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

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 [Ca2+] ([Ca2+](e)) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca2+](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 [Ca2+](e) in GPCs in CaR-/- mice.