Cover Story Current Issue

White adipose tissue (WAT) is a complex organ that plays a central role in systemic energy balance through its interrelated metabolic, endocrine, and immune functions. Adipocytes, the parenchymal cells of adipose tissue, have diverse functions that include storage and mobilization of lipids. They also release endocrine signals that report energy status to the brain, regulating metabolic functions in peripheral organs. Importantly, the metabolic character of white adipocytes is flexible, with cells capable of assuming distinct anabolic and catabolic/thermogenic phenotypes, often within the same adipose tissue depot

Elizabeth A. Rondini, Vanesa D. Ramseyer, Rayanne B. Burl, Roger Pique-Regi, James G. Granneman

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Current Issue

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

Emma Rose McGlone, Yusman Manchanda, Ben Jones, Phil Pickford, ... Alejandra Tomas

 

Objectives

Receptor 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 (GCG) is important for carbohydrate and lipid metabolism.

Methods

Subcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking and radioligand binding assaysin human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking the Wiskott-Aldrich Syndrome protein and scar homologue (WASH) complex and the trafficking inhibitor monensin were used to investigate the effect of halted recycling of internalised proteins on GCGR subcellular localisation and signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on the recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 upregulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied.

Results

GCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular accumulation of GCG-stimulated GCGR in cells lacking the WASH complex or in the presence of monensin indicates that activated GCGR undergoes continuous cycles of internalisation and recycling, despite apparent GCGR plasma membrane localisation up to 40 min 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 β-arrestin-2 recruitment coupled with increased activation of Gαs 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 a minor disturbance of carbohydrate metabolism was observed in mice with upregulated hepatic RAMP2.

Conclusions

By retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted.

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

Emma Rose McGlone, Yusman Manchanda, Ben Jones, Phil Pickford, ... Alejandra Tomas

 

Objectives

Receptor 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 (GCG) is important for carbohydrate and lipid metabolism.

Methods

Subcellular localisation of GCGR in the presence and absence of RAMP2 was investigated using confocal microscopy, trafficking and radioligand binding assaysin human embryonic kidney (HEK293T) and human hepatoma (Huh7) cells. Mouse embryonic fibroblasts (MEFs) lacking the Wiskott-Aldrich Syndrome protein and scar homologue (WASH) complex and the trafficking inhibitor monensin were used to investigate the effect of halted recycling of internalised proteins on GCGR subcellular localisation and signalling in the absence of RAMP2. NanoBiT complementation and cyclic AMP assays were used to study the functional effect of RAMP2 on the recruitment and activation of GCGR signalling mediators. Response to hepatic RAMP2 upregulation in lean and obese adult mice using a bespoke adeno-associated viral vector was also studied.

Results

GCGR is predominantly localised at the plasma membrane in the absence of RAMP2 and exhibits remarkably slow internalisation in response to agonist stimulation. Rapid intracellular accumulation of GCG-stimulated GCGR in cells lacking the WASH complex or in the presence of monensin indicates that activated GCGR undergoes continuous cycles of internalisation and recycling, despite apparent GCGR plasma membrane localisation up to 40 min 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 β-arrestin-2 recruitment coupled with increased activation of Gαs 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 a minor disturbance of carbohydrate metabolism was observed in mice with upregulated hepatic RAMP2.

Conclusions

By retaining GCGR intracellularly, RAMP2 alters the spatiotemporal pattern of GCGR signalling. Further exploration of the effects of RAMP2 on GCGR in vivo is warranted.

2020 impact factor: 7.4

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