Orlandi, CesareAlabdali, Rana2025-01-272024https://hdl.handle.net/20.500.14154/74751G Protein Coupled Receptors (GPCRs) are the largest group of membrane receptors in mammals. They are seven transmembrane proteins that couple and transduce signals through heterotrimeric G proteins and β-arrestins which further regulate various downstream signaling pathways. In this context, biased signaling is defined as the ligand-dependent selectivity for certain signal pathways when compared to a reference ligand acting on the same receptor. The most classical example of biased signaling is the functional selectivity between G proteins and β- arrestin. However, signaling bias can also be generated in response to distinct activation of individual Gα proteins leading to different downstream effectors. Therefore, we hypothesize that agonist structure, accessory molecules, and post- transcriptional modifications are three major contributors of G protein signaling bias that in turn regulates unique intracellular effectors and responses. Using cell- based assays, I investigated the effect of agonist structure on serotonin 1A receptor (5-HT1AR) coupling profile using our optimized G protein nanoBRET assay. Also, I examined the effect of accessory lipid such as cholesterol on G protein coupling using 5-HT1AR as model in transfected HEK293 cells. Additionally, I explored the effect of the interaction with the accessory protein receptor component protein (RCP) on the G protein coupling profile of CGRP receptor. Finally, I studied how RNA edited isoforms of serotonin 2C receptor (5- HT2CR) could tune its G protein coupling profile. This would help in understanding how Gα protein preferential selection can potentially alter cellular responses.161en-USGPCRsbiased signaling5-HTMolecular biasesBRET assayG proteinsThesis