Site-Directed Mutagenesis of the Serotonin 5-Hydroxytryptamine2C Receptor: Identification of Amino Acids Responsible for Sarpogrelate Binding

Muntasir, Habib Abul; Takahashi, Jyuniti; Rashid, Mamunur; Ahmed, Maruf; Komiyama, Tomoyoshi; Hossain, Murad; Kawakami, Jun; Nashimoto, Masayuki; Nagatomo, Takafumi

doi:10.1248/bpb.29.1645

Cited by 12 publications

(11 citation statements)

References 29 publications

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“…K D values derived from saturation binding isotherms of [ 3 H]-mesulergine labeled WT, S3.36A and Y7.43A 5-HT 2C receptors. No specific binding of [ 3 H]-mesulergine to the D3.32A point-mutated 5-HT 2C receptor was detected (not shown), consistent with results previously reported (Muntasir et al, 2006). Affinity of [ 3 H]-mesulergine at the WT, S3.36A and Y7.43A point-mutated receptors was not significantly different (mean (S.E.M.)…”

Section: Resultssupporting

confidence: 92%

Drug discovery targeting human 5-HT2C receptors: Residues S3.36 and Y7.43 impact ligand—Binding pocket structure via hydrogen bond formation

Canal

Córdova-Sintjago

Villa

et al. 2011

European Journal of Pharmacology

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Specific activation of serotonin (5-HT) 5-HT2C G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. Mutagenesis coupled with computational and molecular modeling experiments based on the human β2 adrenergic receptor structure were employed to delineate the interactions of different ligands at human 5-HT2C residues D3.32, S3.36 and Y7.43. No binding of the tertiary amine radioligand ([3H]-mesulergine) could be detected when the 5-HT2C D3.32 residue was mutated to alanine (D3.32A). The S3.36A point-mutation greatly reduced affinity of primary amine ligands, modestly reduced affinity of a secondary amine, and except for the 5-HT2C-specific agonist N(CH3)2-PAT, affinity of tertiary amines was unaffected. Molecular modeling results indicated that the primary amines form hydrogen bonds with the S3.36 residue, whereas, with the exception of N(CH3)2-PAT, tertiary amines do not interact considerably with this residue. The Y7.43A point-mutation greatly reduced affinity of 5-HT, yet reduced to a lesser extent the affinity of tryptamine that lacks the 5-hydroxy moiety present in 5-HT; modeling results indicated that the 5-HT 5-hydroxy moiety hydrogen bonds with Y7.43 at the 5-HT2C receptor. Additional modeling results showed that 5-HT induced a hydrogen bond between Y7.43 and D3.32. Finally, modeling results revealed two low-energy binding modes for 5-HT in the 5-HT2C binding pocket, supporting the concept that multiple agonist binding modes may stabilize different receptor active conformations to influence signaling. Ligand potencies for modulating WT and point-mutated 5-HT2C receptor-mediated phospholipase C activity were in accordance with the affinity data. Ligand efficacies, however, were altered considerably by the S3.36A mutation only.

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Section: Resultssupporting

confidence: 92%

Drug discovery targeting human 5-HT2C receptors: Residues S3.36 and Y7.43 impact ligand—Binding pocket structure via hydrogen bond formation

Canal

Córdova-Sintjago

Villa

et al. 2011

European Journal of Pharmacology

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“…Recently, similar results for α 1b -AR were obtained in our laboratory (27). Mutation of the same conserved aspartate residue on 5-HT 2C and 5-HT 2B also showed no ligand binding or 5-HT stimulated IP formation in our laboratory (28,29). These results may be extrapolated to the conserved aspartate at position 176 in α 1d -AR.…”

Section: Agonist-stimulated Ip Accumulationsupporting

confidence: 87%

Amino Acids of the Human α1d-Adrenergic Receptor Involved in Antagonist Binding

et al. 2008

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Abstract. Computer simulations of the human α 1d -adrenergic receptor (α 1d -AR) based on the crystal structure of rhodopsin have been combined with experimental site-directed mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Our results indicate that the amino acids Asp176 in the third transmembrane domain (TMD), Glu237 in TMD IV, and Ser258 in TMD V of α 1d -AR were directly involved in prazosin and tamsulosin binding. The Asp176Ala mutant did not exhibit any affinity for [ Competition binding experiments showed that prazosin affinity had increased to 5-fold and 3-fold in the Glu237Ala and Ser258Ala mutants, respectively, versus wild-type; and tamsulosin affinity only increased in the Ser258Ala mutant (2-fold vs wild-type). It seems that these two residues constrain the receptor by interaction with other residues and this disruption of the interaction increased the receptor's binding affinity towards antagonists. However, the Glu237Ala and Ser258Ala mutant receptors retained the ability to stimulate the formation of myo-[ 3 H]inositol but had activities lower than that of the wild-type receptor. The present results provide direct evidence that these amino acid residues are responsible for the interactions between α 1d -AR and the radioligand [

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“…With the help of molecular modeling and mutagenesis studies of 5-HT 2B receptors, we previously identified the important binding sites of sarpogrelate to 5-HT 2A -and 5-HT 2C receptors (6,7). Molecular modeling of sarpogrelate to the 5-HT 2B receptor predicted that sarpogrelate makes strong electrostatic interactions towards aspartic acid (Asp) 135 in transmembrane helix (TMH) 3 of the 5-HT 2B receptor (8).…”

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confidence: 99%

Identification of a Key Amino Acid of the Human 5-HT2B Serotonin Receptor Important for Sarpogrelate Binding

et al. 2007

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Based on radio-ligand binding and molecular modeling studies, sarpogrelate shows a moderate selectivity for 5-HT(2B) versus 5-HT(2A) receptors. To confirm the modeling data of sarpogrelate to 5-HT(2B) receptors predicting interaction of sarpogrelate towards Asp135 in helix 3 of 5-HT(2B) receptors, we constructed and characterized the mutation of this residue by site-directed mutagenesis. The Asp135Ala mutant did not exhibit any affinity for [(3)H]rauwolscine. Therefore, it was not possible to find sarpogrelate affinity to the mutant using [(3)H]rauwolscine. The mutation also abolished agonist-stimulated inositol phosphates formation. These results provide evidence that Asp135 is important for the interaction between 5-HT(2B) receptors and sarpogrelate.

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Site-Directed Mutagenesis of the Serotonin 5-Hydroxytryptamine2C Receptor: Identification of Amino Acids Responsible for Sarpogrelate Binding

Abstract: Site-directed mutagenesis was used to investigate the molecular interactions involved in sarpogrelate binding

Cited by 12 publications

References 29 publications

Drug discovery targeting human 5-HT2C receptors: Residues S3.36 and Y7.43 impact ligand—Binding pocket structure via hydrogen bond formation

Drug discovery targeting human 5-HT2C receptors: Residues S3.36 and Y7.43 impact ligand—Binding pocket structure via hydrogen bond formation

Amino Acids of the Human α1d-Adrenergic Receptor Involved in Antagonist Binding

Identification of a Key Amino Acid of the Human 5-HT2B Serotonin Receptor Important for Sarpogrelate Binding

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