RAMPs regulate signalling bias and internalisation of the GIPR

Abstract

Gastric inhibitory polypeptide (GIP) receptor is a class B1 GPCR, that responds to GIP and physiologically potentiates glucose-stimulated insulin secretion. Like most class B1 GPCRs, GIPR has been shown to interact with RAMPs, yet the effects of RAMPs on its signalling and trafficking remain poorly understood. We demonstrate that RAMPs modulate G protein activation and GIPR internalisation profiles. RAMP3 reduced GIPR Gs activation and cAMP production but retained GIPR at the cell surface, and this was associated with prolonged ERK1/2 phosphorylation and β-arrestin association. By contrast, RAMP1/2 reduced Gq/11/15 activation of the GIPR. Through knockout mice studies, we show that RAMP1 is important to the normal physiological functioning of GIPR to regulate blood glucose levels. Thus, RAMPs act on G protein/β-arrestin complexes, having both acute and chronic effects on GIPR function, while this study also raises the possibility of a more general role of RAMP3 to enhance GPCR plasma membrane localisation.

Determining the Effects of Differential Expression of GRKs and β-arrestins on CLR-RAMP Agonist Bias

“…We then determined the suitability of the C-terminal Nluc-tagged CLR with individual FLAG-tagged RAMPs for measuring cAMP accumulation. FLAG-RAMPs have previously been shown to signal comparably to other N terminally tagged RAMPs when coexpressed with CLR ( Harris et al, 2021 ). When compared to HA-CLR, which has previously been used to characterise G protein signalling of the CLR-RAMP complexes ( Weston et al, 2016 ; Harris et al, 2021 ), CLR-Nluc displayed a reduced potency (∼10-fold compared to HA-CLR), but the same rank order of potency of the three peptides at the different RAMP complexes was observed (Compare Figure 1A with Supplementary Figure S1B ).…”

“…The log Potency ratios (as determined from cAMP accumulation assays) are defined as log(EC50 AM2/EC50 agonist). Data compiled from Weston et al, (2016) , Garelja et al, (2020) , Clark et al, (2021) and Harris et al, (2021) . HEK293T cells expressing CLR-Nluc are shown in cyan , HEK293 expressing CLR-Nluc are shown as magenta and HEK293T cells expressing HA-CLR are shown in brown .…”

“…These studies have significance for other investigations into GPCR β-arrestin recruitment/internalisation. If the magnitude of β-arrestin recruitment is weak to the GPCR of choice, addition of GRKs is often used to increase the signal ( Mackie et al, 2019 ; Harris et al, 2021 ). As demonstrated for CLR, this is not always appropriate as it can lead to the opposite effects if agonist-independent phosphorylation of GPCRs occurs.…”

“…CLR containing a direct C-terminal inframe fusion to NanoLuc was generated in pcDNA3.1(−) (pcDNA3.1(−)-CLR-Nluc) using standard molecular cloning techniques by Sabrina Carvalho (University of Cambridge). pcDNA3.1(+)FLAG-RAMPs and pcDNA3.1-HA-CLR have been described previously ( Weston et al, 2016 ; Harris et al, 2021 ). pcDNA3.1(+)-hGRKs (Patel et al .…”

Cancer-Associated Mutations Enhance The Sensitivity Of The Trupath GαQ/11 System

“…SmBiT-VPACR1 was generated via PCR amplification of VPACR1 sequence, without the native signal peptide, which was ligated into a pcDNA3.1(+)-sigSmBiT backbone, containing the signal peptide of the murine 5-HT 3 receptor. The remaining receptor constructs and FLAG-tagged RAMPs were used as previously described (Harris et al, 2021). Plasmid pNL[NFAT-RE-NlucP-Hygro] was purchased from Promega (UK) to determine NFAT-mediated transcription by Gα q/11 -coupled receptors.…”