Human calcium-sensing receptor can be suppressed by antisense sequences.
We have evaluated the ability of an antisense cDNA sequence, directed to the amino-terminus of the human calcium-sensing receptor (CaR), to reduce the expression and function of an EGFP-tagged CaR (CaR-EGFP) in HEK293 cells. Confocal microscopy and Western blot analysis showed a significant and selective reduction of the expression of CaR-EGFP by the antisense construct. Measurements of changes in intracellular calcium induced by CaR agonists showed that CaR-EGFP function was significantly reduced by the antisense sequence, as was agonist-evoked phosphorylation of extracellular signal-regulated protein kinases (ERK1,2). A sense construct directed to the same region of the receptor had no effect, confirming the specificity of the antisense construct. Our results indicate that a CaR antisense cDNA reduces both the expression and function of the receptor. In the absence of strong, specific pharmacological inhibitors of CaR, the antisense approach will be helpful to elucidate contributions of the CaR to cell physiology.
Maldonado-Pérez
David
D
School of Biological Sciences, University of Manchester, Manchester, UK.
Breitwieser
Gerda E
GE
Gama
Lucio
L
Elliott
Austin C
AC
Ward
Donald T
DT
Riccardi
Daniela
D
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D016376:Oligonucleotides, Antisense; D044169:Receptors, Calcium-Sensing; D049452:Green Fluorescent Proteins; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium