Molecular determinants of positive allosteric modulation of the human metabotropic glutamate receptor 2

Abstract

Background and purpose: The activation of the metabotropic glutamate receptor 2 (mGlu2 ) reduces glutamatergic transmission in brain regions where excess excitatory signalling is implicated in disorders such as anxiety and schizophrenia. Positive allosteric modulators (PAMs) can provide a fine-tuned potentiation of these receptors' function and are being investigated as a novel therapeutic approach. An extensive set of mutant human mGlu2 receptors were used to investigate the molecular determinants that are important for positive allosteric modulation at this receptor.

Experimental approach: Site-directed mutagenesis, binding and functional assays were employed to identify amino acids important for the activity of nine PAMs. The data from the radioligand binding and mutagenesis studies were used with computational docking to predict a binding mode at an mGlu2 receptor model based on the recent structure of the mGlu1 receptor.

Key results: New amino acids in TM3 (R635, L639, F643), TM5 (L732) and TM6 (W773, F776) were identified for the first time as playing an important role in the activity of mGlu2 PAMs.

Conclusions and implications: This extensive study furthers our understanding of positive allosteric modulation of the mGlu2 receptor and can contribute to improved future design of mGlu2 PAMs.

Figure 1

Structures of mGlu₂ PAMs used in this study.

Figure 2

(A) Displacement of [³H]LY341495 binding from mGlu₂-wt (stably and transiently transfected) and mGlu₂ mutants by glutamate. Results are presented as percentage of specific binding. The affinity of glutamate is not altered by any of the mutations; total binding levels varied for the different membrane pools. (B) Glutamate-induced [³⁵S]-GTPγS binding. Glutamate concentration–response curves were determined on membranes from CHO-K1 cells expressing mGlu₂-wt (stably and transiently transfected) and five representative mutant receptors (transiently transfected). Results are expressed as % ± SD of the response to 1 mM glutamate. Both data sets are from one experiment performed in triplicate. Similar data were found for the additional set of mutants.

Figure 3

Effect of the PAMs used in this study on glutamate-induced [³⁵S]-GTPγS binding in mGlu₂-wt and a subset of mGlu₂ mutants. Concentration-dependent enhancement of 4 μM glutamate-induced [³⁵S]-GTPγS binding by nine PAMs used in this study. Results are expressed as % ± SD of the response to 1 mM glutamate, and refer to one experiment performed in triplicate.

Figure 4

Displacement curves of [³H]-JNJ-40068782 binding to membranes prepared from CHO-K1 cells stably expressing the hmGlu₂ receptor. Results are expressed as a % of specific binding and are from one representative experiment. The same results were found in at least one independent experiment.

Figure 5

(A) Amino acids, R635, L639, F643, S644, S688, G689, V700, H723, L732, N735, W773, F776, highlighted in the mGlu₂ receptor model built using the mGlu₁ receptor X-ray structure as template, the docked binding mode of JNJ-40068782 shown in sphere representation, coloured turquoise. View between TM5 and TM6 (cut-away) of the docked binding modes of (B) JNJ-40068782, (C) JNJ-40297036, (D) JNJ-46281222, (E) LY2607540 (THIIC). Selected important amino acids from experimental mutagenesis are shown in green. Amino acids and TM annotation is provided in (B) and consistent for (C), (D) and (E).