Chavez-Abiega, S.; Bruggeman, F. J.; Goedhart, J.

Genetically encoded biosensors allow us to study events in single cells in real time. Here, we used biosensors with a read-out based on Forster resonance energy transfer (FRET) or intensity to investigate the main signaling pathways activated immediately downstream of the calcium sensing receptor (CaSR) in HEK-293 cells. CaSR responds to its endogenous ligand, calcium, to activate Gq and Gi protein dependent pathways. We demonstrate that CaSR activates Gq to promote intracellular calcium mobilization, and we monitored these two events simultaneously in the same cells. We also observed that CaSR activates the 3 Gi subtypes and decreases cAMP levels in a Gi-dependent manner. Moreover, the use of negative allosteric modulator NPS-2143 can inhibit these signaling events and also rapidly disrupts them if added after receptor activation by calcium stimulation. In addition, the increases in calcium and cAMP levels were respectively enhanced when activation of Gi or Gq proteins was prevented. Finally, we provide evidence that CaSR does not couple to G13 proteins, and that activation of RhoA by CaSR is solely dependent on Gq/G11 activity.