Figure 2: Identification of synaptamide receptor and associated G-protein.

Biotinylated synaptamide analogue G1 (a) increased cAMP production in DIV3 neurons after 10 min stimulation (b). After mouse fetal brains or NSCs were lysed in PBS containing 0.5% Triton X-100, treated with G1, affinity-purified using streptavidin beads and subjected to SDS/PAGE, the entire gel was cut into several bands for tryptic digestion and protein identification (ID) by mass spectrometric analysis (c). The MS/MS spectra of GPR110 peptides G(432–442)R and G(460–471)K detected from the gel band in the 100–130 kD molecular weight region are shown (d). Binding of G1 to GPR110 was confirmed by the western blot detection of mGPR110-HA expressed in HEK cells at ∼100 kD after streptavidin pull-down while synaptamide dose-dependently decreased the mGPR110-HA recovered after G1-streptavidin pull-down (e). Among fatty acid ethanolamides, only synaptamide displaced the G1 binding to mGPR110 (f). GPR110 expression in the plasma membrane was detected (g). The cAMP response induced by 10 nM synaptamide increased in a GPR110 gene-dose-dependent manner (h). GPR110 interaction with Gαs was identified by co-immunoprecipitation of overexpressed mGPR110-HA with endogenous Gαs (i). Syn, synaptamide; n3, omega-3; n6, omega-6; DPEA, N-docosapentaenoylethanolamine; OEA, N-oleoylethanolamine; AEA, N-arachidonylethanolamine. Results in d and i represent two independent experiments. Data in (b,e,f,h) are means±s.e.m. of biological triplicates (n=3), representing three independent experiments. **P<0.01, ***P<0.001 versus control, and ^#P<0.05, ^##P<0.01, ^###P<0.001 versus G1 binding by unpaired Student t-test. ¶, relative to control. Scale bar, 5 μm (g).