Enhanced agonist residence time, internalization rate and signalling of the GIP receptor variant [E354Q] facilitate receptor desensitization and long‐term impairment of the GIP system

Abstract

In patients with type 2 diabetes mellitus (T2DM), the insulinotropic action of the GIP system is desensitized, whereas this is not the case for the GLP-1 system. This has raised an interesting discussion of whether GIP agonists or antagonists are most suitable for future treatment of T2DM together with GLP-1-based therapies.Homozygous carriers of the GIP receptor (GIPR) variant, [E354Q], display lower bone mineral density, increased bone fracture risk and slightly increased blood glucose. Here, we present an in-depth molecular pharmacological phenotyping of GIPR-[E354Q]. In silico modelling suggested similar interaction of the endogenous agonist GIP(1-42) to [E354Q] as to GIPR wt. This was supported by homologous competition binding in COS-7 cells revealing GIPR wt-like affinities of GIP(1-42) with K d values of ~2 nmol/L and wt-like agonist association rates (K on ). In contrast, the dissociation rates (K off ) were slower, resulting in 25% higher agonist residence time for GIPR-[E354Q]. Moreover, in G αs signalling (cAMP production) GIP(1-42) was ~2-fold more potent and more efficacious on GIPR-[E354Q] compared to wt with 17.5% higher basal activity. No difference from GIPR wt was found in the recruitment of β-arrestin 2, whereas the agonist-induced internalization rate was 2.1-to 2.3fold faster for [E354Q]. Together with the previously described impaired recycling of [E354Q], our findings with enhanced signalling and internalization rate possibly explained by an altered ligand-binding kinetics will lead to receptor desensitization and down-regulation. This could explain the long-term functional impairment of the GIP system in bone metabolism and blood sugar maintenance for [E354Q] carriers and may shed light on the desensitization of the insulinotropic action of GIP in patients with T2DM. K E Y W O R D SGIP receptor, GIPR-[E354Q], internalization, signalling

Arrestin‐independent constitutive endocytosis of GPR125/ADGRA3

“…The BRET experiments were carried out as previously described . Briefly, 1 day after seeding cells in a 6‐well plate, 60–80% confluent parental HEK293 cells and stably transduced GPR125 KD cells were transiently transfected with the following plasmids: GPR125, the human glucagon‐like peptide‐1 (GLP‐1) receptor (GLP‐1R, positive control), or empty pcDNA3.1(+) vector (negative control), in combination with the BRET donors Renilla luciferase−fused arrestins RLuc8–arrestin‐2–Sp1 or RLuc8–arrestin‐3–Sp1, the BRET acceptor mem‐linker‐citrine‐SH3, and GPCR kinases 2 or 6 (GRK2 or GRK6) to facilitate β‐arrestin‐1 and β‐arrestin‐2 recruitment.…”

“…To our knowledge, little is known about intracellular trafficking of aGPCRs compared with class A, B1, and C receptors, apart from certain aGPCR's interaction with β‐arrestin and endosomal proteins . The arrestin family consists of four members: arrestin‐1 and arrestin‐4, also known as visual arrestins, are exclusively expressed in the retina, whereas the two β‐arrestin isoforms, β‐arrestin‐1 and β‐arrestin‐2 (also named arrestin‐2 and arrestin‐3, respectively), are ubiquitously expressed in most tissues …”

Spatiotemporal regulation of GIPR signaling impacts glucose homeostasis as revealed in studies of a common GIPR variant

“…Substitution of glutamine for a glutamic acid at position 354 of GIPR, which is in the 6 transmembrane domain, does not affect the affinity for GIP nor GIP-stimulated increase in cAMP, demonstrating the substitution does not affect the most receptor proximal aspects of GIP-stimulated GIPR signaling (Abdullah et al, 2016;Almind et al, 1998;Gabe et al, 2019;Mohammad et al, 2014). However, we have demonstrated postactivation trafficking differences between GIPR-E354 and GIPR-Q354.…”

“…Activation of GIPR is linked, via Gs, to elevated cAMP, which is required for GIP enhancement of GSIS. The affinity of GIP for GIPR as well as cAMP generated downstream of GIPR are not significantly affected by the Q354 substitution (Abdullah et al, 2016;Almind et al, 1998;Gabe et al, 2019;Mohammad et al, 2014). However, Q354 substitution has been shown to impact GIPR trafficking (Abdullah et al, 2016;Mohammad et al, 2014).…”

G protein‐coupled receptor pharmacology—The next generation

“…These aspects are covered by two articles from the Rosenkilde and Madsen groups. The former provides a detailed characterization of a common E354Q genetic variant of the GIP receptor, which provides a potential mechanism for the long‐term functional impairment of the GIP receptor system in carriers of this variant . This is relevant not only as a naturally occurring receptor variant with phenotypic alterations, but also due to the long‐term desensitization of the GIP system in patients with type 2 diabetes mellitus, where hyperglycaemia and enhanced levels of the endogenous agonist GIP seem to play a role .…”