Distinct roles of the extracellular surface residues of glucagon-like peptide-1 receptor in β-arrestin 1/2 signaling

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with β-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in β-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated β-arrestin 1/2 recruitment for diverse ligands, and β-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased β-arrestin 1 recruitment but increased β-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected β-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in β-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive β-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in β-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in β-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on β-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without β-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and β-arrestins. Our study offers valuable information about ligand induced β-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.