A SINGLE POINT MUTATION (PHE-340-]LEU-340) OF A CONSERVED PHENYLALANINE ABOLISHES 4-[I-125]IODO-(2,5-DIMETHOXY)PHENYLISOPROPYLAMINE AND [H-3] MESULERGINE BUT NOT [H-3] KETANSERIN BINDING TO 5-HYDROXYTRYPTAMINE(2) RECEPTORS

The molecular processes by which agonists and antagonists bind to sero tonin2 [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 inte ractions with the 5-HT2 receptor. To test these models we made single point mutations (Phe339 --> Leu339 and Phe340 --> Leu340) of two aroma tic residues that are conserved among all guanine nucleotide-binding p rotein-coupled 5-HT receptors and a single point mutation (Phe125 --> Leu125) that exchanges a 5-HT2 for a 5-HT1c sequence. [H-3] Mesulergin e binding was abolished by Phe340 --> Leu340 and unchanged with the Ph e339 --> Leu339 and Phe125 --> Leu125 mutations, whereas [H-3]ketanser in binding affinity was diminished by the Phe339 --> Leu339 mutation a nd unchanged by Phe340 --> Leu340 and Phe125 --> Leu125. We also found that the affinities of three ergot derivatives (mesulergine, methyser gide, and lisuride) were decreased by 88-1079-fold with only the Phe34 0 --> Leu340 mutation. We also discovered that 4-[I-125]iodo-2,5-(dime thoxy)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. Add itionally, the K(i) values for several agonists and partial agonists ( 5-HT, DOI, m-chlorophenylpiperazine, trifluoromethylphenylpiperazine, bufotenine, and MK-212) were greatly diminished (26-14,000-fold decrea se) only with the Phe340 --> Leu340 receptor mutation. Finally, the Ph e340 --> Leu340 mutant receptors displayed an attenuated or abolished ability to augment phosphoinositide hydrolysis in COS-7 cells with fou r separate agonists (5-HT, MK-212. bufotenine, and quipazine). Taken t ogether, these results are consistent with the idea that agonists and certain ergot derivatives anchor to 5-HT2 receptors, in part, via spec ific interactions with aromatic residue Phe340 located in transmembran e region VI.