Can SUMOylation of the Beta-2-Adrenergic receptor influence cell signalling and cardiac myocyte physiology?

Ling, Jiayue (2021) Can SUMOylation of the Beta-2-Adrenergic receptor influence cell signalling and cardiac myocyte physiology? PhD thesis, University of Glasgow.

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The beta 2 adrenergic receptor (β2AR) is a transmembrane, G protein-coupled receptor that can be modified post-translationally by phosphorylation, ubiquitination, palmitoylation and glycosylation. These modifications regulate β2AR signalling and desensitisation. SUMOylation is a post-translational modification that is related to ubiquitin, hence the name “SUMO” (small ubiquitin-like modifier). SUMO is a small protein which is covalently attached to substrate proteins on lysine residues following activation of specific SUMO enzyme cascades. Following the discovery that the cardiac signalling protein sarcoplasmic Reticulum Ca2+ ATPase 2a (SERCA2a) could be modified by SUMO and since there are more than 1000 SUMO substrates in nature (Hay, 2013), it is possible that SERCAR2a may not be the only cardiac protein that could be SUMOylated. The Baillie group has used peptide array technology to identify putative SUMOylation sites by inducing in vitro SUMOylation of 15 mer peptides which contained potential SUMO conjugation motifs (Frank, 2002). Baillie group has confirmed putative SUMOylation sites on multiple cardiac specific proteins such as ryanodine receptor (RyR), L-type Ca2+ channel (LTCC), myosin binding protein C, cardiac troponin I (cTnI) and β2AR via the consensus motif ψKxE/D. Therefore, I hypothesised that the β2AR could also be SUMOylated and that this action is a novel point of regulation for β2AR receptor signalling.

In my thesis,

1. I confirm that β2AR is a substrate for SUMOylation and that β2AR SUMOylation can be promoted by the SUMO E3 ligase PIASγ.

2. I show that enhanced SUMOylation driven by PIASγ overexpression rapidly declined after agonist treatment suggesting that SUMOylation is involved in early signalling events.

3. I compare the physiological responses of WT and SUMOylation-null mutants of β2AR in NRVMs using adenoviral gene transfer. Following transfection of the receptors to NRVMs I evaluated five different parameters of contractility using the CellOPTIQ® platform. Using these techniques, I report that untransfected NRVMs displayed expected short-term enhancements in the contractile response to isoproterenol. However, NRVMs transfected with WT and mutant β2AR did not respond as expected. Counter-intuitive data resulting from excessive levels of over-expression of WT and a SUMOylation-null mutant of β2AR was recorded.

Conclusively, I report the novel finding that β2AR is a substrate of SUMOylation and β2AR SUMOylation can be promoted by the SUMO E3 ligase PIASγ. A first-in-class SUMO-β2 antibody was tested in my work in different cells and tissues confirming that SUMOylation is ubiquitous. I also reported that SUMOylation is engaged in early β2AR signalling events. However, using a variety of different model systems and techniques I was unable to definitively characterise the function of this modification. Functional studies via adenovirus vector in animal models to study the role of β2AR SUMOylation in heart failure are the future direction to this project.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
Supervisor's Name: Baillie, Prof. George S. and Graham, Dr. Delyth
Date of Award: 2021
Depositing User: Theses Team
Unique ID: glathesis:2021-82652
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Jan 2022 14:07
Last Modified: 05 Dec 2022 11:06
Thesis DOI: 10.5525/gla.thesis.82652

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