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Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca²⁺-Sensing Receptor in Growth Plate Chondrocytes

Endocrine Research Unit, Department of Medicine, Department of Veterans Affairs Medical Center, University of California, San Francisco, California 94121

Address all correspondence and requests for reprints to: Wenhan Chang, Endocrine Research Unit, 111N, San Francisco Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121. E-mail: bambam{at}itsa.ucsf.edu.

The extracellular Ca²⁺-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.

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