Abstract

The molecular processes by which agonists and antagonists bind to serotonin2 [5-hydroxytryptamine (5-HT2)] receptors are currently unknown. Three molecular models have proposed that conserved aromatic residues help to anchor the phenyl ring of 5-HT via stacking or pi-pi-type interactions with the 5-HT2 receptor. To test these models we made single point mutations (Phe339-->Leu339 and Phe340-->Leu340) of two aromatic residues that are conserved among all guanine nucleotide-binding protein-coupled 5-HT receptors and a single point mutation (Phe125-->Leu125) that exchanges a 5-HT2 for a 5-HT1c sequence. [3H]Mesulergine binding was abolished by Phe340-->Leu340 and unchanged with the Phe339-->Leu339 and Phe125-->Leu125 mutations, whereas [3H]ketanserin binding affinity was diminished by the Phe339-->Leu339 mutation and unchanged by Phe340-->Leu340 and Phe125-->Leu125. We also found that the affinities of three ergot derivatives (mesulergine, methysergide, and lisuride) were decreased by 88-1079-fold with only the Phe340-->Leu340 mutation. We also discovered that 4-[125I]iodo-2,5-(dimethoxy)phenylisopropylamine (DOI) binding was abolished in COS-7 cells expressing 5-HT2 (Phe340-->Leu340) receptors but maintained in cells expressing the Phe339-->Leu339 and Phe125-->Leu125 mutations. Additionally, the Ki values for several agonists and partial agonists (5-HT, DOI, m-chlorophenylpiperazine, trifluoromethylphenylpiperazine, bufotenine, and MK-212) were greatly diminished (26-14,000-fold decrease) only with the Phe340-->Leu340 receptor mutation. Finally, the Phe340-->Leu340 mutant receptors displayed an attenuated or abolished ability to augment phosphoinositide hydrolysis in COS-7 cells with four separate agonists (5-HT, MK-212, bufotenine, and quipazine). Taken together, these results are consistent with the idea that agonists and certain ergot derivatives anchor to 5-HT2 receptors, in part, via specific interactions with aromatic residue Phe340 located in transmembrane region VI.