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Functional specialization of calreticulin domains.

Nakamura K, Zuppini A, Arnaudeau S, Lynch J, Ahsan I, Krause R, Papp S, De Smedt H, Parys JB, Muller-Esterl W, Lew DP, Krause KH, Demaurex N, Opas M, Michalak M - J. Cell Biol. (2001)

Bottom Line: Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity.Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells.We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

View Article: PubMed Central - PubMed

Affiliation: Canadian Institutes of Health Research Group in Molecular Biology of Membranes and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

ABSTRACT
Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

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Ca2+ release in saponin-permeabilized calreticulin-deficient cells. Wild-type (K41) and calreticulin-deficient (K42) cells were loaded with a fluorescent Ca2+ indicator fluo-3 and permeabilized with saponin. (A) InsP3 (10 μM) was added at the time indicated in the figure. (B) Basal [Ca2+]c in InsP3 incubated wild-type (white bar) and calreticulin-deficient (black bar). Data are means ± SD (n = 3).
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fig8: Ca2+ release in saponin-permeabilized calreticulin-deficient cells. Wild-type (K41) and calreticulin-deficient (K42) cells were loaded with a fluorescent Ca2+ indicator fluo-3 and permeabilized with saponin. (A) InsP3 (10 μM) was added at the time indicated in the figure. (B) Basal [Ca2+]c in InsP3 incubated wild-type (white bar) and calreticulin-deficient (black bar). Data are means ± SD (n = 3).

Mentions: We have shown that expression of the InsP3R is reduced in calreticulin-deficient (K42) cells, whereas expression of the bradykinin receptor is not. Therefore, we wanted to determine whether the altered expression of the InsP3R was responsible for the impairment of bradykinin-induced Ca2+ release in these cells. To test this hypothesis, we loaded wild-type and calreticulin-deficient cells with fluo-3, and then we added saponin to permeabilize the plasma membrane (Favre et al., 1994). Subsequently, exogenous InsP3 was added to the digitonin-permeabilized cells, and Ca2+ release from the ER was monitored by changes in fluo-3 fluorescence. Fig. 8 shows that InsP3-induced Ca2+ release from the ER was indistinguishable in the wild-type (K41) and calreticulin-deficient (K42) cells, indicating that there is no difference in function of the InsP3Rs in the different cell types.


Functional specialization of calreticulin domains.

Nakamura K, Zuppini A, Arnaudeau S, Lynch J, Ahsan I, Krause R, Papp S, De Smedt H, Parys JB, Muller-Esterl W, Lew DP, Krause KH, Demaurex N, Opas M, Michalak M - J. Cell Biol. (2001)

Ca2+ release in saponin-permeabilized calreticulin-deficient cells. Wild-type (K41) and calreticulin-deficient (K42) cells were loaded with a fluorescent Ca2+ indicator fluo-3 and permeabilized with saponin. (A) InsP3 (10 μM) was added at the time indicated in the figure. (B) Basal [Ca2+]c in InsP3 incubated wild-type (white bar) and calreticulin-deficient (black bar). Data are means ± SD (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196195&req=5

fig8: Ca2+ release in saponin-permeabilized calreticulin-deficient cells. Wild-type (K41) and calreticulin-deficient (K42) cells were loaded with a fluorescent Ca2+ indicator fluo-3 and permeabilized with saponin. (A) InsP3 (10 μM) was added at the time indicated in the figure. (B) Basal [Ca2+]c in InsP3 incubated wild-type (white bar) and calreticulin-deficient (black bar). Data are means ± SD (n = 3).
Mentions: We have shown that expression of the InsP3R is reduced in calreticulin-deficient (K42) cells, whereas expression of the bradykinin receptor is not. Therefore, we wanted to determine whether the altered expression of the InsP3R was responsible for the impairment of bradykinin-induced Ca2+ release in these cells. To test this hypothesis, we loaded wild-type and calreticulin-deficient cells with fluo-3, and then we added saponin to permeabilize the plasma membrane (Favre et al., 1994). Subsequently, exogenous InsP3 was added to the digitonin-permeabilized cells, and Ca2+ release from the ER was monitored by changes in fluo-3 fluorescence. Fig. 8 shows that InsP3-induced Ca2+ release from the ER was indistinguishable in the wild-type (K41) and calreticulin-deficient (K42) cells, indicating that there is no difference in function of the InsP3Rs in the different cell types.

Bottom Line: Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity.Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells.We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

View Article: PubMed Central - PubMed

Affiliation: Canadian Institutes of Health Research Group in Molecular Biology of Membranes and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

ABSTRACT
Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

Show MeSH
Related in: MedlinePlus