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

AimsG 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 GiprHalo/Halo mouse model to gain insights into endogenous glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signalling, an important modulator of glucose and appetite homeostasis. MethodsGiprHalo/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 GiprWt/Wt and GiprHalo/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. ResultsGiprHalo/Halo mice displayed comparable body weights and responses to oral glucose administration as GiprHalo/Wt and GiprWt/Wt littermates. However, GiprHalo/Halo mice only responded to high dose human GIP during intraperitoneal glucose tolerance testing, and cAMP responses to GIP were impaired in GiprHalo/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 GiprHalo/Wt pancreatic islets displayed detectable signal. ConclusionsWhile the GiprHalo/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.