Receptor Activity-Modifying Protein 2 (RAMP2) alters glucagon receptor trafficking in hepatocytes with functional effects on receptor signalling

Abstract

ObjectivesReceptor Activity-Modifying Protein 2 (RAMP2) is a chaperone protein which allosterically binds to and interacts with the glucagon receptor (GCGR). The aims of this study were to investigate the effects of RAMP2 on GCGR trafficking and signalling in the liver, where glucagon is important for carbohydrate and lipid metabolism. MethodsSubcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking assays and radioligand binding assays in human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking Wiskott Aldrich Syndrome protein and scar homologue (WASH) complex were used to investigate the effect of a halt in recycling of internalised proteins on GCGR signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 up-regulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied. ResultsGCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular retention of glucagon-stimulated GCGR in cells lacking WASH complex indicates that activated GCGRs undergo continuous cycles of internalisation and recycling despite apparent GCGR plasma membrane localisation up to 40 minutes post-stimulation. Co-expression of RAMP2 induces GCGR internalisation both basally and in response to agonist-stimulation. The intracellular retention of GCGR in the presence of RAMP2 confers a bias away from {beta}-arrestin-2 recruitment coupled to increased activation of Gs proteins at endosomes. This is associated with increased short-term efficacy for glucagon-stimulated cAMP production, although long-term signalling is dampened by increased receptor lysosomal targeting for degradation. Despite these signalling effects, only minor disturbance of carbohydrate metabolism was observed in mice with up-regulated hepatic RAMP2. ConclusionsBy retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted. Graphical abstractIcons sourced from [1] O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY