Valentina di Liberto (fourth from the left) pictured with Professor Francisco Ciruela's lab team.
Valentina Di Liberto (Italy) from the Università degli Studi di Palermo shares below her IBRO-PERC InEurope Short Stay final report on the “Identification of novel Guanosine receptor complex,” the research project she completed at the Universitat de Barcelona (Spain) in Francisco Ciruela’s laboratory.
I worked on the identification of novel receptor complexes responsive to Guanosine. According to the two tasks described in the grant proposal, my work aimed to study the oligomerization between GPR23 and Adenosine receptors (especially the A2AR subtype) and the direct binding of Guanosine to GPR23 and/or to Adenosine receptors.
Concerning the first task, HEK293 cells were transiently transfected with NanoLuc-GPR23 and YFP-A2AR, and incubated for 1 hour with APECAlexa-647, a fluorescent A2AR agonist. The NanoBRET signal, index of receptor-receptor interaction, was detected using a POLAStar microplate reader following coelenterazine addition, the NanoLuc substrate.
Preliminary experiments revealed a low NanoBRET signal, that could prove a low-grade interaction (in terms of number of heterocomplexes or proximity) between the two receptors. These preliminary observations, although potentially encouraging, need further investigations. To uncover whether Guanosine binds to GPR23 and/or to adenosine receptors, HEK293 cells were transiently transfected with NanoLuc-GPR23, or NanoLuc-A1R, or NanoLuc A2AR, and treated for 1 hour with Rhodamine-conjugated Guanosine.
NanoBRET signal, index of binding between Guo and the mentioned receptors, was monitored using POLAStar microplate reader following coelenterazine addition. Interestingly, in contrast to negative data obtained with cells transfected with GPR23 or A1R, a specific NanoBret signal was observed in HEK293 cells transfected with A2AR, thus suggesting a direct binding of Guanosine to this receptor.
In order to confirm this result, further NanoBret experiments are currently ongoing in the host laboratory. Moreover, with the purpose of validating these data using different techniques, plasmatic membranes from HEK293 cells, transiently transfected with NanoLuc-GPR23, or NanoLuc-A1R, or NanoLuc A2AR, were extracted.
Currently, I’m using these protein extracts in 3 H-Guanosine Radioligand binding assays. Comparison of the 3 H-Guanosine binding between the four different membrane extracts, including HEK293 not-transfected membranes, will reveal the contribute of each receptor in Guanosine binding.
In conclusion, the IBRO-PERC short stay grant allowed me to learn and set up advanced bioluminescence resonance energy transfer-based techniques to visualize receptor heterodimers and ligand-receptor interactions to successfully carry on my research project. The data obtained in the host laboratory, although preliminary and to be confirmed, encourage further investigations, already in progress, about the novel role of A2A receptors in Guanosine binding.
IBRO congratulates Valentina on completing her short stay collaboration in Spain and wishes her continued success.
For more information on IBRO-PERC Short Stay Grants, click here.