Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5‑HT_2C Receptor Underlying the Pharmacology of Distinct Ligands

While exploring the structure–activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT_2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT_2C receptors. In HEK293 cells expressing human 5-HT_2C‑INI receptors, for example, (−)-<i>trans</i>-3′-Br-PAT and (−)-<i>trans</i>-3′-Cl-PAT are agonists regarding Gα_q-inositol phosphate signaling, whereas (−)-<i>trans</i>-3′-CF₃-PAT is an inverse agonist. To investigate the ligand–receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT_2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (<i>K</i>_i) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC₅₀ functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (−)-<i>trans</i>-3′-CF₃-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (−)-<i>trans</i>-3′-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (<i>K</i>_D) of the antagonist radioligand [³H]­mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT_2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT_2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand