A novel system for controlling dimeric receptor composition to discover unique heterodimer pharmacology

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Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors that function as dimers. While mGluRs are known to form homodimers, more recent work has shown that they can also heterodimerize, but not promiscuously. Because mGluRs exhibit widespread expression in the brain and regulate excitability and plasticity, they have become candidates as druggable targets for a variety of pathologies. To date however, excitement generated by preclinical data has not resulted in mGluR-targeting therapies in the clinic, despite a wealth of available ligands with good selectivity targeting these receptors. Our recent work examining mGluR2/4 heterodimers provides a possible explanation: ligands that are highly efficacious when targeting homodimeric receptors are often without effect when the same receptor is expressed as a heterodimer with another mGluR. Further complicating matters, these changes in pharmacological responses observed in mGluR2/4 heterodimers are not generalizable to all mGluR heterodimers, or even all mGluR2 containing heterodimers. Thus, to understand how any mGluR ligand will function in the brain, we must examine the pharmacological responses of each possible heterodimer pair in isolation. But this is complicated because every mGluR can also form homodimers, so any pair of expressed mGluR will have an unknown propensity to homo- and heterodimerize. To solve this problem, we have designed a novel dimer composition control system using a combination of ER retention sequences paired with orthogonal, split inteins, self-excising protein sequences, that will allow expression of pure populations of nearly wild type mGluR dimers of known composition. We plan to generate a comprehensive ligand vs. mGluR dimer atlas to be used to not only aid in interpretation of experimental data but also to improve therapeutic strategies targeting mGluRs for a range of pathologies. To accomplish these goals, we will pursue the following Specific Aims: 1, T To build and characterize the full complement of tagged mGluRs using the split intein-ER retention strategy, 2, To employ an adapted CODA-RET approach to obtain parallel heterodimer specific G protein recruitment data, and 3, To systematically assess the pharmacological responses of each probable mGluR dimer pair to selective agonists, competitive antagonists, PAMs and NAMs.