BRET assays

For G protein coupling experiments cells were washed twice with permeabilization buffer (KPS) containing 140 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 0.1 mM KEGTA, 20 mM NaHEPES (pH 7.2), harvested by trituration, permeabilized in KPS buffer containing 10 μg ml^-1 high purity digitonin, and transferred to opaque black 96-well plates. Measurements were made from permeabilized cells supplemented either with 100 μM GDP or 2U ml^-1 apyrase. For arrestin and trafficking experiments cells were washed twice in PBS and harvested by trituration in DPBS. For all experiments 5 μM coelenterazine h was used as a substrate. For the experiments shown in Fig 1, permeabilized cells were supplemented with apyrase, and GDP (100 μM) was injected during continuous recording using a Polarstar Optima plate reader (BMG Labtech, Offenburg, Germany). All other measurements were made using a Mithras LB940 photon-counting plate reader (Berthold Technologies GmbH, Bad Wildbad, Germany). Raw BRET signals were calculated as the emission intensity at 520–545 nm divided by the emission intensity at 475–495 nm. Net BRET signals were calculated as the raw BRET signal minus the raw BRET signal measured from cells expressing only the Rluc8 donor.

(A) Cartoon representation of the experimental design. Constitutively-active GPCRs fused to Renilla luciferase (Rluc8) form spontaneous active-state complexes with nucleotide-free G protein heterotrimers fused (via the Gβγ subunit) to the fluorescent protein Venus in the absence of activating ligands. Addition of GDP (100 μM) disrupts these complexes, decreasing BRET between GPCR-Rluc8 and Gαβγ-Venus. (B) Representative experiments of this type with GPR82 and GPR174. Traces represent the mean ± SD of 16 technical replicates from a single experiment, and each trace is normalized to the basal BRET observed for that particular G protein. GDP was injected where indicated by the horizontal bar.