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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 [Ca2+]ER responses in bradykinin- and thapsigargin-stimulated cells. Wild-type (K41) and calreticulin-deficient (K42) cells were transiently transfected with YC4ER expression vector. A and B show the time-course of spatially averaged [Ca2+]ER responses in cells stimulated with bradykinin and thapsigargin (BK + TG) or with thapsigargin alone. The responses were measured in Ca2+-free medium. When indicated, ionomycin (1 μM) was added to maximally deplete ER Ca2+ stores. (C) [Ca2+]ER levels measured shortly after stimulation with bradykinin/thapsigargin or after full depletion of stores with thapsigargin. The absolute values were: 80 ± 12 μM (K41) and 114 ± 18 μM (K42) for BK + TG, and 44 ± 5 μM (K41) 53 ± 7 μM (K42) for depletion with TG. BK, bradykinin; TG, thapsigargin; iono, ionomycin. Data are mean ± SE. n, number of experiments.
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fig5: Free [Ca2+]ER responses in bradykinin- and thapsigargin-stimulated cells. Wild-type (K41) and calreticulin-deficient (K42) cells were transiently transfected with YC4ER expression vector. A and B show the time-course of spatially averaged [Ca2+]ER responses in cells stimulated with bradykinin and thapsigargin (BK + TG) or with thapsigargin alone. The responses were measured in Ca2+-free medium. When indicated, ionomycin (1 μM) was added to maximally deplete ER Ca2+ stores. (C) [Ca2+]ER levels measured shortly after stimulation with bradykinin/thapsigargin or after full depletion of stores with thapsigargin. The absolute values were: 80 ± 12 μM (K41) and 114 ± 18 μM (K42) for BK + TG, and 44 ± 5 μM (K41) 53 ± 7 μM (K42) for depletion with TG. BK, bradykinin; TG, thapsigargin; iono, ionomycin. Data are mean ± SE. n, number of experiments.

Mentions: In further experiments, we used a cameleon reporter to make time-resolved measurements of free [Ca2+]ER in wild-type and calreticulin-deficient cells. Bradykinin by itself had little effects on [Ca2+]ER (unpublished data) but caused a rapid [Ca2+]ER decrease when thapsigargin was included to prevent ER refilling (Fig. 5 A). A pronounced Ca2+ release was observed in wild-type cells, the [Ca2+]ER decreasing to 80 ± 12 μM within 150 s. (Fig. 5 C). Subsequent addition of ionomycin had little effects on the kinetics of the [Ca2+]ER response (Fig. 5 A), indicating the ER Ca2+ permeability was maximally activated by the combination of bradykinin and thapsigargin. In contrast, the addition of bradykinin and thapsigargin to calreticulin-deficient cells decreased [Ca2+]ER only to 114 ± 18 μM (Fig. 5 C), and subsequent addition of ionomycin caused a further decrease in [Ca2+]ER (Fig. 5 A). No differences were observed when thapsigargin was added alone, the [Ca2+]ER decreasing with similar kinetics and to similar levels in wild-type and calreticulin-deficient cells (Fig. 5, B and C). The higher [Ca2+]ER measured in calreticulin-deficient cells stimulated with bradykinin and thapsigargin thus likely reflects the failure of bradykinin to increase the ER Ca2+ permeability. These observations are in keeping with the fura-2 measurements presented in Fig. 4 and indicate that bradykinin-induced Ca2+ release from the ER is impaired in the calreticulin-deficient K42 cells.


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 [Ca2+]ER responses in bradykinin- and thapsigargin-stimulated cells. Wild-type (K41) and calreticulin-deficient (K42) cells were transiently transfected with YC4ER expression vector. A and B show the time-course of spatially averaged [Ca2+]ER responses in cells stimulated with bradykinin and thapsigargin (BK + TG) or with thapsigargin alone. The responses were measured in Ca2+-free medium. When indicated, ionomycin (1 μM) was added to maximally deplete ER Ca2+ stores. (C) [Ca2+]ER levels measured shortly after stimulation with bradykinin/thapsigargin or after full depletion of stores with thapsigargin. The absolute values were: 80 ± 12 μM (K41) and 114 ± 18 μM (K42) for BK + TG, and 44 ± 5 μM (K41) 53 ± 7 μM (K42) for depletion with TG. BK, bradykinin; TG, thapsigargin; iono, ionomycin. Data are mean ± SE. n, number of experiments.
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Related In: Results  -  Collection

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fig5: Free [Ca2+]ER responses in bradykinin- and thapsigargin-stimulated cells. Wild-type (K41) and calreticulin-deficient (K42) cells were transiently transfected with YC4ER expression vector. A and B show the time-course of spatially averaged [Ca2+]ER responses in cells stimulated with bradykinin and thapsigargin (BK + TG) or with thapsigargin alone. The responses were measured in Ca2+-free medium. When indicated, ionomycin (1 μM) was added to maximally deplete ER Ca2+ stores. (C) [Ca2+]ER levels measured shortly after stimulation with bradykinin/thapsigargin or after full depletion of stores with thapsigargin. The absolute values were: 80 ± 12 μM (K41) and 114 ± 18 μM (K42) for BK + TG, and 44 ± 5 μM (K41) 53 ± 7 μM (K42) for depletion with TG. BK, bradykinin; TG, thapsigargin; iono, ionomycin. Data are mean ± SE. n, number of experiments.
Mentions: In further experiments, we used a cameleon reporter to make time-resolved measurements of free [Ca2+]ER in wild-type and calreticulin-deficient cells. Bradykinin by itself had little effects on [Ca2+]ER (unpublished data) but caused a rapid [Ca2+]ER decrease when thapsigargin was included to prevent ER refilling (Fig. 5 A). A pronounced Ca2+ release was observed in wild-type cells, the [Ca2+]ER decreasing to 80 ± 12 μM within 150 s. (Fig. 5 C). Subsequent addition of ionomycin had little effects on the kinetics of the [Ca2+]ER response (Fig. 5 A), indicating the ER Ca2+ permeability was maximally activated by the combination of bradykinin and thapsigargin. In contrast, the addition of bradykinin and thapsigargin to calreticulin-deficient cells decreased [Ca2+]ER only to 114 ± 18 μM (Fig. 5 C), and subsequent addition of ionomycin caused a further decrease in [Ca2+]ER (Fig. 5 A). No differences were observed when thapsigargin was added alone, the [Ca2+]ER decreasing with similar kinetics and to similar levels in wild-type and calreticulin-deficient cells (Fig. 5, B and C). The higher [Ca2+]ER measured in calreticulin-deficient cells stimulated with bradykinin and thapsigargin thus likely reflects the failure of bradykinin to increase the ER Ca2+ permeability. These observations are in keeping with the fura-2 measurements presented in Fig. 4 and indicate that bradykinin-induced Ca2+ release from the ER is impaired in the calreticulin-deficient K42 cells.

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