Ancestral reconstruction of the ligand-binding pocket of Family C G protein-coupled receptors

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Abstract
The metabotropic glutamate receptors (mGluRs) within the Family C subclass of G protein-coupled receptors are crucial modulators of synaptic transmission. However, their closest relatives include a diverse group of sensory receptors whose biological functions are not associated with neurotransmission, raising the question of the evolutionary origin of amino acid-binding Family C receptors. A common feature of most, if not all, functional Family C receptors is the presence of an amino acid-binding site localized within the large extracellular Venus flytrap domain. Here, we used maximum likelihood methods to infer the ancestral state of key residues in the amino acid-binding pocket of a primordial Family C receptor. These residues were reconstructed in the background of the fish 5.24 chemosensory receptor, a broad-spectrum amino acid-activated receptor. Unlike the WT 5.24 receptor, which was not activated by mGluR agonists and displayed low sensitivity toward l-glutamate, the reconstructed ancestral receptor possessed a pharmacological profile characterized by high affinity for both l-glutamate and selective Group I mGluR agonists. This pharmacological phenotype could be largely recapitulated by mutating only two residues in the 5.24 receptor-binding pocket. Our results suggest that this primordial Family C receptor may have arisen early in metazoan evolution and that it already was preadapted as a glutamate receptor for its later use at excitatory synapses in glutamate-mediated neurotransmission.