as dimers, which also applies to HER2. We report here on the linear spatial arrangement of HER2 detected in HER2-overexpressing breast cancer cell lines.…”

Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity

“…Afterward, we obtained the b and g factors from the FRET versus stoichiometry 2D ALEX histograms ( Figures S8A and S8B). Using a Fö rster radius of 56 Å for Cy3-Cy5 (Greife et al, 2016), the corrected FRET values were obtained from the histograms.…”