The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor.

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptor are important targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. Originally identified for their role in desensitization, internalization and recycling of G protein-coupled receptors (GPCRs), arrestins have since been shown to act as scaffolding proteins that allow GPCRs to signal in a G protein-independent manner. While GLP-1R has been reported to interact with arrestins, this aspect of cell signaling remains controversial for GIPR. Using a (FRET)-based assay we have previously shown that yellow fluorescent protein (YFP)-labeled GIPR does not recruit arrestin. This GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was not present in the modified GIPR-SYFP2 used in subsequent FRET and bioluminescence resonance energy transfer (BRET) assays. However, its removal results in the introduction of a serine residue adjacent to the end of GIPR's C-terminal tail which could potentially be a phosphorylation site. The resulting receptor was indeed able to recruit arrestin. To find out whether the serine/arginine (SR) coded by the XbaI site was indeed the source of the problem, it was substituted with glycine/glycine (GG) by site-directed mutagenesis. This substitution abolished arrestin recruitment in the BRET assay but only significantly reduced it in the FRET assay. In addition, we show that the presence of a N-terminal FLAG epitope and influenza hemagglutinin signal peptide were also required to detect arrestin recruitment to the GIPR, most likely by increasing receptor cell surface expression. These results demonstrate how arrestin recruitment assay configuration can dramatically alter the result. This becomes relevant when drug discovery programs aim to identify ligands with "biased agonist" properties.

A Dual GLP-1/GIP Receptor Agonist Does Not Antagonize Glucagon at Its Receptor but May Act as a Biased Agonist at the GLP-1 Receptor.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism, making their receptors (GLP-1R and GIPR) attractive targets in the treatment of type 2 diabetes mellitus (T2DM). GLP-1R agonists are used clinically to treat T2DM but the use of GIPR agonists remains controversial. Recent studies suggest that simultaneous activation of GLP-1R and GIPR with a single peptide provides superior glycemic control with fewer adverse effects than activation of GLP-1R alone. We investigated the signaling properties of a recently reported dual-incretin receptor agonist (P18). GLP-1R, GIPR, and the closely related glucagon receptor (GCGR) were expressed in HEK-293 cells. Activation of adenylate cyclase via Gαs was monitored using a luciferase-linked reporter gene (CRE-Luc) assay. Arrestin recruitment was monitored using a bioluminescence resonance energy transfer (BRET) assay. GLP-1, GIP, and glucagon displayed exquisite selectivity for their receptors in the CRE-Luc assay. P18 activated GLP-1R with similar potency to GLP-1 and GIPR with higher potency than GIP. Interestingly, P18 was less effective than GLP-1 at recruiting arrestin to GLP-1R and was inactive at GCGR. These data suggest that P18 can act as both a dual-incretin receptor agonist, and as a G protein-biased agonist at GLP-1R.