Abstract

G protein-coupled receptors (GPCRs) transduce a diverse variety of extracellular stimuli into intracellular signaling. These receptors are the most clinically productive drug targets at present. Despite decades of research on the signaling consequences of molecule-receptor interactions, conformational components of receptor-effector interactions remain incompletely described. The β₂-adrenergic receptor (β₂AR) is a prototypical and extensively studied GPCR that can provide insight into this aspect of GPCR signaling thanks to robust structural data and rich pharmacopeia. Using BRET-based biosensors, second messenger assays, and biochemical techniques we characterize the properties of β₂AR-F193A. This single point mutation in extracellular loop 2 of the β₂AR is sufficient to intrinsically bias the β₂AR away from b-arrestin interaction and demonstrates altered regulatory outcomes downstream of this functional selectivity. This study highlights the importance of extracellular control of intracellular response to stimuli and suggests a previously undescribed role for the extracellular loops of the receptor and the extracellular pocket formed by transmembrane domains 2, 3, and 7 in GPCR regulation that may contribute to biased signaling at GPCRs.

Significance Statement The role of extracellular GPCR domains in mediating intracellular interactions is not well understood. Here we characterized the effects of extracellular loop mutations on agonist-promoted interactions of GPCRs with G protein and β-arrestin. Our studies reveal that F193 in extracellular loop 2 in the β₂-adrenergic receptor mediates interactions with G protein and β-arrestin with a biased loss of β-arrestin binding. These results provide new insight on the role of the extracellular domain in differentially modulating intracellular interactions with GPCRs.