Modifying Ligand-Induced and Constitutive Signaling of the Human 5-HT4 Receptor

Abstract

G protein–coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT_4b receptor, a GPCR with high constitutive G_s signaling and strong ligand-induced G-protein activation of the G_s and G_s/q pathways. The first receptor in this series, 5-HT₄-D¹⁰⁰A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G_s signaling, but only a few (e.g., zacopride) also induced signaling via the G_q pathway. Zacopride-induced G_q signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT_2C receptor. Additional point mutations (D⁶⁶A and D⁶⁶N) blocked constitutive G_s signaling and lowered ligand-induced G_q signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT_1A conferred ligand-mediated G_i signaling. This G_i-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G_s or G_q pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.