Fig. 6. The non-canonical G_s coupling mode is determined by a larger hydrophobic residue at position 5.65 of TM5.

a Receptors that adopt canonical Gs coupling mode have a small hydrophobic residue A/V at position 5.65, while receptors that adopt non-canonical Gs coupling prefer a large hydrophobic residue (L/C^5.65). b Comparison of the binding interface between β2AR–Gα_s and GPR174–Gα_s. The hook of Gαs is distorted when bound to GPR174 due to a potential steric clash between L212^5.65 and the tri-leucine pocket formed by L394^(-1), L393⁽⁻²⁾, and L388⁽⁻⁷⁾. c Mutation of L212^5.65 in GPR174 remarkably reduces its basal activity. Each data point represents mean ± SEM from three independent experiments. Source data are provided as a Source Data file. d The effects of mutations in the GPR174‒G_s interface on the potency and maximum effect of lysoPS in the context of the Y99^3.33A mutant evaluated by the cAMP assay. Each data point represents mean ± SEM from three independent experiments. Source data are provided as a Source Data file. e Detailed interactions between GPR174 and Gα_s. f and g The presence of a larger hydrophobic residue (L or C) at 5.65 in GPR52 (f) and CCK1R (g) leads to a non-canonical G_s coupling mode.