Metabotropic glutamate receptors (mGluRs) are a diverse and abundant class of receptors that mediate neuromodulatory actions of glutamate, the most abundant and ubiquitous transmitter in the vertebrate CNS. Based on the important role these receptors have in neuronal development, synaptic plasticity, and the modulation of synaptic transmission, it is likely that appropriate functioning of these receptors is a critical determinant in the cell biology underlying mental health and disease. To understand the molecular basis for mGluR function, we have been performing biochemical and molecular biological analyses of the structure of mGluRs, concentrating on mGluR5, a developmentally regulated receptor coupled to phosphatidylinositol hydrolysis and Ca2+ mobilization. We have recently discovered a novel feature of mGluR5 structure, that it is a disulfide-linked dimer. Moreover, there is evidence that this structural feature is shared by the other mGluRs. In order to test the hypothesis that dimerization has important functional consequences and may also be a regulated mechanism, three lines of experimentation are proposed. First, in order to elucidate the molecular basis for dimerization, cysteine residues in the mGluR5 sequence will be systematically changed to serines to identify which cysteines participate in the requisite disulfide bond(s). Moreover, because the closely related mGluR1a does not form heterodimers with mGluR5, chimeras containing increasing lengths of mGluR1a sequence in place of mGluR5 will be constructed, and their ability to dimerize with wild type mGluR5 will be assessed to determine the structural basis for this homomeric specificity. Second, to analyze the functional significance of mGluR5 dimerization, the activity of non-dimerizing, mutant receptors will be compared with wild type ones using binding assays, phosphatidylinositol turnover assays, and assays of intracellular Ca2+ mobilization. Third, to determine whether dimerization is a regulated process, the state of mGluR5 dimerization in different brain regions and at different developmental periods will be examined. Furthermore, the occurrence of dimers following procedures leading to receptor activation will be determined in primary cultures of cerebellar granule cells and transfected cell systems. By studying the basis and significance of mGluR dimerization, we will deepen our understanding of neural communication and signal transduction, processes which underlie brain function and dysfunction.
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