Molecular Characterization of a Purified 5-HT4 Receptor

Abstract

Serotonin 5-HT 4(a) receptor, a G-protein-coupled re-ceptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecu-lar 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 extracellu-lar 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 bind-ing 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-oc-cupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluores-cence emission profile of the purified receptor con-firmed 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 demon-strates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selec-tion of a mixture of different conformational states of the receptor. G-protein-coupled receptors are versatile biological sensors. They are responsible for the majority of cellular responses to hormones and neurotransmitters, as well as sight, smell, and taste senses (1, 2). Signal transduction is specifically associated with GPCR 1 activation. Although significant progress has been made within the last few years in dissecting GPCR-mediated