Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors

© FASEB. Type 4 metabotropic glutamate (mGlu<inf>4</inf>) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu<inf>4</inf>-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu<inf>4</inf> PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu<inf>4</inf> biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ<inf>B</inf>) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na⁺ binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu<inf>4</inf> PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes.