Expression and functional assessment of an alternatively spliced extracellular Ca2+-sensing receptor in growth plate chondrocytes

Endocrinology. 2005 Dec;146(12):5294-303. doi: 10.1210/en.2005-0256. Epub 2005 Sep 15.

Abstract

The extracellular Ca(2+)-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR(-/-)) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [(Exon5(-))CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR(-/-) mice express (Exon5(-))CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of (Exon5(-))CaR in growth plates from CaR(-/-) mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human (Exon5(-))CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human (Exon5(-))CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR(-/-) GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR(-/-) mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably (Exon5(-))CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR(-/-) mice, high extracellular [Ca(2+)] ([Ca(2+)](e)) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca(2+)](e) also promoted the differentiation of CaR(-/-) GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of (Exon5(-))CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca(2+)](e) in GPCs in CaR(-/-) mice.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alternative Splicing*
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cell Differentiation
  • Cells, Cultured
  • Chondrocytes / metabolism*
  • Cricetinae
  • Cricetulus
  • Exons
  • Extracellular Fluid / metabolism*
  • Growth Plate / cytology
  • Growth Plate / metabolism*
  • Humans
  • Mice
  • Mice, Knockout
  • Molecular Sequence Data
  • Receptors, Calcium-Sensing / deficiency
  • Receptors, Calcium-Sensing / genetics*
  • Receptors, Calcium-Sensing / metabolism*
  • Signal Transduction

Substances

  • Receptors, Calcium-Sensing