Cusabio G-protein coupled Transducer Recombinants

Abstract

Although several recent studies have reported that GPCRs adopt multiple conformations, it is unclear how subtle conformational changes translate into divergent downstream responses. In this study, we report a new class of FRET-based sensors that can detect ligand/mutagenic stabilization of GPCR conformations that promote G-protein interactions in living cells. These sensors are based on the well-characterized interaction between a GPCR and the C-terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved RY (E/D) motif on GPCR conformation.

Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signalling. Therefore, it is unclear whether ionic interactions formed by the (E/D)RY (ion-locking) motif are necessary to stabilize G-protein coupled Transducer Recombinants (GPCR) basal states. We found that β2-AR(E/D)RY ion-blocking mutagenesis enhances the interaction with Gs. However, only Glu/Asp, but not Arg, mutants increase G-protein activation. In contrast, ion-blocking mutagenesis of opsin (E/D)RY does not alter its interaction with transducin.

Instead, opsin-specific ionic interactions centred at residue Lys-296 are necessary and sufficient to promote interactions with transducin. Efficient suppression of basal β2-AR activity by the inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with Gs and promotes Gi binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.

Keywords: Motive (E/D)RY; 7-Helix receptor; Phone signal; To worry; Functional Selectivity; G protein-coupled receptors (GPCRs); G proteins.

Cell and Calcium Log

HEK293 cells were recently obtained from ATCC (catalogue no. CRL-1573; Middlesex, UK) for reproducibility with GPCR, G-protein, and GAP expression data from the previous studies16. Cells were grown in DMEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FCS and penicillin-streptomycin at 37°C and 5% CO2.

 

Trypsinized cells were seeded on poly-L-lysine-coated coverslips for 24 min and then loaded with 5 µM Fura-2 AM (Biotium, Fremont, CA) in DPBS without calcium and magnesium (Thermo Fisher Scientific) for 30 min, followed by a wash in HBSS supplemented with 10 mM HEPES pH 7.4 (HBSS-HEPES; Thermo Fisher Scientific). Cells were mounted in a perfusion chamber (Warner Instruments, Hamden, CT) and recorded (excitation (100 ms): 340, 380 nm, emission 520 nm) at one frame per second on a VisiScope Cell Explorer System (Visitor Systems, Puchheim, Germany) equipped with a Plan Neofluar ×40/1.3 oil objective on an Axio Observer.A1 microscope (Zeiss, Jena, Germany), a CoolSNAP HQ2 CCD camera (Photometrics, Tucson, AZ), a VisiChrome polychromator with a 75-watt xenon lamp, and MetaFluor Fluorescence Ratio Imaging software (Molecular Devices, Sunnyvale, CA).

Solutions were changed with the flow controller (Cell MicroControls, Norfolk, VA) and Solution Changer Manifold MSC-200 (Bio-Logic, Seyssinet-Pariset, France), and cells were stimulated with seven different acetylcholine (Ach; Sigma-Aldrich, St. Louis, MO) concentrations in HBSS-HEPES: 100nM, 250nM, 500nM, 750nM, 1.5μM, 3μM, and 10μM. This set of concentrations was selected empirically. It allows detecting a minimum observable cellular response at lower concentrations and reaching a maximum cellular response plateau for higher concentrations.

Ten-second stimulations with each concentration of Ach were repeated five times with an interval of 110 s in which the cells were rinsed with HBSS-HEPES. This temporal protocol was selected taking into account the kinetics of the cell’s Ach response: with the experimental setup used, it takes between 7 and 9 s for a cell to reach the maximum value of the calcium response. Ionomycin (2 μM; Sigma-Aldrich) was added at the end of some experiments.