Failure to detect the extracellular calcium-sensing receptor (CasR) in human osteoblast cell lines.
Whether the known calcium-sensing receptor (CasR) is present in osteoblasts is a source of considerable controversy. Prior studies failed to detect CasR in osteoblasts, but more recent investigations purport the detection of CasR in several osteoblast cell lines by immunoblot analysis with polyclonal anti-CasR antisera (4637) and low stringency reverse transcriptase-polymerase chain reaction (RT-PCR). To explain these disparate findings, we performed immunoblot analysis with the 4637 anti-CasR antisera and a highly specific monoclonal antibody to CasR (ADD), and we compared the ability of low and high stringency RT-PCR to amplify CasR transcripts. We found that the ADD antibody detected the anticipated CasR immunoreactive bands, including a approximately 165 kDa and approximately 140 kDa glycosylated doublet and a >250 kDa dimerized receptor, in positive control mouse kidney, human parathyroid, and human embryonic kidney (HEK) 293 cells transfected with rat CasR, but we did not detect these bands in either wild-type HEK 293 cells or Saos2, MG-63, or U-2 OS osteoblast-like cell lines. Standard two-step RT-PCR using CasR-specific primers confirmed these results by detecting CasR transcripts in positive controls but not in negative control HEK 293 cells or osteoblast cell lines. In contrast, the 4637 antisera did not recognize CasR by immunoblot analysis under the conditions studied and our low stringency RT-PCR procedure amplified nonspecific products in wild-type HEK 293 cells and osteoblasts. Since we failed to detect CasR in human osteoblast cell lines using either the highly specific ADD antibody or RT-PCR under standard conditions, it is possible that the cation response in osteoblasts is mediated by a functionally similar but molecularly distinct calcium sensing receptor.
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Duke University Medical Center, Durham, North Carolina 27710, USA.
Hinson
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Quarles
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; C473605:extracellular calcium cation-sensing receptor, rat; D002118:Calcium