Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT2c Receptor Underlying the Pharmacology of Distinct Ligands

While exploring the structure activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT2c receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT2c receptors. In HEK293 cells expressing human 5-HT2c-INI receptors, for example, (-)-trans-3'-CF3-PAT and (-)-trans-3'-CI-PAT are agonists regarding G alpha(q)-inositol phosphate signaling, whereas (-)-trans-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 5HT(2c) receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that 53.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (K-D) of all PATs tested, we also found that F6.44, M6.47, C7.45, and 57.46 are primarily involved in regulating EC/IC50 functional potencies of PATs. We discovered that when residue 55.43, N6.55, or both are mutated to alanine, (-)-trans-3',CF3-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (-)-trans-3'-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (K-D) of the antagonist radioligand [H-3]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT2c ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT2c receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.