UC San Diego

G Protein-Coupled Receptors (GPCRs) represent one of the largest and physiologically relevant families of cell surface receptors which mediate extracellular signals through the direct and selective interaction with heterotrimeric G proteins. GPCRs have also been observed coupling to multiple G protein families, yet the structural drivers of this type of selectivity, or promiscuity, haven’t been fully explored. Current models in the field suggest that selectivity by the GPCR is primarily driven by the carboxy-terminal end of the G subunit of the heterotrimeric G protein complex, but more recent studies have identified other important regions to explore i.e., the Gamino terminus. To address this, we generated a library of chimeric Gs subunits through site-directed mutagenesis in which the carboxy-termini and/or amino-termini from different families were swapped and replaced to explore each motif’s influence on the GPCR-G protein interaction. The activity of the chimeras were indirectly measured using a FRET-based assay to detect the downstream second messenger cAMP using HEK293 cells in which endogenous Gs subunits have been disrupted. While the results of the data collected show a synergistic effect when both regions are replaced, an extended literature review has inarguably revealed the amino-terminus to be among a group of important structural players influencing selectivity of GPCRs, along with the N/1 hinge, the 23 loop, and the carboxy-terminus, which all have varying influences depending on the receptor. Overall, the findings herein provide important insights into the selectivity determinants related to the GPCR-G protein selection process and identify motifs outside of the carboxy-termini.