Closure of the Venus flytrap module of mGlu8 receptor and the activation process: Insights from mutations converting antagonists into agonists

Abstract

Ca 2؉ , pheromones, sweet taste compounds, and the main neurotransmitters glutamate and ␥-aminobutyric acid activate G proteincoupled receptors (GPCRs) that constitute the GPCR family 3. These receptors are dimers, and each subunit has a large extracellular domain called a Venus flytrap module (VFTM), where agonists bind. This module is connected to a heptahelical domain that activates G proteins. Recently, the structure of the dimer of mGlu1 VFTMs revealed two important conformational changes resulting from glutamate binding. First, agonists can stabilize a closed state of at least one VFTM in the dimer. Second, the relative orientation of the two VFTMs in the dimer is different in the presence of glutamate, such that their C-terminal ends (which are connected to the G protein-activating heptahelical domain) become closer by more than 20 Å. This latter change in orientation has been proposed to play a key role in receptor activation. To elucidate the respective role of VFTM closure and the change in orientation of the VFTMs in family 3 GPCR activation, we analyzed the mechanism of action of the mGlu8 receptor antagonists ACPT-II and MAP4. Molecular modeling studies suggest that these two compounds prevent the closure of the mGlu8 VFTM because of ionic and steric hindrance, respectively. We show here that the replacement of the residues responsible for these hindrances (Asp-309 and Tyr-227, respectively) by Ala allows ACPT-II or MAP4 to fully activate the receptors. These data are consistent with the requirement of the VFTM closure for family 3 GPCR activation.

Molecular Determinants Involved in the Allosteric Control of Agonist Affinity in the GABAB Receptor by the GABAB2 Subunit

“…Such domains are well known to adopt either an open conformation (VFT o ) stabilized by antagonists (25,26) or a closed conformation (VFT c ) stabilized by agonists (24,26,27). VFT can oscillate between the VFT o and VFT c states with an equilibrium constant of K 1 ϭ VFT c /VFT o .…”

“…The membranes were prepared as described previously (26). For each sample, 50 g of total protein was subjected to SDS-PAGE using 10% polyacrylamide gels, transferred to nitrocellulose membrane (Hybond-C, Amersham Biosciences), and probed with mouse anti-HA monoclonal antibody 12CA5 (Roche Applied Science) at 0.1 g/ml.…”

“…Modeling and site-directed mutagenesis studies indicate that ligands bind in the cleft that separates both lobes of GB1 VFT, as observed for ligand binding in many similar protein modules (23), including mGlu1 VFT (24,25). Antagonists are expected to prevent the closure of GB1 VFT (19), as observed in mGlu receptors (25,26). Conversely, agonists interact with residues from both lobes of GB1 VFT, and they stabilize a closed form of this domain (16,19).…”

Structure and ligand recognition of class C GPCRs

“…In the presence of ligand, glutamate interacts with lobe 1 in the open form of the VFT and then stabilizes a closed form through additional contacts with lobe 2. Competitive antagonists inhibit receptor activation by preventing VFT closure [35] , whereas locking the VFT in a closed conformation with an artificial disulfide bond results in a constitutively active receptor [36] . VFTs form constitutive dimers.…”

“…Both competitive agonists and antagonists interact with this site and induce significant conformational changes in the VFT: binding of a full agonist stabilizes a closed conformation [35] , whereas binding of competitive antagonists stabilizes an open conformation [33,34,43] . Binding of partial agonists results in a partial or a complete, yet unstable, closure of the VFT domain [35,58] . In contrast, the allosteric sites are topographically distinct from the orthosteric sites in any given receptor.…”