An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism

Abstract

GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.

Fig. 1. Cryo-EM structure of active-state GPR61-G protein complex.

a Map (left) and model (right) of active-state GPR61-G protein complex with scFv16, colored by subunit. Grey lines indicate the position of the plasma membrane. b Highlighted by inset from a, left panel. Left panel, Extracellular face of GPR61, with ECL2 (extracellular loop 2) highlighted in orange and residues participating in the conserved disulfide shown in stick representation. Right panel, The orthosteric pocket of GPR61 is highlighted with a red dotted line. Key residues of ECL2 (orange) blocking access to the lower portion of the pocket are shown in stick representation. Transmembrane helices are labeled with numbered TM notations. c–f. Key residues involved in GPR61-Gαs interaction, corresponding to the highlighted region of A, right panel. c Polar interactions d Hydrogen bond network underlying selectivity for Gαs. e D/ERY motif hydrophobic stacking interactions with Gαs. f Hydrophobic interactions. g A disulfide bridging GPR61 TM6 and TM7, with nearby motifs involved in activation switching highlighted.

Fig. 2. Inverse agonist compound structure and characterization.

a Chemical structure of Compound 1. b Relative activity in the cAMP assay of wild-type (WT) GPR61 and HiBit-tagged GPR61 used for measuring cell surface expression. c Total and surface expression of GPR61 as measured using HiBit-tagged GPR61. d Compound 1 cyclic adenosine monophosphate (cAMP) assay inhibition curves and IC₅₀ values for GPR61 (Untagged and HiBit-tagged). e General schematic indicating the expected directions of concentration-dependent responses to agonist, antagonist, and inverse agonist ligands, respectively, by a generic constitutively active receptor. In panels b–d, bar plots and error bars represent the mean ± SEM. N = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 3. Structural and functional analysis of compound 1 binding to GPR61_IA.

a Cryo-EM map of apo GPR61_IA, colored by subunit. The sequence inserted into GPR61 (comprising BRIL and A₂AR-derived linker sequences) is colored in cyan. b Cryo-EM map of GPR61 _IA bound to compound 1, colored as in a. The compound 1 binding site is indicated by the dotted box. Inset shows compound 1, colored in magenta, fitted into its corresponding map density. c A comparison of apo and compound 1-bound GPR61_IA conformations, showing the conformational changes induced by binding of compound 1. Ribbon diagrams, with TM6 highlighted, are shown at left, with a cutaway of the corresponding surface representation shown to the right. d Compound 1 (Cpd 1) is shown in magenta with its binding site, with key interaction residues shown in stick representation. Map density for an ordered water is shown as dark gray mesh. Hydrogen bonds are indicated by dashed lines. e GPR61 cAMP IC₅₀ curves and values for WT GPR61 and the indicated mutants. Parenthetical values in the table represent 95% CI and asterisks indicate statistical significance. f Basal activity of GPR61 WT and mutants normalized to relative surface expression (total activity and expression data are included in Supplementary Fig. 7a, b). Bar plots and error bars represent the mean ± SEM. In panels e, f, statistical significance is indicated with asterisks (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) and was assessed using one-way ANOVA with one-sided Dunnett’s post hoc test. N = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 4. Analysis of compound 1 inverse agonist mechanism.

a Key residue clashes and conformational changes induced by binding of compound 1 (Cpd 1) to GPR61. The structure of active GPR61 (light grey) is overlaid with the compound 1 (magenta)-bound structure of inactive GPR61 (blue), with key residues highlighted in stick representation. Clashes with the compound are indicated by red stars, while clashes with Gαs induced by compound binding are indicated by yellow stars. b Compound 1 defines an unusual allosteric site and mechanism. The structure of compound 1-bound GPR61_IA is shown in ribbon representation, with published exemplars^,– representing the known allosteric sites of class A GPCRs superimposed, colored as indicated. c Compound 1-bound GPR61_IA and vercirnon-bound CCR9 structures, colored as indicated, are superimposed. Vercirnon occupies the known allosteric site that lies nearest to that of compound 1. The different conformations of TM6 induced by these two inverse agonists are highlighted.

Fig. 5. A G protein-competitive mechanism of allosteric GPCR inverse agonism.

In its constitutively active state (top panel), GPR61 adopts a conformation that allows binding and nucleotide exchange of the G protein complex (bottom left panel) to stimulate cyclic AMP-mediated signaling through activation of adenylate cyclase. Inverse agonist compound 1 (magenta) binds to an intracellular allosteric pocket overlapping that bound by Gαs and acts as a wedge to remodel the flanking helices (yellow arrow), destroying the Gαs-binding pocket and creating direct clashes that prevent potential Gαs binding.