Molecular Determinants of Beta-Arrestin Binding to the Glucagon Receptor

Abstract ID 130707 Poster Board 169 Activation of the heptahelical glucagon receptor (GCGR) by the peptide hormone glucagon raises blood glucose levels during hypoglycemia. GCGR activation contributes to additional pathways including lipid and amino acid metabolism, as well as secretion of insulin in islets. Manipulation of GCGR agonism shows promise for the development of novel therapies for serum glucose management as well as for obesity and heart failure. GCGR couples to stimulatory heterotrimeric G proteins (G_s) to increase cAMP levels and signaling via Protein Kinase A. The endocytic adaptors βarrestin1 (βarr1) and βarrestin2 (βarr2) are also recruited at the activated GCGRs to attenuate G protein signaling. During GPCR signal quenching, βarrs can assume a secondary role as scaffolds to promote signaling via GPCR-βarr megaplexes. We discovered that polyubiquitin chains appended to lysine333 (K333) in the GCGR act as a primer for efficient G protein coupling. Elimination of this ubiquitin signature in the GCGR drastically reduces G protein coupling while simultaneously enhancing βarr1 recruitment and p38 MAPK scaffolding, thus producing ubiquitination-driven βarr1-biased signaling. Using GCGR-wild type (WT) and ubiquitin-deficient GCGR-K333R as model systems for unbiased and βarr biased receptors, and bioluminescence resonance energy transfer (BRET) assays, we tested whether G protein expression, and βarr1 single nucleotide polymorphisms (SNPs) modulate agonist-induced recruitment of βarr1 and GCGR internalization. In parental HEK-293 cells, βarr1 recruitment to GCGR-K333R is significantly increased both in terms of agonist potency and efficacy compared to GCGR-WT. However, in HEK-293 cells lacking G_s/G_q/G₁₂ subfamilies (G6-KO cells), βarr1 recruitment to GCGR-WT improved to levels obtained with GCGR-K333R, and furthermore the increased association of βarr1 with GCGR-K333R was recovered when G_sα was overexpressed in G6-KO cells. This suggests that G proteins could potentially inhibit the association of βarr and ubiquitinated GCGR, acting as reciprocal inhibitors to βarr-induced steric occlusion of the G protein binding site. Four SNPs and resulting amino acid mutations in βarr1, namely I158V, T275I, T370M and G411S have been recently correlated with improvement in glycemic index in diabetic patients treated with GLP1R agonism. These four SNPs have not been linked with βarr1 functions thus far and when tested in angiotensin type 1 receptor expressing cells, they behaved similarly as WT βarr1 in terms of recruitment and endocytic potential. However, the SNPs showed differential effects when tested in cells expressing GCGR-WT or GCGR-K333R: their binding to the activated receptor was reduced, whereas their endocytic potential was increased as compared with WT βarr1. These data provide insights into specific regulation of βarr1 in mediating glucagon signaling and might ultimately lead to testing and development of glucagon agonists biased toward specific transducers: G_s and βarr1. 23TPA1062201 (American Heart Association); HL160029 (National Institutes of health)