Metabotropic glutamate receptors (mGluRs) are a diverse and abundant class of G protein-coupled receptors (GPCRs) that mediate the transmitter and modulatory actions of glutamate, the most abundant and ubiquitous transmitter in the vertebrate CNS. Based on the important role these receptors have in synaptic plasticity, neuronal development, and the modulation of synaptic transmission, it is likely that appropriate functioning of these receptors is a critical determinant of the cell biology underlying mental health and disease. In this application, we propose a series of experiments to continue investigating the structural basis and functional consequences of mGluR dimerization. We discovered this phenomena, shown it is due to a homodisulfide between cys129 of two mGlu5 polypeptides, and have demonstrated that, surprisingly, covalent dimerization is not critically essential for agonist binding or signal transduction through mGlu5, but is important for receptor stability. To explore in more detail the functional role of covalent mGluR dimerization, a series of in vitro and in vivo experiments are proposed here. We will undertake a detailed comparison between the biochemical, kinetic, and cell biological properties of wt and dimerization-deficient receptors in heterologous expression systems. We will produce transgenic mouse lines in which the mutant receptor incapable of covalent dimerization replaces the wt. These animals will be used for in vivo studies on the function of the receptor, including the responses of the animals to hyperthermia and ischemia, and as a source of tissue and cells expressing the mutant receptor in situ, which will be used for in vitro experiments. We have also shown that there are non-covalent associations between mGlu5 polypeptides. Here we propose a set of experiments to ascertain the specific parts of the molecule mediating this dimerization. Related studies will explore how the cell achieves subtype-specific assembly of mGluR dimers. We anticipate that these investigations of mGluR dimerization will deepen our understanding of neural communication and signal transduction, processes that underlie brain function and dysfunction.
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