Pharmacological profile of engineered 5-HT₄ receptors and identification of 5-HT₄ receptor-biased ligands.

G protein-coupled receptors (GPCRs) can activate simultaneously multiple signaling pathways upon agonist binding. The combined use of engineered GPCRs, such as the receptors activated solely by synthetic ligands (RASSLs), and of biased ligands that activate only one pathway at a time might help deciphering the physiological role of each G protein signaling. In order to find serotonin type 4 receptor (5-HT₄R) biased ligands, we analyzed the ability of several compounds to activate the Gs and G(q/11) pathways in COS-7 cells that transiently express wild type 5-HT₄R, the 5-HT₄R-D(100)A mutant (known also as 5-HT₄-RASSL, or Rs1) or the 5-HT₄R-T(104)A mutant, which modifies agonist-induced 5-HT₄R activation. This analysis allowed completing the pharmacological profile of the two mutant 5-HT₄Rs, but we did not find any biased ligand for the mutant receptors. Conversely, we identified the first biased agonists for wild type 5-HT₄R. Indeed, RS 67333 and prucalopride acted as partial agonists to induce cAMP accumulation, but as antagonists on inositol phosphate production. Moreover, they showed very different antagonist potencies that could be exploited to study the activation of the G(s) pathway, with or without concomitant block of G(q/11) signaling. This article is part of a Special Issue entitled Optogenetics (7th BRES).