Examining the contribution of ghrelin receptor constitutive activity to different signalling pathways

Rachel Thomas, Sina Tavakol, Nick Holliday. University of Nottingham, Queen's Medical School, Nottingham, NG7 2UH, UK

The stomach peptide ghrelin stimulates appetite via the G-protein coupled ghrelin receptor (ghrelinR /GHSR1a), which has become a therapeutic target for obesity1. The ghrelinR can couple to multiple G proteins as well as G-protein independent β-arrestin pathways, and displays an unusually high level of constitutive activity2. To explore whether the extent of spontaneous ghrelinR activation depends on the response measured, we have compared signalling profiles of the wild type (WT) ghrelinR and two non-constitutively active ghrelinR mutants (F221A and F279N2,3) using G-protein coupled calcium mobilisation, G-protein independent SNAP-tagged receptor internalisation and nuclear ERK translocation. All data are expressed as means ± s.e.m. for at least three independent experiments.

Calcium mobilisation, in HEK293 cells stably transfected with SNAP-tagged ghrelinR cDNAs, was assessed by measuring peak changes in Fluo4 fluorescence on an MDC FlexStation. In this assay ghrelin (pEC50 8.28 ± 0.22), L692585 (pEC50 8.59 ± 0.13) and GHRP-6 (pEC50 8.74 ± 0.09) were equipotent agonists (n = 3). The inverse agonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P (SP-A) decreased basal Fluo4 fluorescence at 1 µM. F221A and F279N mutations prevented the inverse agonist response in this assay, and also increased the maximum response to agonists (e.g. fold increase over basal with ghrelin: WT 2.5 ± 0.3; F221A 15.4 ± 3.6*; F279N 11.3 ± 3.8; n = 4-6, * p < 0.05, one-way ANOVA).

Internalisation of SNAP-tagged ghrelin receptors was assessed using a confocal plate reader (IX Ultra) combined with automated image analysis4. Ghrelin, GHRP-6 and L692585 stimulated WT ghrelinR internalisation (30 min. stimulation), with respective pEC50 values of 8.10 ± 0.24, 8.30 ± 0.51 and 8.66 ± 0.10 (n = 3-5). In contrast SP-A decreased constitutive ghrelinR internalisation with a pIC50 of 7.25 ± 0.12 (n = 7). As observed for calcium responses, F221A and F279N mutations prevented basal internalisation and abolished the effect of SP-A, but did not alter ghrelin potency (pEC50 8.07 ± 0.15 and 7.73 ± 0.24 for F221A and F279N, respectively).

Finally, the ability of ghrelin to stimulate ERK nuclear translocation was assessed using single cell confocal microscopy in dual SNAP-ghrelinR ERK2-GFP stable transfected cells. Following overnight serum starvation, stimulation of the WT ghrelinR with ghrelin (1µM) caused nuclear accumulation of ERK-GFP within 4 minutes resulting in approx. 1.4 fold increase in nuclear fluorescence (n ≥ 10 cells). F221A and F279N mutations did not prevent ghrelin stimulated nuclear ERK translocation via these receptors in a similar manner to WT (n ≥ 10 cells).

Thus the F221A and F279N mutations selectively reduce ghrelinR constitutive activity and reveal its contribution to both G protein dependent (calcium signalling) and G protein independent (internalisation) responses investigated in this study.

1. Chen C.Y. et al. (2009) Pharmacol. Rev. 61; 430

2. Holst B et al. (2004) J. Biol. Chem. 279, 53806

3. Holst B et al. (2010) J. Biol. Chem. 285, 3973

4. Sivertsen et al. (2011) J. Biol. Chem. 286, 20845