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Calcium‐sensing Receptor Internalization is β‐arrestin‐dependent and Modulated by Allosteric Ligands
Author(s) -
BräunerOsborne Hans,
Mos Iris,
Jacobsen Stine Engesgaard,
Foster Simon Richard
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.lb30
Subject(s) - internalization , g protein coupled receptor , microbiology and biotechnology , arrestin , endosome , receptor , dynamin , chemistry , g protein coupled receptor kinase , calcium sensing receptor , endocytosis , biology , signal transduction , calcium , intracellular , biochemistry , calcium metabolism , organic chemistry
G protein‐coupled receptor (GPCR) internalization is a complex process responsible for the transport of GPCRs from the cell‐surface into intracellular compartments. Receptor internalization is crucial for the termination of GPCR activity and in some cases linked to the initiation of G protein‐independent signalling pathways and endosomal receptor signalling. Internalization has been widely studied in class A GPCRs, where the most prevalent mechanism after receptor activation is initiated by kinase‐mediated phosphorylation of intracellular loops and the C‐terminus, followed by β‐arrestin recruitment and dynamin driven internalization via clathrin‐coated pits. However, the mechanism and clinical relevance in class B and C GPCRs remain largely unknown. The extracellular calcium‐sensing receptor (CaSR) is a prototypical class C GPCR that plays a pivotal role in calcium homeostasis due to continuous sensing of small changes in blood ionized calcium (Ca 2+ ) levels. Impressively, CaSR is able to tightly control its activity despite being constantly exposed to its endogenous agonist. Previous studies investigating CaSR internalization were based on functional assays, and microscopy‐ or flow cytometry‐based analyses. However, there is no general agreement on the pathway(s) involved in CaSR internalization. Overall, the mechanism of CaSR remains to be poorly understood. In this study, we aimed to investigate CaSR internalization using the recently developed TR‐FRET‐based real‐time internalization assay for the first time. We demonstrate that Ca 2+ induces CaSR internalization in a concentration‐dependent manner with an EC 50 of 3.3 mM, which aligns well with the IP 1 response with an EC 50 of 3.2 mM. We show that internalization can be positively modulated by NPS R‐568 and negatively modulated by NPS 2143. Through the usage of CRISPR‐Cas9 edited HEK293 cells, we demonstrate that CaSR internalization is β‐arrestin dependent. Support or Funding Information This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement NO 675228, the Lundbeck Foundation, the Carlsberg Foundation, the Augustinus Foundation and the Toyota Foundation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .