Overexpression of Calreticulin Increases Intracellular Ca Storage and Decreases Store-operated Ca Influx

doi:10.1074/jbc.271.16.9332

Overexpression of Calreticulin Increases Intracellular Ca Storage and Decreases Store-operated Ca Influx (*)

From the (1)Division of Infectious Diseases, University Hospital, 1211 Geneva 14, Switzerland, the
(2)Medical Research Council Group in Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada, and the
(3)Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

^§ To whom correspondence should be addressed. Tel.: 41-22-3729818; Fax: 41-22-3729830; kkrause@cmu.unige.chandMery{at}dminov1.hcuge.ch.

Abstract

The widely distributed and highly conserved Ca-binding protein calreticulin has been suggested to play a role as a Ca 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 Ca content of calreticulin-overexpressing and control cells, as assessed by equilibrium Ca uptake, was 141 ± 8 and 67 ± 6 pmol of Ca/10⁶ cells, respectively (i.e. a 2.1 ± 0.2-fold increase in the Ca content of calreticulin-overexpressing cells). Over 80% of the increased Ca content was found within thapsigargin-sensitive Ca 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 [Ca] elevations due to Ca release were enhanced when either ATP or a combination of ionomycin and thapsigargin was used as a stimulus. In contrast, thapsigargin-induced Ca and Mn influxes from the extracellular space were markedly diminished in calreticulin-overexpressing cells, suggesting an active involvement of calreticulin in the regulation of store-operated Ca influx.

Footnotes

↵^¶ Postdoctoral Fellow of the Heart and Stroke Foundation of Canada.
↵** Alberta Heritage Foundation for Medical Research Senior Scholar and Medical Research Council Scientist.
↵* This work was supported by Swiss National Foundation Grant 32 30161.90 and by grants from the Medical Research Council of Canada, the Carlos and Elsie de Reuter Foundation, the Sandoz Foundation, the Ernst and Lucie Schmidheiny Foundation, the Société Académique de Genève, the Helmut Horten Foundation, and the Zyma Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

[Ca]

cytosolic free calcium concentration

DTPA

diethylenetriaminepentaacetic acid

fura-2/AM

fura-2 acetoxymethyl ester

PBS

phosphate-buffered saline

PIPES

1,4-piperazinediethanesulfonic acid.
↵²Since submission of this manuscript, a publication by Bastianutto et al.(25) showed an increase in the capacity of rapidly exchanging Ca stores through transient overexpression of calreticulin in HeLa cells; the authors also observed an inhibition of Ca influx in calreticulin-transfected cells.
- Received August 10, 1995.
- Revision received November 30, 1995.