Human GIP(3-30)NH2 inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors

GIP(3-30)NH₂ is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting Insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH₂ in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH₂ among Other human and animal GPCRs. cAMP accumulation and β-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A β-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human ¹²⁵I-GIP(3-30)NH₂. The selectivity of human GIP(3-30)NH₂ was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH₂ inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA₂ and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH₂ inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (K_d values of 2.0, 2.5, 31.6 and 100 nM, respectively). In conclusion, human GIP(3-30)NH₂ is a selective and species-specific GIPR antagonist with broad inhibition of signaling and internalization in transfected cells as well as in human adipocytes.

Antagonist; GIP receptor; Glucose-dependent insulinotropic polypeptide (GIP); Human subcutaneous adipocytes; Signaling.