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 Gq/11 pathways in COS-7 cells that transiently express wild type 5-HT4R, the 5-HT4R-D100A mutant (known also as 5-HT4-RASSL, or Rs1) or the 5-HT4R-T104A 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 Gs pathway, with or without concomitant block of Gq/11 signaling.This article is part of a Special Issue entitled Optogenetics (7th BRES)

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► We tested the major 5-HT4R ligands with WT, D100A and T104A 5-HT4Rs.
► We examined 5-HT4R-induced Gs and Gq/11 activation.
► We measured cAMP and inositol phosphate accumulation upon 5-HT4R activation.
► We identified molecules that activate the Gs but not the Gq/11 pathway.
► We identified the first biased 5-HT4R ligands: RS 67333 and prucalopride.