Calcium-Sensing Receptor Internalization Is β-Arrestin–Dependent and Modulated by Allosteric Ligands

Abstract

G protein-coupled receptor (GPCR) internalization is crucial for the termination of GPCR activity, and in some cases is 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 (Ca 21) levels by sensing minute changes in extracellular free Ca 21. It is thus necessary that the activity of the CaSR is tightly regulated, even while continuously being exposed to its endogenous agonist. Previous studies have used overexpression of intracellular proteins involved in GPCR trafficking, 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 the CaSR. We have used a state-of-the-art time-resolved fluorescence resonance energy transfer-based internalization assay to directly measure CaSR internalization in real-time. We demonstrate that the CaSR displays both constitutive and concentration-dependent Ca 21-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 N-(3-[2-chlorophenyl]propyl)-(R)-a-methyl-3-methoxybenzylamine (NPS R-568) and 2-chloro-6-[(2R)-2hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl)amino] propoxy]benzonitrile (NPS 2143), respectively. In addition, we provide compelling evidence that CaSR internalization is b-arrestin-dependent while interestingly being largely independent of G q/11 and G i/o protein signaling. SIGNIFICANCE STATEMENT A novel highly efficient cell-based real-time internalization assay to show that calcium-sensing receptor (CaSR) internalization is b-arrestin-dependent and sensitive to modulation by allosteric ligands.

International Union of Basic and Clinical Pharmacology. CVIII. Calcium-Sensing Receptor Nomenclature, Pharmacology, and Function

“…Similarly, agonist-induced CaSR internalization requires b-arrestin, which is in contrast with the GABA B and mGluRs, which function independently of b-arrestin (Pin and Bettler, 2016). However, constitutive and agonist-induced CaSR internalization is largely independent of G q/11 and G i/o in HEK293 cells (Mos et al, 2019), thus indicating that G protein-mediated activation of PKC is not involved in this cell line. Studies of the internalization mechanisms of endogenously expressed CaSRs in nonrecombinant cells are still lacking because of the technical difficulty of performing such experiments.…”

“…In heterologous expression systems, the CaSR undergoes constitutive internalization (Reyes-Ibarra et Gorvin et al, 2018c;Mos et al, 2019) and agonistinduced internalization (Lorenz et al, 2007;Zhuang et al, 2012;Nesbit et al, 2013b). Furthermore, CaSR internalization is increased by the CaSR PAM, NPS R-568, and agonist-induced but not constitutive internalization is inhibited by the NAM, NPS 2143 (Mos et al, 2019).…”

“…In heterologous expression systems, the CaSR undergoes constitutive internalization (Reyes-Ibarra et Gorvin et al, 2018c;Mos et al, 2019) and agonistinduced internalization (Lorenz et al, 2007;Zhuang et al, 2012;Nesbit et al, 2013b). Furthermore, CaSR internalization is increased by the CaSR PAM, NPS R-568, and agonist-induced but not constitutive internalization is inhibited by the NAM, NPS 2143 (Mos et al, 2019). GPCR internalization usually involves desensitization by kinase phosphorylation and subsequent b-arrestin binding followed by recruitment to clathrin-Calcium-Sensing Receptor Pharmacology and Function coated pits by b-arrestin and the clathrin-binding AP2 heterotetramer (Hanyaloglu and von Zastrow, 2008).…”

Constitutive Activation and Inactivation of Mutations Inducing Cell Surface Loss of Receptor and Impairing of Signal Transduction of Agonist-Stimulated Eel Follicle-Stimulating Hormone Receptor

“…In recent years, GPCR signal transduction has been studied in detail with respect to cell surface receptor loss, constitutive internalization, constitutive endocytosis, recycling, and β-arrestin-dependent internalization [ 3 , 33 , 34 , 35 , 36 ]. Many studies have elucidated several features of the post-endocytotic trafficking of FSHR [ 7 , 37 ].…”

The extracellular calcium‐sensing receptor promotes porcine egg activation via calcium/calmodulin‐dependent protein kinase II

“…Whether or not CASR activity is involved in egg activation is unknown. Therefore, the present study was conducted to examine the role of CASR during egg activation by using CASR agonist NPS R‐568 and the antagonist NPS2390 function which has been studied in many different cells types (Aggarwal et al, 2016; Chen, Li, Wu, Ma, & Gu, 2019; Mos, Jacobsen, Foster, & Brauner‐Osborne, 2019). The intracellular signaling cascades mediating the CASR response were also investigated to demonstrate the mechanism of CASR in egg activation.…”