Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-hydroxytryptamine-4 receptors.

The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R(*). However, different structural active R(*) complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R(*) states of 5-hydroxytryptamine-4 (5-HT(4)) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R(*)basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive g- to active g+ and Trp6.48 from inactive g+ to active t). Mutation of either threonine or tryptophan to alanine resulted in a lowering of the activity of the R(*)basal similar to the Rg. The T3.36A mutation shows that the Thr3.36 toggle switch plays a minor role in the stabilization of R(*) induced by 5-HT (R(*)-5-HT) and BIMU8 (R(*)-BIMU8) and is fully required in the stabilization of R(*) induced by (S)-zacopride, cisapride, and 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butyl-4-piperidinyl)-1-propanone (RS 67333) (R(*)-benzamides). Thus, benzamides stabilize R(*)-benzamides by forming a specific hydrogen bond with Thr3.36 in the active g+ conformation. Conversely, R(*)-BIMU8 was probably the result of a direct conformational transition of Trp6.48 from inactive g+ to active t by hydrogen bonding of this residue to a carboxyl group of BIMU8. We were surprised that the Trp6.48 toggle switch was not necessary for receptor activation by the natural agonist 5-HT. R(*)-5-HT is probably attained through other routes of activation. Thus, different conformational arrangements occur during stabilization of R(*)basal, R(*)-5-HT, R(*)-benzamides, and R(*)-BIMU8.

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).