For my HotStart Placement, I spent eight weeks working under the supervision of Dr Dawn Thompson, alongside India D’Orsi, on a lab-based project investigating cellular signalling responses linked to the prostaglandin f (FP) receptor and phosphoinositide 3-kinase (PI3K).
The FP receptor is a G-protein coupled receptor (GPCR) which is activated by prostaglandins, it is particularly important in female reproductive function and is involved in regulating blood pressure. PI3K is an enzyme involved in cellular responses to insulin and growth factors.
One of the first parts of the project involved investigating how the FP receptor interacts with different signalling proteins. To do this, we generated constructs that could be temporarily transfected into HEK293 cells before carrying out bioluminescence resonance energy transfer (BRET) assays to investigate the recruitment of different G proteins (Gq, Gs, Gi) and Arrestin 3 to the activated FP receptor. Our results showed that the FP receptor strongly recruits Gq, while showing much more limited recruitment of Gs, Gi and Arr3.
We then investigated how the FP receptor responds to prolonged stimulation with prostaglandin F2α (PGF2α), an agonist of the receptor. By stimulating the cells over different time points, we could see how receptor signalling changed over time. We also investigated how the strength of the cellular response changed when the concentration of PGF2α was altered. These experiments allowed us to build a better understanding of how the FP receptor responds to stimulation and how this response changes over time and at different concentrations.
Another part of my project focused on PI3K and how it interacts with different receptors. I generated different constructs which were transfected into HEK293 cells and used BRET assays to investigate the behaviour of PI3K following stimulation of the FP receptor and formyl peptide receptor 2 (FPR2), another GPCR involved in processes such as tissue repair. These experiments allowed us to investigate whether PI3K was recruited towards the cell membrane or moved away from it following receptor activation.
Overall, I found this placement to be an incredibly valuable experience. I have developed a wide range of practical lab skills including PCR, cloning, preparing and streaking LB-Agar plates, growing bacterial cultures, extracting bacterial DNA, cell culture, gel electrophoresis, western blotting and BRET assays as well as gaining a greater appreciation for molecular pharmacology. This experience has pushed me out of my comfort zone and has helped me to develop skills which will be essential for my honours project and further studies.
