Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca2+ ([Ca2+]o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site (Site 1) in the ECD-mGluR1α using a recently developed computational algorithm. This predicted site (D318, E325, D322 and the bound L-Glu) is situated in the hinge region in the ECD-mGluR1α adjacent to the reported Glu-binding site. Mutagenesis studies indicated that binding of L-Glu and Ca2+ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca2+-sensing capability largely intact. Mutating the predicted Ca2+-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca2+]o and [Gd3+]o but also, in some cases, to Glu. In addition, the Ca2+ effects on drugs targeting mGluR1α were investigated. Ca2+ enhances L-Quis response of the receptor by increasing L-Quis binding to ECD-mGluR1α and promotes the potency of Ro 67-4853, a positive allosteric modulator of mGluR1α. Increasing Ca2+ concentration, the inhibitory effects of a competitive antagonist ((s)-MCPG) and a non-competitive negative allosteric modulator (CPCCOEt), were eliminated. Furthermore, we also identified another potential Ca2+ binding pocket (Site 2) consists of S165, D208, Y236 and D318, which completely overlapped with L-Glu. Thapsigargin (TG) induced ER Ca2+ depletion reduced surface expression of mGluR1α, and D208I and Y236I also decreased the receptor trafficking to plasma membrane suggesting the role of Ca2+ binding in protein folding and trafficking in the ER. Further, to measure ER Ca2+, a series of genetically encoded biosensors were designed by placing a Ca2+ binding pocket at the chromophore sensitive region of red florescent protein mCherry. The designed sensors are able to bind Ca2+ and monitor Ca2+ concentration change both in vitro and in cells. The findings in this dissertation open up new avenues for developing allosteric modulators of mGluR function that target related human diseases.
| Identifer | oai:union.ndltd.org:GEORGIA/oai:digitalarchive.gsu.edu:chemistry_diss-1070 |
| Date | 01 August 2012 |
| Creators | Jiang, Yusheng |
| Publisher | Digital Archive @ GSU |
| Source Sets | Georgia State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Chemistry Dissertations |
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