Optical Probing of Metabotropic Glutamate Receptor Assembly and Cooperativity

Abstract

Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors that are found throughout the nervous system where they respond to the major excitatory neurotransmitter, glutamate, to modulate synaptic transmission and plasticity via a variety of effectors. mGluRs are class C GPCRs which are characterized by their large, bi-lobed extracellular ligand binding domains. Studies of the assembly of mGluRs have been limited to classical biochemistry and recently, bulk FRET, and have failed to fully describe how mGluRs specifically assemble as homo or heteromers and interact with G-proteins in the cellular context. We used a combination of optical techniques to examine mGluR stoichiometry and to explore how these receptors activate in response to ligand binding. Using single molecule fluorescence photobleaching in the plasma membrane of Xenopus oocytes we confirm that mGluRs form both homo- and heteromeric dimers. Furthermore, we have used truncations, mutations, and chimeras to determine the mechanism of dimerization and heterodimer specificity. Purification of full-length receptors from mammalian cells followed by immobilization on a surface and single molecule fluorescence counting have confirmed the results. In addition to studies of mGluR stoichiometry we have used light-gated mGluRs which are based on the covalent attachment of photoswitchable tethered ligands (PTLs) that work as agonists or antagonists to study receptor cooperativity. PTLs provide the ability to probe single subunit contributions to receptor activity using a ligand with an extremely high local concentration that may be isomerized in less than 1 ms. We will report on experiments that use photoswitching in one or both subunits of a dimer to determine how ligand binding couples to receptor activation. Finally, we have used the absolute subtype specifiicity afforded by PTLs to analyze the signal transduction properties and pharmacology of mGluR2/3 heterodimers.