Calcium-sensing receptor internalization is β-arrestin-dependent and modulated by allosteric ligands
2019
G protein-coupled receptor (GPCR) internalization is crucial for the termination of GPCR activity and in some cases associated with G protein-independent signaling and endosomal receptor signaling. To date, internalization has been studied in great detail for class A GPCRs, whereas it is not well established to what extent the observations can be generalized to class C GPCRs including the extracellular calcium-sensing receptor (CaSR).
The CaSR is a prototypical class C GPCR that maintains stable blood calcium (Ca2+) levels by sensing minute changes in extracellular free Ca2+. It is thus necessary that the activity of CaSR is tightly regulated, even whilst continuously being exposed to its endogenous agonist. Previous studies have utilized protein overexpression, pathway inhibitors and cell-surface expression or functional desensitization as indirect measures to investigate CaSR internalization. However, there is no general consensus on the processes involved and the mechanism of CaSR internalization remains poorly understood.
The current study provides new insights into the internalization mechanism of CaSR. We have utilized a state-of-the-art TR-FRET-based internalization assay to directly measure CaSR internalization in real-time. We demonstrate that CaSR displays both constitutive and concentration-dependent Ca2+-mediated internalization. For the first time, we conclusively show that CaSR internalization is sensitive to immediate positive and negative modulation by the CaSR-specific allosteric modulators NPS R-568 and NPS 2143, respectively. In addition, we provide compelling evidence that CaSR internalization is β-arrestin-dependent while interestingly being largely independent of Gq/11 and Gi/o protein signaling. SIGNIFICANCE STATEMENT A novel highly efficient cell-based real-time internalization assay to show that calcium-sensing receptor (CaSR) internalization is β-arrestin-dependent and sensitive to modulation by allosteric ligands.
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