Inactive 5‐HT7 Receptors Associate with Inactive and Active Gs Heterotrimers

Abstract

G protein‐coupled receptors (GPCRs) interact cooperatively with agonists and heterotrimeric G proteins to form ternary complexes, leading to activation of G proteins. Following exchange of GDP for GTP the complex is thought to rapidly disassemble as nucleotide‐bound G proteins adopt conformations that have low affinity for GPCRs, and heterotrimers dissociate into Gα and Gβγ subunits. Unlike most GPCRs, serotonin 5‐HT7 receptors are known to preassemble with GDP‐bound Gs heterotrimers prior to agonist binding. Here we show that these receptors also interact with GTP‐bound Gs heterotrimers. We used bioluminescence resonance energy transfer (BRET) and luciferase complementation assays to assess the effects of ligands and nucleotides on 5‐HT7‐Gs coupling. We find that stimulation with agonist (5‐HT) in intact cells promotes net dissociation of 5‐HT7‐Gs complexes, whereas inverse agonists (e.g. methiothepin) produce net association. To determine how nucleotide binding by Gs affects interactions with receptors, we replaced endogenous nucleotides with the non‐hydrolyzable analogs GTPγS or GDPβS in permeabilized, apyrase‐treated HEK293 cells. Unexpectedly, ligand‐mediated effects on 5‐HT7‐Gs complexes were largely maintained in the various nucleotide conditions. Agonist‐induced 5‐HT7‐Gs dissociation still occurred when only GDPβS was present, indicating that dissociation does not require Gs activation. Similarly, inverse agonist‐induced 5‐HT7‐Gs association still occurred when only GTPγS was present, suggesting that activated Gs heterotrimers retain affinity for inactive 5‐HT7 receptors. Moreover, we found that inverse agonist‐bound 5‐HT7 sequestered free Gβγ in the presence of GTPγS, suggesting that inactive 5‐HT7 receptors stabilized active Gs heterotrimers. These results reveal unconventional coupling of inactive 5‐HT7 receptors to both inactive and active Gs heterotrimers. This represents a significant departure from the standard allosteric model of GPCR‐G protein coupling, wherein G proteins interact primarily with active GPCRs, and GTP‐bound Gα subunits dissociate from both receptors and Gβγ subunits.Support or Funding InformationSupported by GM130142 (to N.A.L.)