Abstract
The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca(2+)](o)) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca(2+)](i)) was increased by an increment of [Ca(2+)](o). This [Ca(2+)](i) increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca(2+)](o)-induced [Ca(2+)](i) increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR-related disorders in the body.
Highlights
Extracellular calcium is essential for a number of vital processes, including bone mineralization, blood coagulation, regulation of enzymatic activity, and the modulation of permeability and excitability of the plasma membranes
Vol 37(2), 91- 100, 2005 calcium concentration in the resorption lacunae suppresses osteoclastic bone-resorbing activity through an increase in intracellular calcium; this is mediated through a ryanodine-like receptor (Zaidi et al, 1989; 1991; 1993; Adebanjo et al, 1994; Shin et al, 2003) and the calcium sensing receptor (CaSR) regulates osteoclastic bone resorption (Kameda et al, 1998)
Our results show that CaSR protein is co-localized together with Cav-1 in the caveolae-rich membrane fractions
Summary
Extracellular calcium is essential for a number of vital processes, including bone mineralization, blood coagulation, regulation of enzymatic activity, and the modulation of permeability and excitability of the plasma membranes. For these reasons, the calcium concentration in extracellular fluids is under strict control by a complex homeostatic system that includes the bones, kidney, intestines, parathyroid and thyroid glands (Brown, 1991). Recent studies have shown that functional proteins including receptors are localized in the caveolae by being anchored through the caveolins (Li et al, 1995; GarciaCardena et al, 1996; Couet et al, 1997; Lee et al, 2001; Cha et al, 2004). Immuno-cytochemical confocal microscopic examinations supported that CaSR is co-localized with Cav-1 in the plasma membrane of Saos-2 cells
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