A single point mutation (Phe340-->Leu340) of a conserved phenylalanine abolishes 4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine and [3H]mesulergine but not [3H]ketanserin binding to 5-hydroxytryptamine2 receptors.
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.
Department of Psychiatry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
M S
MS
Choudhary
S
S
Craigo
B L
BL
Roth
D000595:Amino Acid Sequence; D000662:Amphetamines / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004873:Ergolines / Q000378:metabolism; D006868:Hydrolysis; D007650:Ketanserin / Q000378:metabolism; D007930:Leucine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D010716:Phosphatidylinositols / Q000378:metabolism; D017354:Point Mutation; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000378:metabolism
D000662:Amphetamines; D004873:Ergolines; D010716:Phosphatidylinositols; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D010649:Phenylalanine; D007650:Ketanserin; D007930:Leucine; C015952:4-iodo-2,5-dimethoxyphenylisopropylamine; C031649:mesulergine