The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

Abstract

Background and ObjectivesGlucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.MethodsGLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET).ResultsGIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.ConclusionsGIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist

“…Moreover, our results agree with those reported during the preparation of this manuscript, indicating no significant recruitment b-arrestin2 by the GIPR in HEK293 cells (Al-Sabah et al, 2014). On the other hand, confocal microscopy observations strongly support participation of the AP-2 complex in GIPR internalization, a result in line with evidence showing that AP-2 is essential, if not required, in clathrin-coat pit formation (Boucrot et al, 1059;Motley et al, 2003).…”

“…However, contradictory results were reported about interaction of GRK2 with GIPR (Tseng and Zhang, 2000;Al-Sabah et al, 2014). Indeed, recruitment assay using FRET could not detect any binding of GRK2 to the GIPR whereas GRK2 over-expression caused drops in GIPR-dependant cAMP production and insulin secretion (Tseng and Zhang, 2000;Al-Sabah et al, 2014).…”

Forskolin-free cAMP assay for Gi-coupled receptors

“…For instance, prominent examples of GPCRs lacking the ability to bind arrestins include the b 3 -adrenoceptor, the relaxin family peptide receptor 1 (RXFP1) and 2 (RXFP2), or the glucose-dependent insulinotropic polypeptide (GIP) receptor [35][36][37][38]. More surprisingly, some GPCRs such as the atypical chemokine receptor ACKR3 or receptors involved in stem cells biology (LGR5) seem to lack G-protein-coupling [39,40].…”

Pathogenetic significance of single nucleotide polymorphisms in the gastric inhibitory polypeptide receptor gene for the development of carbohydrate metabolism disorders in obesity

“…Доказано, что снижение «инкретинового эффекта» является ранней характеристикой СД2 [6]. Ис-следованиями многих авторов выявлена значительная лиганднезависимая или базальная активность рецептора GIPR, что может играть важную роль в регуляции уровня глюкозы в крови [13][14][15][16]. Предполагают, что о функци-ональной роли GIPR можно судить не только по пост-прандиальной секреции гормонов поджелудочной железы (инсулина и С-пептида), а также и по их уров-ням натощак, что косвенно может отражать базальную активность GIPR [13,14,15].…”

“…Согласно данным научной периодики, у пациентов с СД2 снижа-ется секреция инсулина, стимулированная питатель-ными веществами [6]. Многочисленные исследования направлены на изучение причин нарушения секреции инкретинов или снижения чувствительности рецепторов к ним при нарушениях углеводного обмена [7]. Так, до-клинические исследования показали, что снижение от-вета на терапию GIP может быть вызвано пониженной экспрессией или дефектами его рецептора [8].…”