SUMOylation of the B2AR influences receptor internalisation, desensitisation and downstream signalling

Wills, Lauren (2017) SUMOylation of the B2AR influences receptor internalisation, desensitisation and downstream signalling. PhD thesis, University of Glasgow.

Abstract

The beta 2 adrenergic receptor (β2AR) is a GPCR that is susceptible to multiple post-translational modifications (PTMs) including phosphorylation, ubiquitination, palmitoylation and glycosylation, which can alter how the β2AR orchestrates downstream intracellular signals. Following the discovery of SUMOylation of the cardiac signalling protein SERCA2a, we hypothesised that the β2AR, which is also involved in cardiac signalling, may be a substrate for SUMOylation. This notion was supported by previous findings that five different GPCRs have been identified as susceptible to SUMOylation, including metabotropic glutamate receptors (mGluR), a cannabinoid receptor, a serotonin receptor and a receptor involved in basal cell carcinoma known as smo.

For the first time, we confirm the susceptibility of the β2AR to SUMOylation by identifying SUMO accepting lysines, interaction sites between the β2AR and the enzymes of the SUMOylation cascade, and the traditional “ghost” band, which is indicative of protein SUMOylation. SUMOylation has been shown to influence receptor signalling, and we now uncover a possible role for SUMOylation in β2AR signalling. I report that SUMOylation of the β2AR (mediated via overexpression of the E3 ligase PIASγ) reduces β2AR phosphorylation by PKA altering the receptor driven phospho-ERK response, inhibits β2AR ubiquitination and degradation, and delays β2AR internalisation. These changes could be associated with steric effects of the bulky SUMO modification and ubiquitin-SUMOylation competition for available surface associated lysines.

With the importance of SUMOylation in multiple disease states (including cardiovascular disease, neurodegenerative disease and cancer), we worked in conjunction with the antibody production company Badrilla ® to produce a SUMO-substrate specific antibody for a site within the third intracellular loop of the β2AR. The antibody we have produced is successful in recognising the SUMOylated form of the receptor in both cell and tissue lysate, making it the first antibody of its kind to be generated. In conjunction with the Hajjar group at Mount Sinai Cardiovascular Research Centre (New York, USA) we used the SUMO-β2AR specific antibody to assess the influence of the SUMO modification in two animal models of heart failure (HF); transverse aortic constriction (TAC) pressure overload HF model in mice and the left anterior descending (LAD) artery balloon occlusion ischemic HF model in pigs. Prior work within the Hajjar group has revealed that SUMOylation of SERCA2a is reduced in HF, and restoring this modification to SERCA2a via SUMO-1 adeno-associated viral-mediated gene delivery is beneficial in restoring cardiac function. We hypothesised that SUMOylation of the β2AR would also be reduced in HF, however unexpectedly; the SUMOylated form of the β2AR was increased in the LAD artery balloon occlusion ischemic HF model in pigs.

The role of the β2AR in HF is unclear. There are studies which both report a cardio-protective and a cardio-toxic role of the receptor. To ascertain the role SUMOylation of the β2AR plays, in vivo studies promoting SUMOylation of the β2AR in the HF models described above will help to determine if enhanced SUMOylation of the receptor worsens the HF phenotype or prevents its development.

To conclude, we present the first evidence that the β2AR can be modified by SUMOylation, which acts to influence the receptors downstream signalling, desensitisation and degradation. We have designed a first-in-class SUMO-β2AR antibody – which paves the way for a panel of SUMO specific antibodies – and utilised it to assess SUMOylation of the β2AR in model cellular systems and animal models of HF. Similarly, to SERCA2a, SUMOylation does influence β2AR in the HF phenotype, although not a decrease in SUMOylation as was expected, but an increase. Future work in animal models promoting SUMOylation of the receptor is vital to assess the role of this highly novel post-translational modification of the β2AR.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: SUMOylation, β2AR.
• Subjects: Q Science > Q Science (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Baillie, Prof. George
• Date of Award: 2017
• Depositing User: Dr Lauren Wills
• Unique ID: glathesis:2017-8564
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 02 Nov 2017 11:54
• Last Modified: 29 Oct 2018 13:11
• URI: https://theses.gla.ac.uk/id/eprint/8564