Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT2C Receptor Underlying the Pharmacology of Distinct Ligands
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- 作者：Yue Liu ; Clinton E. Canal ; Tania C. Cordova-Sintjago ; Wanying Zhu ; Raymond G. Booth
- 刊名：ACS Chemical Neuroscience
- 出版年：2017
- 出版时间：January 18, 2017
- 年：2017
- 卷：8
- 期：1
- 页码：28-39
- 全文大小：649K
- ISSN：1948-7193
文摘
While exploring the structure–activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT<sub>2Csub> receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT<sub>2Csub> receptors. In HEK293 cells expressing human 5-HT<sub>2C-INIsub> receptors, for example, (−)-trans-3′-Br-PAT and (−)-trans-3′-Cl-PAT are agonists regarding Gα<sub>qsub>-inositol phosphate signaling, whereas (−)-trans-3′-CF<sub>3sub>-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<sub>2Csub> 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<sub>isub>) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC<sub>50sub> functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (−)-trans-3′-CF<sub>3sub>-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<sub>Dsub>) of the antagonist radioligand [<sup>3sup>H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT<sub>2Csub> ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT<sub>2Csub> receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.