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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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Free ER Ca2+ luminal concentration in K41 wild-type and K42 calreticulin-deficient cells. Cells were transiently transfected with YC4ER and the ratio fluorescence calibrated to [Ca2+]ER using calibration curves obtained in situ (Arnaudeau et al., 2001). (A) Intracellular distribution of ER-targeted cameleon in wild-type (K41) and calreticulin-deficient (K42) cells. (A) Single wavelength cameleon fluorescence (emission at 535 nm) imaging of transfected cells revealing reticular pattern reminiscent of the ER. (B) Free [Ca2+]ER in K41 wild-type (white bars) and K42 calreticulin-deficient (gray bars) cells was measured with ER-targeted cameleon as described under Materials and methods. TG, thapsigargin; iono, ionomycin. Data are derived from 15–22 independent experiments.
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fig3: Free ER Ca2+ luminal concentration in K41 wild-type and K42 calreticulin-deficient cells. Cells were transiently transfected with YC4ER and the ratio fluorescence calibrated to [Ca2+]ER using calibration curves obtained in situ (Arnaudeau et al., 2001). (A) Intracellular distribution of ER-targeted cameleon in wild-type (K41) and calreticulin-deficient (K42) cells. (A) Single wavelength cameleon fluorescence (emission at 535 nm) imaging of transfected cells revealing reticular pattern reminiscent of the ER. (B) Free [Ca2+]ER in K41 wild-type (white bars) and K42 calreticulin-deficient (gray bars) cells was measured with ER-targeted cameleon as described under Materials and methods. TG, thapsigargin; iono, ionomycin. Data are derived from 15–22 independent experiments.

Mentions: To determine the free [Ca2+]ER in calreticulin-deficient cells, we transfected wild-type (K41) and calreticulin-deficient (K42) mouse embryonic fibroblasts with the ER-targeted cameleon YC4ER (Arnaudeau et al., 2001), a Ca2+ indicator, which relies on fluorescent proteins and calmodulin (Miyawaki et al., 1997). Fig. 3 A shows that the cameleon was expressed in both cell types and was localized in a reticular pattern consistent with labeling of the ER. Fig. 3 B shows that the free [Ca2+]ER did not differ significantly in the wild-type cells (290 ± 18 μM Ca2+; mean ± SE; n = 18) and calreticulin-deficient cells (288 ± 20 μM Ca2+; mean ± SE; n = 22). In further studies, we used thapsigargin and ionomycin to completely deplete cellular Ca2+stores. Under these conditions, free [Ca2+]ER was reduced to 27 ± 3 μM Ca2+ (mean ± SE; n = 15) in the wild-type cells and to 23 ± 3 μM Ca2+ (mean ± SE) in the calreticulin-deficient cells (Fig. 3 B). Our results indicate that although the total Ca2+ content of the ER is significantly decreased in calreticulin-deficient cells, the free [Ca2+]ER is unaltered both when Ca2+ stores are full and when they are depleted.


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)

Free ER Ca2+ luminal concentration in K41 wild-type and K42 calreticulin-deficient cells. Cells were transiently transfected with YC4ER and the ratio fluorescence calibrated to [Ca2+]ER using calibration curves obtained in situ (Arnaudeau et al., 2001). (A) Intracellular distribution of ER-targeted cameleon in wild-type (K41) and calreticulin-deficient (K42) cells. (A) Single wavelength cameleon fluorescence (emission at 535 nm) imaging of transfected cells revealing reticular pattern reminiscent of the ER. (B) Free [Ca2+]ER in K41 wild-type (white bars) and K42 calreticulin-deficient (gray bars) cells was measured with ER-targeted cameleon as described under Materials and methods. TG, thapsigargin; iono, ionomycin. Data are derived from 15–22 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Free ER Ca2+ luminal concentration in K41 wild-type and K42 calreticulin-deficient cells. Cells were transiently transfected with YC4ER and the ratio fluorescence calibrated to [Ca2+]ER using calibration curves obtained in situ (Arnaudeau et al., 2001). (A) Intracellular distribution of ER-targeted cameleon in wild-type (K41) and calreticulin-deficient (K42) cells. (A) Single wavelength cameleon fluorescence (emission at 535 nm) imaging of transfected cells revealing reticular pattern reminiscent of the ER. (B) Free [Ca2+]ER in K41 wild-type (white bars) and K42 calreticulin-deficient (gray bars) cells was measured with ER-targeted cameleon as described under Materials and methods. TG, thapsigargin; iono, ionomycin. Data are derived from 15–22 independent experiments.
Mentions: To determine the free [Ca2+]ER in calreticulin-deficient cells, we transfected wild-type (K41) and calreticulin-deficient (K42) mouse embryonic fibroblasts with the ER-targeted cameleon YC4ER (Arnaudeau et al., 2001), a Ca2+ indicator, which relies on fluorescent proteins and calmodulin (Miyawaki et al., 1997). Fig. 3 A shows that the cameleon was expressed in both cell types and was localized in a reticular pattern consistent with labeling of the ER. Fig. 3 B shows that the free [Ca2+]ER did not differ significantly in the wild-type cells (290 ± 18 μM Ca2+; mean ± SE; n = 18) and calreticulin-deficient cells (288 ± 20 μM Ca2+; mean ± SE; n = 22). In further studies, we used thapsigargin and ionomycin to completely deplete cellular Ca2+stores. Under these conditions, free [Ca2+]ER was reduced to 27 ± 3 μM Ca2+ (mean ± SE; n = 15) in the wild-type cells and to 23 ± 3 μM Ca2+ (mean ± SE) in the calreticulin-deficient cells (Fig. 3 B). Our results indicate that although the total Ca2+ content of the ER is significantly decreased in calreticulin-deficient cells, the free [Ca2+]ER is unaltered both when Ca2+ stores are full and when they are depleted.

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