Defining the role of receptor phosphorylation on mGlu5 metabotropic glutamate receptor signal transduction

Strellis, Bethany (2025) Defining the role of receptor phosphorylation on mGlu5 metabotropic glutamate receptor signal transduction. PhD thesis, University of Glasgow.

Abstract

The type 5 metabotropic glutamate receptor (mGlu5) is a G protein-coupled receptor (GPCR) located on excitatory neurons, and its dysfunction has been linked to multiple neuropathologies such as Alzheimer’s disease (Abd-Elrahman et al., 2020). Previous data form our laboratory on phosphoproteomic analysis of the hippocampus of wildtype mice showed that global mGlu5 phosphorylation increases following a fear conditioning learning and memory test, but the specific role of phosphorylation on signal transduction was not identified. To elucidate the role of mGlu5 phosphorylation, the aim of this thesis was to dissect the signal transduction pathways downstream of G protein versus phosphorylation dependent pathways.

Phosphodeficient mutants of mGlu5 were generated by synthesising a mouse ortholog mGlu5 C-terminus with either all serine residues (mGlu5-PD), or all serine and threonine residues (total phosphodeficient mutant, mGlu5-TPD) mutated to alanine. Signal transduction pathways were assessed by generating stable cell lines expressing either wildtype mGlu5, mGlu5-PD or mGlu5-TPD. In βarrestin 2 recruitment assays, removal of putative phosphorylation sites within the C-terminus of mGlu5 was found to negatively impact the ability of the receptor to recruit β-arrestin 2. In calcium mobilisation, IP1 accumulation, and G protein dissociation assays assessing the Gαq dependent transduction pathway, there was no difference in responses between wildtype and phosphodeficient mutant receptors following agonist stimulation. However, the basal activity was reduced with removal of putative C-terminal phosphorylation sites. To further examine G protein activation, a bioluminescent resonance energy transfer (BRET)-based single genetically encoded biosensor was generated to compare the impact of phosphorylation on mGlu5 G protein activation. Findings with this biosensor revealed a reduced level of G protein activation with the phosphodeficient mutants compared to wildtype receptor.

These results demonstrate that phosphorylation of the mGlu5 C-terminus appears to predominantly modulate ligand-independent signalling through the G protein-coupled pathway, whilst phosphorylation of C-terminal serine and threonine residues impacts ligand-dependent and ligand-independent β-arrestin 2 recruitment. Therefore, understanding the impact of mGlu5 phosphorylation on receptor signalling is likely to be crucial when considering the therapeutic potential of this receptor.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: R Medicine > RB Pathology; R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Hudson, Dr. Brian
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-84922
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 25 Feb 2025 16:48
• Last Modified: 25 Feb 2025 16:51
• Thesis DOI: 10.5525/gla.thesis.84922
• URI: https://theses.gla.ac.uk/id/eprint/84922

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