Calbindin-D28k and calcium sensing receptor cooperate in MCF-7 human breast cancer cells.
The metastases of breast cancer cells to bone result in osteolysis and release of Ca2+. Ca2+ as a primary signal transducer can regulate the expression patterns of cell signaling systems. The extracellular calcium ion concentration sensing receptor CaR is a 123 kDa G-protein coupled membrane protein that resides within caveolin-rich regions as a dimer. CaR is involved in regulating several cellular processes such as proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a 28 kDa high affinity calcium-binding protein and it is involved in regulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to extracellular calcium ion concentration, [Ca2+]o, and neomycin sulfate, and spatial interactions of CaR with calbindin-D28k in MCF-7 human breast cancer cells were studied. Fura-2 loaded MCF-7 cells were exposed to increasing concentrations of CaCl2 or neomycin sulfate and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 or neomycin sulfate caused an increase in [Ca2+]i. The normalized dose response curves fitting yielded Hill co-efficient values of 4.32+/-0.63 and 1.49+/-0.14 for Ca2+ and neomycin sulfate respectively, thus indicating highly co-operative, 4-5 binding sites for Ca2+ and 1-2 binding site(s) for neomycin sulfate on CaR. The EC50 values were 21+/-1.6 mM and 43+/-3.5 micro M for CaCl2 and neomycin sulfate respectively. The confocal microscopy data, obtained by using a highly sensitive tyramide signal amplification technology for immunofluorescence detection, showed CaR and calbindin-D28k were co-localized when cells were exposed to 200 micro M neomycin sulfate, whereas in control cells there was no co-localization of these two proteins. We hypothesize that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.
Parkash
Jai
J
Department of Anatomy, Cell and Neurobiology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704-9388, USA. parkash@marshall.edu
Chaudhry
Muhammad A
MA
Rhoten
William B
WB
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D020013:Calcium Signaling; D003600:Cytosol; D005260:Female; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D018613:Microscopy, Confocal; D009355:Neomycin / Q000494:pharmacology; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D014407:Tumor Cells, Cultured
C576498:CALB1 protein, human; D064092:Calbindin 1; D064026:Calbindins; D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; D044169:Receptors, Calcium-Sensing; D064030:S100 Calcium Binding Protein G; D009355:Neomycin; D002122:Calcium Chloride; D002118:Calcium; D016257:Fura-2