Differential Role of Phosphorylation in Glucagon Family Receptor Signaling Revealed by Mass Spectrometry.

In response to extracellular ligands, G protein-coupled receptors (GPCRs) undergo conformational changes that induce coupling to intracellular effectors such as heterotrimeric G proteins that trigger various downstream signaling pathways. These events have been shown to be highly regulated by concerted effects of post-translational modifications (PTMs) that occur in a ligand-dependent manner. Most notably, phosphorylation of residues in the C-terminal cytoplasmic tail of GPCRs has been strongly implicated in promoting receptor interactions with β-arrestins (βarrs), which are cytosolic adaptor proteins that modulate G protein coupling, receptor internalization, and perhaps also serve as signaling modules in their own right. Here, we use proteomic methods to identify C-tail residues that are phosphorylated in the glucagon family of class B1 GPCRs (GLP-1R, GCGR, and GIPR) upon agonist addition. We demonstrate that the phosphorylation of GLP-1R and GIPR is a critical determinant in the formation of GPCR-βarr complexes. However, our results suggest that ligand-induced βarr recruitment to GCGR proceeds in a phosphorylation-independent manner. These findings highlight the importance of recognizing phosphorylation as a component in the regulation of class B1 GPCR signaling but also the need to consider how such phenomena may not necessarily yield identical effects on intracellular signaling cascades.