Calcium Modulates MGLUR1 Folding in ER in the Trafficking Process and Regulates the Drug Activity Upon the Receptor Expressing on the Cell Membrane

Author

Yusheng Jiang, Georgia state universityFollow

Date of Award

Summer 8-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jenny Yang

Abstract

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.

DOI

https://doi.org/10.57709/3098550

Recommended Citation

Jiang, Yusheng, "Calcium Modulates MGLUR1 Folding in ER in the Trafficking Process and Regulates the Drug Activity Upon the Receptor Expressing on the Cell Membrane." Dissertation, Georgia State University, 2012.
doi: https://doi.org/10.57709/3098550