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-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, (−)- trans -3′-Br-PAT and (−)- trans -3′-Cl-PAT are agonists regarding Gα q -inositol phosphate signaling, whereas (−)- trans -3′-CF 3 -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 ( K i ) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC 50 functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (−)- trans -3′-CF 3 -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 [ 3 H]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. Graphic Abstract ACS Electronic Supporting Info
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