GPR3 forms a dimer in its active state

(A) Cryo-EM map of the dimeric GPR3-Gs complex, showing the GPR3 dimer (cyan, green), two copies of mini-Gαs (light blue, grey), Gβ (blue), Gγ (magenta), and Nb35 (yellow). (B) Overall structure of the dimeric GPR3-Gs complex, colored according to the scheme in (A). (C) Cryo-EM map of the monomeric GPR3-Gs complex. (D) Extracellular view of the GPR3 dimer structure. (E) EM density showing the two parallel cholesterol molecules at the dimerization interface of GPR3.

(F) Electrostatic potential map of the potential endogenous lipid-binding surface, with the corresponding EM density for the lipid shown.

Putative endogenous lipid-binding site

(A) GPR3 residues in close proximity to the EM density corresponding to endogenous lipids are shown as stick representations. Two polar residues, H962.60 and Y2807.36 are labelled in red. (B-C) Effect of mutations in the putative lipid-binding site on cAMP levels in cells expressing GPR3 (B) or GPR6 (C) mutants, driven by the CMV and SFFV promoters. (D) Effect of mutations in the putative lipid-binding site of GPR12 on cAMP levels in cells expressing GPR12 mutants under the CMV promoter. All cAMP levels were measured using the cAMP accumulation assay and normalized as a percentage of the wild-type (WT) levels.

GPR3 forms a dimer in its inactive state

(A) Cryo-EM map of the GPR3-BRIL fusion protein (cyan, green) in complex with anti-BRIL Fab (blue) and anti-Fab nanobody (yellow). (B) Overall structure of the GPR3-BRIL fusion complex, using the same color scheme as in (A). (C) EM density map of the endogenous lipid (shown in magenta mesh), with key polar residues H962.60, Y2807.36, and the toggle switch W2606.48 represented as sticks. (D) Structural overlay of the active GPR3 monomer (yellow) with a protomer (cyan) of the GPR3 dimer. (E-G) Superposition of dimeric GPR3 structures in the inactive and active states, shown from the extracellular view (E), side view (F), and cytoplasmic view (G).

Dimerization interface of GPR3

(A) Residues involved in GPR3 dimer formation are shown as sticks. Non-conserved residues at the dimer interface in GPR6 are indicated in parentheses. (B) 2D class averages of the GPR6-BRIL fusion protein in complex with anti-BRIL Fab and an anti-Fab nanobody. (C) Fluorescence size-exclusion chromatography profiles of GPR3WT and the GPR3_6 chimera, in which dimer interface residues in GPR3 are replaced with the corresponding residues from GPR6. (D) cAMP levels in Expi293F cells expressing GPR3WT or the GPR3_6 chimera, driven by CMV and SFFV promoters, measured using the GloSensor cAMP accumulation assay.

Structural and functional characterization of AF64394 binding to GPR3

(A) cAMP accumulation assay showing the effect of AF64394 on GPR3, GPR6, GPR12, and D1R in the presence of 1 nM dopamine. (B) Cryo-EM map of AF64394-bound GPR3, highlighting the ligand (magenta), GPR3-BRIL (green and cyan), anti-BRIL Fab (blue) and an anti-Fab nanobody (yellow). The Fab and nanobody are poorly defined in one protomer, with the corresponding map colored in grey. (C) EM density of the endogenous lipid shown in magenta mesh, with key interacting residues H962.60, Y2807.36, and W2606.48 represented as sticks. (D) Close-up view of the AF64394 binding pocket. The EM density map for AF64394 is shown in grey mesh. Yellow dash line indicates hydrogen bonds. (E) cAMP accumulation assay in Expi293F cells expressing WT or mutant GPR3, showing the effect of site-specific mutations on the AF64394 response. (F) Superposition of inactive GPR3, active GPR3PFK, and AF64394-bound GPR3 intermediate structures, revealing conformational changes upon AF64394 binding. (G) Comparison of the active state and the AF64394-bound state of GPR3, showing a shift in ICL2 from an α-helical to a random coil structure. (H) Close-up view of interactions between the α-helical structure of ICL2 with Gαs in the active state. The transition of ICL2 to a random coil structure upon AF64394 binding disrupts Gs coupling. (I) cAMP accumulation assay in Expi293F cells expressing WT or triple-mutant GPR3PFK, demonstrating altered AF64394 response.