Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state

Abstract

Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases. Much hope in drug development come from the discovery of positive allosteric modulators that display subtype selectivity, and act by increasing agonist potency, as well as efficacy in most cases. How such compounds can influence agonist efficacy remains unclear. Here, we explore the structural dynamics of the full-length mGlu2 dimers at submillisecond timescales using single molecule FRET on diffusing receptors in optimized detergent micelles. We show that glutamate binding in the Venus flytrap extracellular domains does not stabilize fully the receptors in their active states. The full activation of all receptors can only be observed in the presence of either a positive allosteric modulator or the Gi protein. Our results provide important new insights on the fast kinetics and the action of the allosteric modulators on mGlu activation at the single molecule level.

Dissecting conformational rearrangements and allosteric modulation in metabotropic glutamate receptor activation

“…In contrast, the PAM that binds in the 7TM domain is strictly required to stabilize the fully reoriented VFTs in the active state, even though this effect to increase efficacy remains dependent of the orthosteric agonist. We note that these observations are perfectly consistent with those we previously obtained using SNAP-tagged receptors, labeled at the level of the upper lobes as well 17 .…”

“…Control LRET titration experiments on SNAP-tag labeled receptors confirmed unimpaired functional integrity over time at room temperature and demonstrated that the C-terminal modifications do not impair receptor stimulation by Glu and allosteric modulation by BINA ( Figure S6a-d ). In addition, the same constructs were analyzed by smFRET, which confirmed that intersubunit VFT reorientation occurs in an identical manner on these C-terminally modified constructs as on wildtype receptors (as in 17 ) in the absence of ligands or when saturated with LY341495, Glu, Glu + Gi and Glu + BINA ( Figure S7a-e ).…”

“…As this strategy requires the modification of the C-terminal tails of mGlu2 we first verified the functionality of these constructs after solubilization. To account for the decreased yields of receptors with PrF incorporated by genetic code expansion, we modified our previously published solubilization protocol 17 . This new strategy relies on an initial solubilization using 1% LMNG + 0.1% CHS Tris followed by dilution into buffer containing GDN (as compared to our previously described protocol using 0.1% LMNG + 0.01% CHS Tris + 0.1% GDN for initial solubilization followed by dilution in buffer without additional detergent).…”

“…LRET measurements of PrF-containing sensors and the SNAP sensor after receptor solubilization were performed on a PHERAstar FS microplate reader (BMG LABTECH) as described 17, 34, 35 . After Lumi4-Tb donor excitation with a laser at 337 nm, the donor emission was collected at 620 nm and the sensitized acceptor emission at 520 nm for green or 665 nm for red in 5 µs intervals for a total of 2.5 s. LRET was calculated as the ratio of the sensitized acceptor emission integrated from 50 µs to 100 µs over 1200 µs to 1600 µs after donor excitation for the pure SNAP sensor and over 300 µs to 500 µs for SNAP with PrF and pure PrF sensors.…”

“…Surprisingly, some early crystal VFT structures showed agonist-bound mGlu3 VFT dimers, adopting a Rcc conformation with the VFTs being closed but the lower lobes remaining separated 10 , implying that agonists alone may not be sufficient for full VFT reorientation. In accordance with this, we recently used single molecule Förster resonance energy transfer (smFRET) on receptors labeled through N-terminal SNAP-tags to show that the combination of an orthosteric agonist with positive allosteric modulators (such as the small organic molecule BINA or the heterotrimeric G protein) is required for promoting a full shift of the equilibrium toward the active state 17 . This SNAP sensor only reported on the reorientation of the N-termini being located on the upper lobes of the VFTs ( Figure 1a ).…”