Selectivity and Evolutionary Divergence of Metabotropic Glutamate Receptors for Endogenous Ligands and G Proteins Coupled to Phospholipase C or TRP Channels

Background: Endogenous ligands and G-proteins regulate responses of metabotropic glutamate receptors. Results: l-serine-O-phosphate activates Group III while antagonizing Group I & II. mGluR1 couples to G(i/o) as well as G(q/11). Conclusion: l-Serine-O-phosphate differentially interacts with mGluR. mGluR1 has a bifurcating G-protein coupling. Significance: Defining the interactions of mGluR with endogenous ligands and G-proteins is important for understanding their roles in human neuronal function and for design of therapeutics. To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of G(i/o) proteins coupled to the ion channel TRPC4. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is l-serine-O-phosphate (l-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas l-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of l-glutamate and l-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both l-glutamate and l-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of l-SOP. Experiments with pertussis toxin and dominant-negative G(i/o) proteins revealed that mGluR1 couples strongly to TRPC4 through G(i/o), in addition to coupling to PLC through G(q/11).