The Role of RAMPs in Modulating Glucagon Receptor Pharmacology

Cathryn Weston1, Simon Dowell2, Graham Ladds1. 1Warwick Medical School, Coventry, UK, 2GlaxoSmithKline, Stevenage, UK

The glucagon receptor (GCGR) is a member of the family B or secretin-like group of G protein-coupled receptors (GPCRs). This family all respond to peptide ligands and predominantly mediate their effects via activation of the Gαs subunit to induce an increase in cAMP production. However, coupling to a range of other effectors has been observed (including Gαi, Gαq and Gαz) (1). Many secretin-like receptors, including GCGR, have been demonstrated to couple to members of the receptor activity-modifying proteins (RAMPs) and this association has been shown to alter their pharmacology: affecting ligand preference and downstream effector recruitment (2). Despite the identification of many RAMP-GPCR interactions the functional consequences have not thus far been identified for the majority of these physiologically important receptors (3). Here we identify, significant changes in ligand-binding and effector activation when GCGR interacts with RAMP2. The method used could be employed to determine the effect of RAMP interactions on other receptors thereby enabling better informed drug design.

Since the GCGR has been demonstrated to activate multiple, competing signalling pathways all of which are present in mammalian cells it is difficult to assess the effect of RAMP2 binding on an individual pathway. We therefore utilised a modified yeast system provided by GSK (4) containing individual Gα chimeras. We have previously demonstrated the use of this system to characterise the effect of RAMP interaction on ligand-directed signal bias for the calcitonin-related-like receptor (5). In this study we expressed the GCGR in combination with RAMP2 in the yeast strains containing either the Gαs or Gαi chimera. Reporter gene activity was measured following 17 h stimulation with ligand. Data was plotted as mean of at least 5 independent experiments and pEC50 values determined through fitting of a 3-parameter concentration-response curve.

The natural GCGR ligand, glucagon activated both Gαs and Gαi subunits giving concentration-response curves (pEC50 values of 8.1 ±0.2 and 7.6 ± 0.3 respectively). Significantly co-expression of the receptor with RAMP2 potentiated both responses, however, their was a greater effect when the receptor was coupled to the Gαs chimera bringing the potency to that seen in mammalian cAMP assays (pEC50 10.1 ±0.15) demonstrating for the first time a functional consequence of the GCGR-RAMP2 interaction. Furthermore, we reveal that the glucagon-like peptide-1 (GLP-1) activates the GCGR in the yeast system with similar potency at both the Gαs and Gαi coupled receptor (pEC50 6.75 ± 0.08). This response is reduced in the presence of the competitive glucagon inhibitor, des-His1-[Glu9)-glucagon (1-29) amide, and significantly is abolished when RAMP2 is co-expressed.

Since GLP-1 and glucagon perform opposing roles in maintaining glucose homeostasis, these data suggest a possible important physiological role for the interaction of GCGR with RAMP2. Further they highlight the importance of considering all accessory protein interactions when developing drugs to target this class of receptors.

1. Grammatopoulos (2012) Br J Pharmacol 166: 85-97

2. Morfis (2008) Endocrinol 149: 5423-5432

3. Christopoulos et al., (2003) J Biol Chem 278: 3293-3297

4. Brown, A. J. et al (2000) Yeast 16:11-22

5. Weston et al., (2013) Proc Brit Pharmaco Soc http://www.pA2online.org/abstracts/Vol11Issue3abst051P