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Controlling the Agonist-Mediated and Constitutive G-Protein Signaling of the Human 5-HT4 Receptor


G-protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and are crucial in many biological processes. The molecular and functional diversity of GPCRs and the lack of ligands with specific signaling effects have complicated studies of their in vivo functions. To better compare the effects of activating different G-protein-signaling pathways through agonist-mediated or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) activating through different G-protein-signaling pathways. These RASSLs are based on the human 5-HT4 receptor, a GPCR with a high level of constitutive Gs signaling and strong agonist-mediated G-protein activation of Gs and Gs/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 (antagonists and inverse agonists for the 5-HT4 receptor). All agonists potently induced Gs signaling, but only a few (e.g., Zacopride) also induced signaling via the Gq pathway. Zacopride-induced Gq signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT2C receptor. Additional point mutations (D66A and D66N) blocked constitutive Gs signaling and lowered agonist-mediated Gq signaling. Finally, replacing the third intracellular loop of Rs1 with that of human 5-HT1A conferred ligand-mediated Gi signaling. This Gi-coupled RASSL, Rs1.3, exhibited no measurable signaling to the Gs or Gq pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection. Here, we describe a new series of RASSLs developed to modify the agonist-mediated and constitutive signaling of the human 5-HT4 receptor. Since none of the inverse agonists for the 5-HT4 receptor works in the presence of the D100A mutation, I also engineered a series of synthetic internal ribosomal entry sites and a single plasmid tetracycline-inducible (tet) system to better control GPCR expression. Combined with a tissue-specific promoter, these RASSLs can be expressed in a tissue-specific manner and at specific levels. With these additional tools, RASSLs will help us better study the effect of constitutive Gs signaling and agonist-mediated Gs, Gs/Gq, and Gi signaling in vivo.

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