An inactive receptor-G protein complex maintains the dynamic range of agonist-induced signaling

Abstract

Agonist binding promotes activation of G protein-coupled receptors (GPCRs) and association of active receptors with G protein heterotrimers. The resulting active-state ternary complex is the basis for conventional stimulus-response coupling. Although GPCRs can also associate with G proteins before agonist binding, the impact of such preassociated complexes on agonist-induced signaling is poorly understood. Here we show that preassociation of 5-HT7serotonin receptors with Gsheterotrimers is necessary for agonist-induced signaling. 5-HT7receptors in their inactive state associate with Gs, as these complexes are stabilized by inverse agonists and receptor mutations that favor the inactive state. Inactive-state 5-HT7–Gscomplexes dissociate in response to agonists, allowing the formation of conventional agonist–5-HT7–Gsternary complexes and subsequent Gsactivation. Inactive-state 5-HT7–Gscomplexes are required for the full dynamic range of agonist-induced signaling, as 5-HT7receptors spontaneously activate Gsvariants that cannot form inactive-state complexes. Therefore, agonist-induced signaling in this system involves two distinct receptor-G protein complexes, a conventional ternary complex that activates G proteins and an inverse-coupled binary complex that maintains the inactive state when agonist is not present.

Structural basis for strychnine activation of human bitter taste receptor TAS2R46

“…We note that the extent of activation may be influenced by the chimeric G protein and stabilization of the receptor. However, the preassociation of TAS2R46 with gustducin without agonist binding might be indicative of flexibility of the receptor that is required for binding and rapid activation by many ligands ( 47 ). In our BRET assay, addition of strychnine decreased bioluminescence resonance energy transfer between labeled TAS2R46 and WT gustducin heterotrimers (fig.…”

(-)-Epicatechin Is a Biased Ligand of Apelin Receptor

“…Moreover, Phe291 seems to be an anchor for the ligand stabilization within the binding cavity; consequently, Trp85 and Ile109 favor the CMF-019 aliphatic chain interaction in the binding site. In the inactive receptor status, the interaction with Asp168 also plays an essential role in the G protein pathway activation; in this condition, the ligand could be performing a G protein preassembly, preparing the system to signal when the conformation turns active [ 49 , 50 ].…”

“…In the first approach of EC binding to APLNR analysis, we found that it reaches a deeper site than the CMF-019 fitting site, which can also be occupied by apelin-13. The main interactions are Tyr182, Tyr264, Pro292, and Tyr299 [ 50 ]. Some of these amino acid residues in both APLNR states are related to the β-arrestin pathway [ 51 ].…”

Optical sensors of heterotrimeric G protein signaling

“…Moreover, the GPCR-binding specificity of mini G proteins can be easily altered by modifying their C-terminal sequence in the same way as for Ga subunits. Mini G proteins have recently been used in BRET [114] and luciferase complementation studies [115], as well as in live-cell imaging [54] of GPCR-G protein interactions. The stable binding of mini G proteins to active GPCRs also makes mini G proteins suitable for studies of localisation and dynamics of endosomal signaling [116].…”

“…This type of interaction is termed preassembly [52]. Using RET techniques, preassembly was shown for G i1 and the platelet‐activating hormone receptor 1 [53], for G s and the serotonin receptor 7 (but not the serotonin receptor 4) [26,27,54], as well as for G q and the ghrelin receptor [25]. Although FRET [13] (and FRAP [55]) experiments detected no complexes between G i1 and the α 2 ‐adrenoceptor, another FRET‐based study showed preassembly [24].…”