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

Author:

Rodriguez Luis,Tu Chialing,Cheng Zhiqiang,Chen Tsui-Hua,Bikle Daniel,Shoback Dolores,Chang Wenhan

Abstract

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.

Publisher

The Endocrine Society

Subject

Endocrinology

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