Serine 3.39 and isoleucine 4.60 are key sites for 5-HT2AR-mediated Gs signaling.

Certain amino acid sites of 5-HT2AR play crucial roles in interacting with various G proteins. Hallucinogens and non-hallucinogens both act on 5-HT2AR but mediate different pharmacological effects, possibly due to the coupling of different G proteins. Therefore, this study identified the binding sites of hallucinogens and non-hallucinogens with 5-HT2AR through molecular docking. We conducted site mutation to examine the impact of these sites on G proteins, in order to find out the sites that can distinguish the pharmacological effects of hallucinogens and non-hallucinogens. Our results indicate that I4.60A and S3.39A did not affect the ability of hallucinogens to activate Gq signaling, but significantly reduced Gs signaling activation by hallucinogens. These results suggest that S3.39 and I4.60 are important for the activation of Gs signaling by hallucinogens.