Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells.
Calreticulin is a Ca2+ binding protein located primarily in the endoplasmic reticulum (ER) lumen of non-excitable cells, where it is considered to be involved mainly in Ca2+ storage and buffering. However, there is increasing evidence to implicate the protein in other facets of Ca2+ signalling. In this study, we sought to establish more clearly the role of the protein in the regulation of intracellular Ca2+ signalling. Generating HeLa cells stably transfected with GFP-tagged calreticulin (GFPCRT) allowed to us to select cells by FACS in which calreticulin was expressed at ten times its endogenous levels. Using transiently expressed aequorin as a Ca2+ indicator in these cells, we investigated the role of calreticulin in intracellular Ca2+ storage, IP3-mediated Ca2+ release, and capacitative Ca2+ entry. The data showed that the capacity of the ionomycin-sensitive Ca2+ store was doubled in over-expressing cells, indicating that although calreticulin has a role in Ca2+ storage within the lumen, other lumenal proteins are also likely to be involved. No difference was observed in the release of Ca2+ from the IP3-sensitive store in response to prolonged single stimulation with histamine in the absence of extracellular Ca2+, but use of short, sequential pulses of histamine and ATP revealed that calreticulin may exert an effect upon IP3-mediated Ca2+ release. Two different experimental approaches indicated that calreticulin participates in the regulation of capacitative Ca2+ entry. In the presence of extracellular Ca2+, the histamine-generated cytosolic Ca2+ signal was significantly lower in GFPCRT cells than those in control cells. Induction of capacitative Ca2+ entry by complete emptying of the store using the SERCA pump inhibitor, cyclopiazonic acid also showed that the influx component was significantly reduced in the GFPCRT cells. Use of ER-targeted apoaequorin acting as a luciferase demonstrated that the resting ER free [Ca2+] in the GFPCRT cells was lower than that in control cells. These data implicate calreticulin in the control of IP3-mediated Ca2+ release and capacitative Ca2+ entry, which may involve direct interaction with Ca2+ signalling components or control of ER free [Ca2+].
Llewelyn Roderick
H
H
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Llewellyn
D H
DH
Campbell
A K
AK
Kendall
J M
JM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000331:Aequorin / Q000378:metabolism; D001059:Apoproteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D037282:Calreticulin; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
D001059:Apoproteins; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C060014:apoaequorin; D049452:Green Fluorescent Proteins; D000331:Aequorin; D015759:Ionomycin; D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium