The physiological impact of an N-terminal Halo-tag on GIPR function in mice

Aims G protein-coupled receptors (GPCRs) modified with self-labelling enzymatic tags (e.g. Halo, SNAP, CLIP) have enabled the in vitro study of receptor expression and trafficking. We developed a Gipr Halo/Halo mouse model to gain insights into endogenous glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signalling, an important modulator of glucose and appetite homeostasis. Methods Gipr Halo/Halo mice were generated through ES cell-based gene targeting and physiologically characterised via body weight, oral glucose tolerance test and intraperitoneal glucose tolerance test analysis. Live cell imaging was used to measure cAMP responses in dispersed pancreatic islets from Gipr Wt/Wt and Gipr Halo/Halo littermates in response to GIP. cAMP accumulation and surface receptor expression were assessed in AD293 cells transiently transfected with mouse and human untagged GIPR and Halo-GIPR. Endogenous pancreatic islet expression of Halo-GIPR was determined by immunohistochemistry. Results Gipr Halo/Halo mice displayed comparable body weights and responses to oral glucose administration as Gipr Halo/Wt and Gipr Wt/Wt littermates. However, Gipr Halo/Halo mice only responded to high dose human GIP during intraperitoneal glucose tolerance testing, and cAMP responses to GIP were impaired in Gipr Halo/Halo islets. Despite unimpaired surface expression, in vitro cAMP responses were lessened in AD293 cells expressing Halo-GIPR compared to untagged GIPR. Anti-Halo staining in mouse Gipr Halo/Wt pancreatic islets displayed detectable signal. Conclusions While the Gipr Halo/Halo mouse model could be used to study endogenous GIPR expression, due to reduced responses we advise against its use for endogenous GIPR trafficking and signalling. Our study highlights the importance of carefully phenotyping mouse models in which modifications to endogenous proteins have been introduced.