Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site.

The metabotropic glutamate receptor 5 (mGlu) is a promising therapeutic target for multiple CNS disorders. Recent mGlu drug discovery has focused on targeting binding sites within the mGlu 7-transmembrane domain (7TM) that are topographically distinct from that of the endogenous ligand. mGlu primarily couples to G proteins leading to mobilization of intracellular Ca (iCa), but also activates iCa independent signaling pathways, with biased agonism/modulation operative for multiple positive allosteric modulator (PAM) and PAM-agonist chemotypes. Although several residues within the common allosteric binding pocket are key determinants of PAM activity, how these residues affect biased modulation is unknown. The current study probed the molecular basis of mGlu PAM biased modulation. Modulation of mGlu activity by four chemically distinct mGlu PAMs (VU0424465, DPFE, VU29 and VU0409551) was assessed across two distinct receptor endpoints (iCa mobilization and ERK1/2 phosphorylation) at mGlu receptors containing single-point mutations of allosteric binding pocket residues informed by computational modeling. Many mutations had differential effects on PAM affinity and cooperativity across signaling endpoints, resulting in gain or reversal of bias at the level of both affinity and functional cooperativity. Additionally, mutants had differential effects on functional cooperativity between the orthosteric ligands, DHPG and glutamate, and the PAMs, DPFE and VU29, but not VU0409551, indicating that probe dependence is linked to orthosteric agonists conferring activation states that differentially influence allosteric ligand-receptor interactions in a chemotype dependent fashion. Collectively, these data provide crucial insight into the residues that govern different activation states adopted by mGlu in order to signal via distinct intracellular pathways when co-bound by orthosteric agonists and PAMs.