Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy.
Serotonin 5-HT(4(a)) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist (partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free (or antagonist-occupied), active (partial- or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, well-defined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.
Banères
Jean-Louis
JL
UMR CNRS 5074, Chimie Biomoléculaire et Interactions Biologiques, Faculté de Pharmacie, 15 Avenue Ch. Flahault, 34093 Montpellier Cedex 05, France. baneres@univ-montp1.fr
Mesnier
Danielle
D
Martin
Aimée
A
Joubert
Lara
L
Dumuis
Aline
A
Bockaert
Joel
J
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002942:Circular Dichroism; D004358:Drug Therapy; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007211:Indoles / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D044422:Receptors, Serotonin, 5-HT4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D012702:Serotonin Antagonists / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000378:metabolism
D001562:Benzimidazoles; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007211:Indoles; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D013449:Sulfonamides; C070189:(endo-N-8-methyl-8-azabicyclo-(3.2.1)oct-3-yl)-2,3-dihydro-3-isopropyl-2-oxo-1H-benzimidazol-1-carboxamide; D044422:Receptors, Serotonin, 5-HT4; C082871:GR 113808