Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx.
The widely distributed and highly conserved Ca(2+)-binding protein calreticulin has been suggested to play a role as a Ca2+ storage protein of intracellular Ca+ stores. To test this hypothesis, we have generated a mouse L fibroblast cell line stably transfected with a calreticulin expression vector. The calreticulin content of the overexpressers was increased by 1.6 +/- 0.2-fold compared with mock-transfected cells. The total cellular Ca2+ content of calreticulin-overexpressing and control cells, as assessed by equilibrium 45Ca+2 uptake, was 141 +/- 8 and 67 +/- 6 pmol of Ca2+/10(6) cells, respectively (i.e. a 2.1 +/- 0.2-fold increase in the Ca2+ content of calreticulin-overexpressing cells). Over 80% of the increased Ca2+ content was found within thapsigargin-sensitive Ca2+ stores. The pattern of calreticulin distribution, revealed by immunofluorescence microscopy, showed an endoplasmic reticulum-like pattern and was identical in overexpressers and control cells. In overexpressers, cytosolic free [Ca2+] elevations due to Ca2+ release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca2+ and Mn2+ influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca2+ influx.
Mery
L
L
Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
Mesaeli
N
N
Michalak
M
M
Opas
M
M
Lew
D P
DP
Krause
K H
KH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D037282:Calreticulin; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006706:Homeostasis; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D007739:L Cells; D008345:Manganese / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012261:Ribonucleoproteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014162:Transfection
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D013729:Terpenes; D008345:Manganese; D015759:Ionomycin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D008985:Monensin; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004121:Dimethyl Sulfoxide