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

内化 逮捕 G蛋白偶联受体 受体 内科学 内分泌学 HEK 293细胞 转染 G蛋白 生物 Gsα亚单位 细胞生物学 信号转导 细胞培养 生物化学 医学 遗传学
作者
M Gabe,Alexander H. Sparre‐Ulrich,Mie Fabricius Pedersen,Lærke S. Gasbjerg,Asuka Inoue,Hans Bräuner‐Osborne,Bolette Hartmann,Mette M. Rosenkilde
出处
期刊:Biochemical Pharmacology [Elsevier]
卷期号:150: 97-107 被引量:76
标识
DOI:10.1016/j.bcp.2018.01.040
摘要

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 (Kd 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.
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