The effect of oxidative stress on the Adenosine A2A Receptor activity and signalling.

These data underpin the results reported in the paper of the same title, published in BBA-Biomembranes https://doi.org/10.1016/j.bbamem.2025.184412.

The adenosine A2A receptor (A2AR) is a G-protein coupled receptor that has important anti-inflammatory effects in response to some agonists and consequently is considered a therapeutic target. It is known that its activity can be affected by the local membrane lipid environment and presence of certain phospholipid classes, as discussed in the article, so studies of its activity should ideally be conducted using extraction methods that form native nanodiscs that retain the local lipids, such as styrene maleic acid co-polymers (SMA).
Oxidative stress often occurs in inflammatory situations, as the innate immune system releases reactive oxygen species. This can oxidize both phospholipids, which can alter membrane properties, and proteins, which affects their structure and activity. Oxidized lipids that are electrophilic can also modify proteins covalently. Currently, little is known about the effect of oxidative stress on the A2AR. We hypothesized that treatment with the radical initiator AAPH would oxidize either the protein or the lipids, or both, while the small reactive compound acrolein would react with nucleophilic sidechains of the A2AR, and these changes would modulate the conformational changes or activity of the receptor.
To investigate this, the A2AR was over-expressed in Pichia pastoris and SMA2000 or DIBMA were used to extract the A2AR from isolated cell membranes. The response of the A2AR in native nanodiscs to agonist NECA or antagonist ZM241385 was tested in the presence or absence of the radical initiator AAPH or reactive aldehyde acrolein. Conformational changes were monitored by tryptophan fluorescence.
Protein stability was measured by a thermal unfolding assay.
The protein was transiently transfected into HEK293 cells to investigate downstream signalling.
Cell viability to treatments with acrolein and AAPH was monitored by MTT assay.
Downstream signalling from the A2AR was assessed by measurement of cAMP.
It was found that SMA-extracted A2AR, but not DBMA_extracted A2AR, was able to undergo conformational changes, measured by tryptophan fluorescence, in response to its ligands. However, oxidative treatments had no effect on the conformational changes or temperature-dependent protein unfolding.
In contrast, in HEK293 cells transiently expressing the A2AR, oxidative treatments increased the cAMP levels in response to the agonist NECA but had no effect on direct activation of adenylate cyclase, as shown by an absence of effect on forskolin induced cAMP levels.
As increased cAMP can downregulate inflammatory processes, oxidant-induced alterations in downstream signalling from the A2AR may represent homeostatic mechanism that abrogates inflammation.

The Metadata file indicates the experiments in the uploaded files. The methods used are described in the published article.