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 [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.
Rodriguez
Luis
L
Endocrine Research Unit, Department of Medicine, Department of Veterans Affairs Medical Center, University of California, San Francisco, 94121, USA.
Tu
Chialing
C
Cheng
Zhiqiang
Z
Chen
Tsui-Hua
TH
Bikle
Daniel
D
Shoback
Dolores
D
Chang
Wenhan
W
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005091:Exons; D045604:Extracellular Fluid / Q000378:metabolism*; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D044169:Receptors, Calcium-Sensing / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction
D044169:Receptors, Calcium-Sensing