Cell-based calcium assays have been a mainstay of GPCR (G protein coupled receptor) drug discovery and signal transduction research since the mid 1990s. However, a calcium readout alone might be ambiguous, because many cellular processes produce calcium. Furthermore, it does not differentiate between signalling pathways routed through the different types of heterotrimeric G proteins. While effects on GPCRs which exhibit effects on Gs proteins lead to an elevated level of the second messaeger cAMP by activating the activity of adenylate cyclase, signalling through Gi proteins inhibits adenylate cyclase and thus leads to a decrease of cAMP. Finally signalling through Gq proteins leads to phospolipase C activation which cleaves Phosphatidylinositol 4,5-bisphosphate (PIP2) to Diacetlyglycerol (DAG) and Inositol trisphosphate (IP3) which leads to an increase in Ca2+ level (for an overview see reference 1).
Dye based methods to measure Ca2+ are very well established, whereas there are no dyes available for most of the other second messengers in these signalling pathways.
Genetically encoded fluorescent biosensors for live cell discovery
That’s why Montana Molecular developed genetically encoded fluorescent biosensors for second messengers such as cAMP, DAG, PIP2, and Ca2+, references 2, 3). [Read more…]