Sensing Cyclic Adenosine Monophosphate and Guanine Nucleotide Exchange Factor Communication Through Rluc-Epac-Citrine2 BRET Sensor in Live GBM Cells

Abstract

Cyclic adenosine 3’,5’-monophosphate (cAMP) is a versatile secondary messenger that communicates with Guanine Nucleotide Exchange Factor (EPAC) to transfer cellular signaling and regulates numerous physiological conditions. Early studies focused on measuring this communication is considered as crucial in GPCR ligand-mediated EPAC activation, where bioluminescence resonance energy transfer (BRET) sensor has been widely used to study the cAMP level in living cells. However, a BRET sensor pairing with the best brightness and photostability for detecting low levels of cAMP in single and whole-cell populations has yet to be developed. Here, we constructed a novel BRET-based cAMP biosensor with Rluc-Epac-Citrine2. A molecular communication study revealed a significant change of over 100° in the phi value for the residues Thr253, Val259, and Thr260 in the presence of cAMP, leading to strong cAMP-Epac-induced dynamics in the ternary complex. Spectrum scanning, luminescence, and fluorescence emission studies on glioblastoma multiforme (GBM) cells demonstrated closer proximity between donor and acceptor, ensuring the cAMP sensor’s activity. This sensor detects changes in endogenous cAMP levels, and the observed BRET signal can be enhanced by increasing the concentration of the substrate, coelenterazine. The sensor also efficiently detects the communication between cAMP and EPAC in live GBM cells over time. We used this sensor to assess the activation of GPR17, a potential biomarker for GBM. The activation of MDL 29,951, a GPR17 agonist, supports the sensor’s ability to detect Gi-coupled protein activation. This study also shows the feasibility of sensor readouts using inexpensive instrumentation such as plate readers and image systems. Overall, this study sheds new light on detecting cAMP communication with EPAC and GPR17 ligand-mediated EPAC in GBM cells, potentially aiding the development of precision therapies.