Modulating GIP Receptor signalling via RAMP association and inverse agonism

The glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in the insulin response upon nutrient uptake. It is rapidly cleaved by the enzyme dipeptidyl peptidase IV to GIP 3-42 (1). Activation of the GIP receptor (GIPR) by GIP 1-42 leads to Gs coupling and cAMP production, resulting in insulin granule exocytosis (2). We first analysed the signalling properties of human GIP 1-42 and GIP 3-42 at the GIPR. The receptor was transiently transfected into HEK293 cells in the presence or absence of receptor activity modifying proteins (RAMPs) and cAMP production measured using a PerkinElmer LANCE assay kit (3). Although the GIPR is known to exhibit basal activity (4), GIP 3-42 has previously been thought to act as an antagonist (1). However, we observed that GIP 3-42 behaved as an inverse agonist at the GIPR (Fig. 1). Despite this, and based upon knowledge about the mode of action of peptide fragments at family B GPCRs, it is most likely that, GIP 3-42 will be competitive with respect to GIP 1-42 for the GIPR. On this assumption we estimate the pKB as 7.68 ± 0.36 (n=4).

Several family B GPCRs have been shown to interact with RAMPs, which can affect ligand specificity and can also influence signal transduction. However, a complete pharmacological analysis of these interactions has not been carried out for many receptors, including the GIPR. We therefore investigated the effects of the RAMPs upon the ability of GIP 1-42 and GIP 3-42 to bind and activate the GIPR to generate cAMP. RAMP3 attenuated GIP 1-42 / GIPR signalling through Gs by significantly reducing the transducer function log tau (GIPR alone = -0.18 ± 0.06; RAMP3 + GIPR = -0.51 ± 0.13, P < 0.05, one-way ANOVA and Dunnett’s test). Interestingly, RAMP3 did not affect the inverse agonism of GIP 3-42.

Information regarding the action of GIP 3-42 as an inverse agonist will be important for the development of drugs for the treatment of diabetes. In addition, these findings further demonstrate the ability of RAMPs to modulate signalling bias at family B GPCRs.

References

(1) Hinke AS et al (2004). Life Sci 75: 1857–1870,

(2) Kashima Y et al (2001). J Biol Chem 276: 46046–46053,

(3) Weston C et al (2015). J Biol Chem jbc.M 114.624601,

(4) Al-Sabah S et al (2014). PLoS One 9: p. e106890.

This research was funded by BBSRC grants BB/M00015X/1 and BB/M000176/1.