Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2).

The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.This work was supported by a Warwick Impact Fund (C.W., G.L.), the BBSRC (G.L. - BB/G01227X/1), (T.S., G.R., D.R. - BB/F008392/1), (D.P. - BB/M007529/1 and BB/M000176/1), Warwick Research Development Fund (C.W., G.L.) grant number (RD13301) and the Birmingham Science City Research Alliance (G.L).This is the final version of the article. It first appeared from ASBMB at http://dx.doi.org/10.1074/jbc.M114.62460

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading.

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y13 receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y13 receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild-type (WT) and P2Y13 receptor knockout (P2Y13 R(-/-) ) mice were subject to non-invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro-computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y13 R(-/-) mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y13 R(-/-) mice compared with controls. In vitro, primary P2Y13 R(-/-) osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y13 R(-/-) osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto-enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y13 receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX-induced bone loss after P2Y13 receptor depletion as previously described, suggests a potential role for P2Y13 receptor antagonist-based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.Journal ArticleFLWINSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mean and SEM values of cell membrane FRET efficiency and fraction of receptor components involved in FRET between the CaSR and RAMPs.

<p>Significant difference in the NFRET value was observed between CaSR+RAMP1 and 3 vs. CaSR+RAMP2, and between CaSR+RAMP1 vs. CaSR +RAMP3 (***p<0.0001, Kruskal-Wallis test, Dunn's multiple comparison test). Significant differences were also observed in the fraction of RAMP3 and RAMP1 donor (Fd) in cell-surface FRET complex (*p<0.05, Mann Whitney test). There was no significant difference between fraction of CaSR acceptor in the FRET complex between RAMPs 1 and 3 (Fa). The table displays combined data of three independent experiments.</p

Effect of RAMP1 mRNA knockdown on TT cells.

<p>(A) mRNA expression levels of RAMP1, RAMP2 and CaSR in TT cells transfected with RAMP1 or scrambled siRNA, 72 hr post-transfection expressed as fold change normalised to Actβ. (B) Representative images from immunofluorescent staining for RAMP1 expression in cells transfected with scrambled siRNA (top panel) and RAMP1 siRNA(bottom panel), 72 hr after transfection. (C) Intracellular calcium response of the RAMP1 siRNA cells to 1µM Cinacalcet in presence of 1.5 mM CaCl₂ and (D) 100µM Neomycin in presence of 2 mM CaCl₂ The responses were decreased by ∼42% and ∼50% respectively compared to scrambled siRNA transfected cells. The data are combined from five independent experiments with a total of 241, 231 and 154 cells analysed in (C), and 354, 354 and 118 cells analysed in (D) for knock-down, control and normal conditions respectively. **** p<0.0001 analysed by two-tailed Mann-Whitney test.</p

Cell surface expression of non-tagged CaSR.

<p>(A) Western blot of membrane and total cell lysate preparations from COS-7 cells transfected with CaSR alone (lane 1), CaSR and RAMP1 (lane 2), and CaSR and RAMP3 (lane 3), incubated with antibody to the CaSR, demonstrating the ability of RAMPs 1 and 3 to traffic the CaSR to the cell surface. (B) FACS analyses of non-permeabilised COS-7 cells expressing CaSR with either RAMPs 1, 2 or 3 showing shift from the IgG control in number of cells with surface fluorescence in RAMP1 and RAMP3 expressing cells, but not RAMP2 expressing cells.</p

Attenuation of CaSR response in TT cells by RAMP1 antibodies.

<p>(A) Concentration-dependant decrease in Cinacalcet-induced intracellular calcium release in presence of 1.5 mM CaCl₂, by RAMP1 antibodies in TT cells. Total cells analysed combined from five independent experiments are 272 (1µM Cinacalcet), 261 (0.0125µg/µl control IgG), 240 (0.025µg/µl control IgG), 252 (0.0025µg/µl RAMP1 Ab), 272 (0.0125µg/µl RAMP1 Ab) and 250 (0.025µg/µl RAMP1 Ab). (B) 0.025µg/µl RAMP1 antibody caused a significant inhibition of 100µM Neomycin response in presence of 2 mM CaCl₂ compared to control IgG. Total cells analysed combined from five independent experiments are 192 (100µM Neomycin), 177 (0.025µg/µl control IgG) and 166 (0.025µg/µl RAMP1 Ab). *** p<0.001 determined by Kruskal-Wallis test, Dunn's multiple comparison post-test, ** p<0.01 two-tailed Mann-Whitney test.</p

Cell-surface FRET efficiencies of CaSR+RAMPs and fraction of receptor components involved in FRET complex.

<p>(A) Cell-surface FRET efficiencies of individual RAMPs with CaSR compared among themselves and also with respective negative control Citrine alone+RAMP-Cerulean. Data are expressed as a percentage of a positive control comprising cells expressing a Citrine-Cerulean fusion protein. *** p<0.001 (2-way ANOVA, Bonferroni post-test) ***p<0.001 (Kruskal-Wallis test, Dunn's multiple comparison test) (B) and (C) Stoichiometric analysis of fraction of donor RAMP (Fd) and acceptor CaSR (Fa) present in FRET complex on the cell-surface, respectively.* p<0.05 Mann Whitney test. The graph represents data from three independent experiments.</p