G protein-coupled receptors (GPCRs) are transmembrane domain proteins. GPCRs activate G protein (Gαs/Gαi/Gαq, Gβγ complex) (Smith and Pack, 2021) and trigger guanosine diphosphate (GDP) exchange on the Gα subunit to guanosine-5′-triphosphate (GTP) leading to Gα and Gβγ subunits dissociation, transducing pleiotropic downstream signaling pathways, including cAMP and protein kinase A (PKA) (Magalhaes et al., 2012; Ritter and Hall, 2009). Activated GPCRs can also bind β-arrestin 1/2 dependent or independent on GPCR phosphorylation by G protein-coupled receptor kinases (GRKs) (Celver et al., 2013; Jorgensen et al., 2011; Latorraca et al., 2020; Takenouchi et al., 2018; Zhang et al., 2005), desensitizing receptors (Takenouchi et al., 2018) and scaffolding cellular components extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), serine/threonine kinase AKT, nuclear factor-κB (NF-κB) and so on (Jean-Charles et al., 2017; Toth et al., 2018). β-arrestin 1/2 have been demonstrated to play central roles for GPCRs in several diseases including Alzheimer's disease (Thathiah et al., 2013), diabetes (Guven et al., 2020; Liu et al., 2016) and colitis (Lee et al., 2013; Srivastava et al., 2015).

Activation of class B1 GPCR glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion from pancreatic β cells, inhibits glucagon secretion from pancreatic α cells, and prolongs gastric emptying resulting in weight loss. GLP-1R has been a successful and hot therapeutic target for type 2 diabetes and obesity (Biggs et al., 2017). The pharmacological functions of GLP-1R are modulated not only by Gαs/Gαi/Gαq (Hallbrink et al., 2001; Oduori et al., 2020; Thompson and Kanamarlapudi, 2015) but also β-arrestins (Jones et al., 2018, 2021). β-arrestin 1 regulates GLP-1 induced antiapoptotic effect through ERK1/2-p90RSK-phosphorylation of BAD protein in β cells (Quoyer et al., 2010). β-arrestin 1 knockdown decreases ERK, cAMP and insulin secretion in cultured INS-1 pancreatic β cells, but not ligand-induced GLP-1R internalization/desensitization (Sonoda et al., 2008). Additionally, β-arrestin 1 knockdown or β-arrestin 1 mutants with c-Src binding deficiency blunt GLP-1 induced proliferation in INS832/13 cells (Talbot et al., 2012). When β-arrestin 2 is specifically knocked out in mouse β cell, acute cAMP increases are impaired and desensitization is reduced in islets (Bitsi et al., 2023). Biased GLP-1 analogue with β-arrestin 2 recruitment reduction reduces receptor endocytosis and elevates insulin secretion over a prolonged stimulation period (Jones et al., 2021), indicating the vital roles of β-arrestin 1/2 for GLP-1R.

Cryo-electron microscopy (Cryo-EM) structures of GLP-1R–Gs complexes have been well studied (Cong et al., 2022; Zhao et al., 2022; Ma et al., 2020; Zhang et al., 2020), but not GLP-1R–β-arrestin complex. Additionally, the extracellular surface of GLP-1R interacts directly with ligands, controls signaling initiation and activates receptors. However, the roles of the extracellular surface in GLP-1R mediated β-arrestin 1/2 recruitment are obscure. Since the roles of 85 residues on the extracellular surface in signaling pathways other than β-arrestin 1/2 recruitment were studied in CHO-Flp-In cells upon stimulation of GLP-1, exendin-4 and oxyntomodulin (Koole et al., 2012a, 2012b; Lei et al., 2018; Wootten et al., 2016), we set up and validated a bioluminescence resonance energy transfer (BRET) assay to detect protein-protein interactions between GLP-1R and β-arrestin 1/2. Combing alanine-scanning mutagenesis and operational model of agonism (Black and Leff, 1983), β-arrestin 1/2 recruitments for the 85 mutated residues were evaluated upon GLP-1, exendin-4 and oxyntomodulin stimulation (Baggio et al., 2004). The contribution of non-conserved residues on β-arrestin 1/2 to receptor interaction was also determined. GLP-1, exendin-4, oxyntomodulin and β-arrestin 1/2 overexpression induced proteomic profiles support β-arrestin 1/2 isoform difference in structure and function. Our results demonstrate that the receptor extracellular surface contributes to ligand activated β-arrestin 1/2 signaling.