Differential signalling by the human incretin receptors.

S Al-Sabah1, M Al-Fulaij1, HA Ahmed1, M Bunemann2, C Krasel2. 1Kuwait University, Kuwait, Kuwait, 2Marburg University, Marburg, Germany

Introduction & Objectives

The incretin effect describes the observation that glucose taken orally elicits a greater insulin response than glucose given by intravenous injection. This effect is mediated by two hormones secreted from the gut; glucose-dependent insulinotropic polypeptide (GIP); and glucagon-like peptide-1 (GLP-1) and has been shown to be impaired in type 2 diabetic patients. Pharmacological levels of GLP-1 can overcome this loss of response whereas it is unclear if the same is true for GIP. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic beta-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 via Gs. As less has been reported regarding these receptors’ G protein-independent signalling properties we compared their interaction with arrestin.

Methods

Signalling through Gs was investigated using a cAMP-responsive luciferase (cre-luc) reporter gene assay. Agonist stimulated arrestin recruitment to the two receptors was investigated using confocal microscopy and fluorescence resonance energy transfer (FRET). Significance was analysed using a two-tailed unpaired Student’s t-test.

Results

GLP-1 and GIP dose dependently stimulated cAMP-responsive luciferase activity in HEK-293 cell transfected with creluc and either GLP-1R or GIPR with a pEC50 value of 8.5 (±0.18) and 8.8 (±0.18) respectively (mean ± S.E.M.,n=4). YFPlabelled arrestin3 displayed a robust translocation to agonist stimulated GLP-1R but not to GIPR. These results were quantified as loss of cytoplasmic fluorescence over time. At 15 minutes the loss of cytoplasmic fluorescence stimulated by GLP-1 was significantly greater (P<0.005) than with GIP. These observations were confirmed in FRET experiments where GLP-1 stimulated recruitment of both CFPlabelled GPCR kinase 2 (GRK2) and CFPlabelled arrestin3 to YFP-labelled GLP-1R. These interactions were not reversed upon agonist washout. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP1, suggesting that GLP-1R is a class A receptor with regard to arrestin binding. In contrast, GIP did not stimulate recruitment of either GRK2 or arrestin3 to its receptor.

Conclusion

Unlike GLP-1R, activated GIPR does not recruit GRK2 or arrestin effectively.