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RECEPTORS AND SIGNAL TRANSDUCTION

Molecular cloning and characterization of a rat sensory nerve Ca²⁺-sensing receptor

¹Cardiovascular Disease Research Program, Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, North Carolina 27707; and ²NPS Pharmaceuticals, Inc., Salt Lake City, Utah 84108

Submitted 22 November 2002 ; accepted in final form 28 February 2003

A full-length cDNA encoding a Ca²⁺-sensing receptor (CaSR) expressed in rat dorsal root ganglia (DRG) was identified using rapid amplification of 5'-cDNA ends and primer extension and then cloned into the plasmid vector pCR3.1. The DNA sequence of the DRG CaSR was 99.9% homologous with published rat kidney CaSR in the coding region and 247 bp upstream of the start site but showed little homology 5' to this site, which maps to exonic junction I/II, supporting the hypothesis that CaSR message arises as a splice variant and showing tissue-to-tissue heterogeneity. Western blot revealed a doublet of 140 and 160 kDa in a thyroparathyroid preparation and a single 140-kDa band in DRG. Deglycosylation using N-glycanase increased the mobility of CaSR protein from both DRG and thyroparathyroid, whereas endo-H was without effect, indicating that the DGR CaSR is a mature form of the receptor. A DRG CaSR-pEGFP fusion product was constructed, and when transfected into HEK-293 cells, it was distributed at the cell membrane and resulted in extracellular Ca²⁺ (0.5–3 mM)-evoked increases in intracellular Ca²⁺, which in some instances exhibited oscillatory behavior. We conclude that DRG CaSR cDNA arises from tissue-specific alternative splicing of a single gene, that the amino acid sequence of DRG CaSR is homologous to other known CaSRs, and that the DRG CaSR undergoes differential posttranslational processing relative to the thyroparathyroid CaSR and is functionally active when transfected into a human-derived cell line.

calcium; dorsal root ganglion; posttranscriptional modification; glycosylation

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