Direct coupling of detergent purified human mGlu5 receptor to the heterotrimeric G proteins Gq and Gs

Abstract

The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu1 and mGlu5 activate Gαq G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism. To study directly the G protein coupling properties of the human mGlu5 receptor homodimer, we purified the full-length receptor, which required careful optimisation of the expression, N-glycosylation and purification. We successfully purified functional mGlu5 that activated the heterotrimeric G protein Gq. The high-affinity agonist-PAM VU0424465 also activated the purified receptor in the absence of an orthosteric agonist. In addition, it was found that purified mGlu5 was capable of activating the G protein Gs either upon stimulation with VU0424465 or glutamate, although the later induced a much weaker response. Our findings provide important mechanistic insights into mGlu5 G protein-dependent activity and selectivity.

D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson’s disease

“…On the other side, the mGlu5 agonist quisqualate induced a similar Ca 2+ response in the presence or absence of coexpressed D1 receptors ( Figure 4C). In agreement with recent reports (24), we noticed that the mGlu5 agonist induced cAMP production when mGlu5 was expressed alone, and this property was not affected by D1 coexpression ( Figure 4E). The application of SKF81297 did not induce Ca 2+ release in cells expressing only D1 receptors (Figure stitutive InsP production induced by increasing expression levels of mGlu5 was higher in the presence than in the absence of D1 receptor ( Figure 3I).…”

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

“…Through a careful optimization of solubilization conditions, we demonstrate that the functional integrity of full-length mGlu2 can be maintained for hours at room temperature (Figure 2d and S9). to the Gq protein (Nasrallah et al 2018). Improved GPCR functionality by the branched nonionic detergent LMNG has been described to result from an enhanced stabilization of the 7TM due to a lower detergent exchange rate as compared to its single aliphatic counterpart DDM, thereby confining receptor motions in a way similar to lipids(S. Lee et al 2020).…”

“…It is not surprising that a functional reconstitution of multidomain, multimeric membrane proteins such as mGlu require adapted characteristics to account for proper folding, ligands binding and activity. The combination of MNG and CHS was shown to be required for a proper activation of purified mGlu5 to the Gq protein(Nasrallah et al 2018). Improved GPCR functionality by the branched nonionic detergent LMNG has been described to result from an enhanced stabilization of the 7TM due to a lower detergent exchange rate as compared to its single aliphatic counterpart DDM, thereby confining receptor motions in a way similar to lipids(S. Lee et al 2020).…”

Cholesterol impacts chemokine CCR5 receptor ligand‐binding activity

“…Solubilization of P. pastoris membranes expressing the CCR5 was performed with a mixture of two detergents, dodecylmaltoside (DDM) and 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) and a Chol analog [cholesterol hemisuccinate (CHS)], reported to improve receptor stability during detergent‐isolation procedures and to increase the amounts of solubilized material for other GPCRs and the CCR5 itself . Additionally, a CCR5 partial agonist, J113863, was included during the solubilization step to improve receptor stability.…”