PhD Scientific Days 2023

Molecular Sciences - Posters B

The investigation of a new BRET system to study protein interactions

Text of the abstract

Titration BRET measurements are commonly used to study dynamic interactions. The disadvantage of this system is that it is cumbersome and time-consuming. Our aim was to develop a system in which static interactions can be studied under dynamic conditions in a simple way.
We investigated the interaction of HTSF1 protein and β-arrestin protein. β-arrestin proteins terminate the G-protein signaling after activation of GPCRs (G-protein coupled receptors) and also orchestrate a new wave of signal transduction steps. They regulate a broad range of cellular processes e.g. migration, proliferation, differentiation, and apoptosis.
A rapamycin system was used in this new method, which binds FRB and FKBP molecular tags upon stimulation. HEKT cells were transfected with; L10-FRB-mVenus, FKBP-Barr2 or FKBP-Barr2-384del (pre-activated form), and HTSF1-RLuc8 or HTSF1mut-RLuc8 (β-arrestin binding S-T pattern is mutated). In our experiments, we also cotransfected various GRKs (G protein-coupled receptor kinases) to promote protein phosphorylation.
Our results show that the static interaction of HTSF1 with β-arrestin2 can be detected with good sensitivity in our BRET system and this system can be used to rapidly investigate interactions that are otherwise difficult to manipulate dynamically.
Supported by the ÚNKP-22-4-I New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development, and Innovation Fund.