Extended Data Fig. 11: Potential structural mechanism of SPE and PEA recognition among TAAR members.

a, The potency and efficacy of Gαs-Gβγ dissociation in receptor (mTAAR2-6, 7d, 8c, and 9)-overexpressing HEK293 cells in response to stimulation with PEA. The heatmap is colored according to the value of pEC50 and Emax from 3 independent experiments (n = 3). ND, signal not detectable. b, Dose response curves of Gαs-Gβγ dissociation in receptor (mTAAR2-6, 7d, 8c, and 9)-overexpressing HEK293 cells in response to stimulation with PEA. Values are represented as mean ± SEM of 3 independent experiments (n = 3). c, Effects of mutations of key residues in SPE-binding hydrophobic pocket of mTAAR9 in response to the stimulation with SPE. The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). Values were from 3 independent experiments (n = 3). d, Dose response curves of mutations of key residues in SPE-binding hydrophobic pocket of mTAAR9 in response to stimulation with SPE. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Consistently, structurally equivalent mutations of these 4 hydrophobic residues of F117^3.37T, F168^4.61V, Y274^6.51C, V297^7.39T/C, and V300^7.42A/G decreased SPE-induced mTAAR9 activation. e-f, Dose response curves of HEK293 cells overexpressing wild type and mutants (F168^4.61L and V300^7.42A) of mTAAR9 (c) or wild type and mutants (L170F^4.61 and A294^7.42V) of mTAAR5 (d) in response to SPE stimulation. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Mutation of F168^4.61L and V300^7.42A in mTAAR9 indeed decreased SPE-induced receptor activation by 5.13 ± 0.66- and 27.2 ± 5.04-fold, respectively (e), whereas mutation of L170^4.61F and A294^7.42V in mTAAR5 increased SPE-stimulated receptor activation by 5.43 ± 0.55- and 9.26 ± 0.68-fold, respectively(f). These results suggest that the binding of these 2 residues contributes to the potency difference of SPE that acts between the 2 mTAAR members. g, Effects of mutations of key residues in PEA-binding pocket of mTAAR9 in response to the stimulation with PEA. The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). Values were from 3 independent experiments (n = 3). h, Dose response curves of mutations of key residues in PEA-binding hydrophobic pocket of mTAAR9 in response to stimulation with PEA. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Notably, mutating the four residues in mTAAR4/6/7d/9 to structurally equivalent residues present in other mTAARs, including F/Y^3.37L/T, Y^6.51C, Y^7.35L, or V^7.39T/C, significantly decreased the PEA-induced mTAAR9 activities. i, Effects of mutations of key residues in PEA-binding pocket of mTAAR7d in response to the stimulation with PEA. The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). Values were from 3 independent experiments (n = 3). j, Dose response curves of mutations of key residues of mTAAR7d in response to stimulation with PEA by Gαs-Gβγ dissociation assay. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Notably, mutating the four residues in mTAAR4/6/7d/9 to structurally equivalent residues present in other mTAARs, including F/Y^3.37L/T, Y^6.51C, Y^7.35L, or V^7.39T/C, significantly decreased the PEA-induced mTAAR7d activities.