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Inactive 5‐HT 7 Receptors Associate with Inactive and Active G s Heterotrimers
Author(s) -
Jang Wonjo,
Lambert Nevin A.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03446
Subject(s) - g protein coupled receptor , g protein , chemistry , gtp' , heterotrimeric g protein , receptor , agonist , nucleotide , gs alpha subunit , biochemistry , biophysics , biology , enzyme , gene
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‐HT 7 receptors are known to preassemble with GDP‐bound G s heterotrimers prior to agonist binding. Here we show that these receptors also interact with GTP‐bound G s heterotrimers. We used bioluminescence resonance energy transfer (BRET) and luciferase complementation assays to assess the effects of ligands and nucleotides on 5‐HT 7 ‐G s coupling. We find that stimulation with agonist (5‐HT) in intact cells promotes net dissociation of 5‐HT 7 ‐G s complexes, whereas inverse agonists (e.g. methiothepin) produce net association. To determine how nucleotide binding by G s 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‐HT 7 ‐G s complexes were largely maintained in the various nucleotide conditions. Agonist‐induced 5‐HT 7 ‐G s dissociation still occurred when only GDPβS was present, indicating that dissociation does not require G s activation. Similarly, inverse agonist‐induced 5‐HT 7 ‐G s association still occurred when only GTPγS was present, suggesting that activated G s heterotrimers retain affinity for inactive 5‐HT 7 receptors. Moreover, we found that inverse agonist‐bound 5‐HT 7 sequestered free Gβγ in the presence of GTPγS, suggesting that inactive 5‐HT 7 receptors stabilized active G s heterotrimers. These results reveal unconventional coupling of inactive 5‐HT 7 receptors to both inactive and active G s 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 Information Supported by GM130142 (to N.A.L.)