Determinants of endogenous ligand specificity divergence among metabotropic glutamate receptors.

To determine the structural origins of diverse ligand response specificities among metabotropic glutamate receptors (mGluRs), we combined computational approaches with mutagenesis and ligand response assays to identify specificity-determining residues in the group I receptor, mGluR1, and the group III receptors, mGluR4 and mGluR7. Among these, mGluR1 responds to L-glutamate effectively, whereas it binds weakly to another endogenous ligand, L-serine-O-phosphate (L-SOP), which antagonizes the effects of L-glutamate. In contrast, mGluR4 has in common with other group III mGluR that it is activated with higher potency and efficacy by L-SOP. mGluR7 differs from mGluR4 and other group III mGluR in that L-glutamate and L-SOP activate it with low potency and efficacy. Enhanced versions of the evolutionary trace (ET) algorithm were used to identify residues that when swapped between mGluR1 and mGluR4 increased the potency of L-SOP inhibition relative to the potency of L-glutamate activation in mGluR1 mutants and others that diminished the potency/efficacy of L-SOP for mGluR4 mutants. In addition, combining ET identified swaps from mGluR4 with one identified by computational docking produced mGluR7 mutants that respond with dramatically enhanced potency/efficacy to L-SOP. These results reveal that an early functional divergence between group I/II and group III involved variation at positions primarily at allosteric sites located outside of binding pockets, whereas a later divergence within group III occurred through sequence variation both at the ligand-binding pocket and at loops near the dimerization interface and interlobe hinge region. They also demonstrate the power of ET for identifying allosteric determinants of evolutionary importance.