Assembly and Cooperativity of Metabotropic Glutamate Receptors

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 characterized by obligate dimerization and the presence of large extracellular ligand binding domains. In this study we used a combination of novel optical techniques to examine mGlu stoichiometry and to explore how these receptors cooperatively activate in response to ligand binding. We first used single molecule fluorescence photobleaching in live Xenopus oocytes and in receptors immobilized from mammalian cell lysate to map the dimer interfaces of homo and heterodimers. This work was complemented by experiments that use a covalently attached photoswitchable tethered ligand (PTL) that works as an agonist to analyze the cooperativity that arises from ligand binding to one or both subunits within a dimer. Finally, intersubunit FRET was used to analyze the conformational changes of mGluRs in order to gain an integrative view of receptor assembly, structural dynamics, and activation.