Abstract
Endogenous satiety hormones provide an attractive target for obesity drugs. Glucagon causes weight loss by reducing food intake and increasing energy expenditure. To further understand the cellular mechanisms by which glucagon and related ligands activate the glucagon receptor (GCGR), we investigated the interaction of the GCGR with receptor activity modifying protein (RAMP)2, a member of the family of receptor activity modifying proteins. We used a combination of competition binding experiments, cell surface enzyme-linked immunosorbent assay, functional assays assessing the Gαs and Gαq pathways and β-arrestin recruitment, and small interfering RNA knockdown to examine the effect of RAMP2 on the GCGR. Ligands tested were glucagon; glucagonlike peptide-1 (GLP-1); oxyntomodulin; and analog G(X), a GLP-1/glucagon coagonist developed in-house. Confocal microscopy was used to assess whether RAMP2 affects the subcellular distribution of GCGR. Here we demonstrate that coexpression of RAMP2 and the GCGR results in reduced cell surface expression of the GCGR. This was confirmed by confocal microscopy, which demonstrated that RAMP2 colocalizes with the GCGR and causes significant GCGR cellular redistribution. Furthermore, the presence of RAMP2 influences signaling through the Gαs and Gαq pathways, as well as recruitment of β-arrestin. This work suggests that RAMP2 may modify the agonist activity and trafficking of the GCGR, with potential relevance to production of new peptide analogs with selective agonist activities.
Highlights
Gut and pancreatic hormones involved in appetite regulation are an attractive target for the development of drugs that aim to cause effective weight loss with minimal side effects
H-RAMP2 was undetectable in the control cell line but expressed in the CHO-K1-glucagon receptor (GCGR)-CFP-RAMP2 cells. 292 RAMP2 reduces specific glucagon binding at the GCGR 293 When specific glucagon binding to the GCGR was compared in RAMP2 positive and negative CHO294 K1 cells, it was found to be 10-fold lower in the presence of RAMP2
Oxyntomodulin and analogue G(X) showed a 7300 fold and 2.5 fold lower affinity for the GCGR than the native peptide, respectively (Figures 1E and F). Similar to glucagon, the presence of RAMP2 had no effect on the binding affinity at the GCGR for glucagon-like peptide-1 (GLP-1), oxyntomodulin or analogue G(X). 304 To ensure that these findings were attributable to co-expression of RAMP2 with the GCGR, a second 305 independent cell line with RAMP2 stably upregulated was investigated (CHO-K1-GCGR-CFP306 RAMP2) and compared to a cell line transfected in parallel with a control plasmid
Summary
Gut and pancreatic hormones involved in appetite regulation are an attractive target for the development of drugs that aim to cause effective weight loss with minimal side effects. 70 The glucagon receptor (GCGR) is a 7 transmembrane class B G-protein coupled receptor (GPCR). It classically activates adenylyl cyclase through Gαs with subsequent activation of protein kinase A (PKA) signalling [5,6]. Work to unpick glucagon signalling pathways has been underway since the 1970s, it has focussed primarily on understanding the interactions involved in the downstream effects in the liver and the pancreas. 85 Understanding the interaction of these pathways may allow ‘biasing’ of signalling to exploit desirable downstream effects [15,16]. The ability of RAMPs to influence downstream signalling pathways is an exciting concept, as it may enable the creation of biased agonists that fully exploit the therapeutic potential of clinically important receptors
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