Extracellular domain-dependent modulation of class B1 G-protein-coupled receptor signaling

The class B1 subfamily of G-protein-coupled receptors (GPCRs) comprises 15 members that respond to endogenous peptides to regulate an array of important physiological processes. Activation by peptide ligands is considered to follow a 2-step binding mechanism that involves an initial interaction with the receptor ectodomain (ECD) followed by engagement with transmembrane regions that collectively result in effector coupling. This model suggests that the ECD acts as an affinity trap that generally does not regulate downstream signaling events. Here, we show that members of the glucagon receptor subfamily of class B1 GPCRs contain a conserved 6-amino acid motif within their ECDs and that individual residues regulate cAMP and/or βarr pathways by either allosterically promoting effector coupling or as determinants required for proper receptor expression and function via modulation of posttranslational receptor glycosylation. We also highlight that this conserved motif surprisingly alters observed signaling phenomena in both a ligand- and receptor-dependent manner. Our study unveils an unexpected role of the ECD in regulating class B1 GPCR signaling properties independently of ligand binding affinity and suggests that the well-established 2-step binding paradigm requires further investigation to understand the nuances of activation mechanisms for this receptor class. SIGNIFICANCE STATEMENT: Class B1 G-protein-coupled receptors respond to endogenous peptide ligands to fine-tune biological processes. These receptors contain the hallmark structural feature of a large ectodomain at their N-termini that has been shown to be critical for ligand binding affinity, but whether this region regulates downstream signaling remains unclear. We identify a conserved site within the glucagon-like peptide-1 receptor, glucagon receptor, and glucose-dependent insulinotropic polypeptide receptor localized to the ectodomain that modulates both effector coupling as well as proper receptor expression and functionality in ligand- and receptor-dependent manners. Given the benefit of targeting these receptors in the treatment of type 2 diabetes and obesity, our findings may provide insights toward a better understanding of their activation mechanisms and facilitate rational drug discovery approaches.