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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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Bradykinin-induced Ca2+ release in calreticulin-deficient cells. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. (A) Represents typical traces showing bradykinin (BK) stimulation of cells in a Ca2+-free medium. Arrows indicate the time of addition of bradykinin. (B) Basal [Ca2+]c in wild-type (white bar), calreticulin- deficient (black bar), and calreticulin-deficient cells transfected with calreticulin expression vector (hatched bar). The absolute values of [Ca2+]c were: 100 ± 12 nM (K41), 148 ± 10 nM (crt−/− K42), and 95 ± 8 nM (crt−/− + CRT, K42CRT). (C) Ca2+ released by bradykinin was significantly different in cell lines investigated. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. Data are mean ± SE (n = 3).
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fig4: Bradykinin-induced Ca2+ release in calreticulin-deficient cells. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. (A) Represents typical traces showing bradykinin (BK) stimulation of cells in a Ca2+-free medium. Arrows indicate the time of addition of bradykinin. (B) Basal [Ca2+]c in wild-type (white bar), calreticulin- deficient (black bar), and calreticulin-deficient cells transfected with calreticulin expression vector (hatched bar). The absolute values of [Ca2+]c were: 100 ± 12 nM (K41), 148 ± 10 nM (crt−/− K42), and 95 ± 8 nM (crt−/− + CRT, K42CRT). (C) Ca2+ released by bradykinin was significantly different in cell lines investigated. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. Data are mean ± SE (n = 3).

Mentions: We next performed experiments with the Ca2+-sensitive fluorescent dye fura-2. Fig. 4 shows that in wild-type (K41) cells, the resting free cytoplasmic Ca2+ concentration ([Ca2+]c) was ∼100 ± 12 nM (mean ± SD; n = 3). In calreticulin-deficient fibroblasts (K42), the resting free [Ca2+]c was significantly increased (148 ± 10 nM; mean ± SD; n = 3). We also performed fura-2 analysis of [Ca2+]c in calreticulin-deficient mouse embryonic fibroblasts, which had been transfected with a calreticulin expression vector (K42CRT cells). These cells, which express recombinant calreticulin, had 108 ± 13 nM (mean ± SD; n = 3) basal [Ca2+]c, indicating that reintroduction of calreticulin to calreticulin-deficient cells lowered the basal [Ca2+]c to the levels observed in the wild-type (K41) cells (Fig. 4, A and B).


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)

Bradykinin-induced Ca2+ release in calreticulin-deficient cells. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. (A) Represents typical traces showing bradykinin (BK) stimulation of cells in a Ca2+-free medium. Arrows indicate the time of addition of bradykinin. (B) Basal [Ca2+]c in wild-type (white bar), calreticulin- deficient (black bar), and calreticulin-deficient cells transfected with calreticulin expression vector (hatched bar). The absolute values of [Ca2+]c were: 100 ± 12 nM (K41), 148 ± 10 nM (crt−/− K42), and 95 ± 8 nM (crt−/− + CRT, K42CRT). (C) Ca2+ released by bradykinin was significantly different in cell lines investigated. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. Data are mean ± SE (n = 3).
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Related In: Results  -  Collection

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fig4: Bradykinin-induced Ca2+ release in calreticulin-deficient cells. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. (A) Represents typical traces showing bradykinin (BK) stimulation of cells in a Ca2+-free medium. Arrows indicate the time of addition of bradykinin. (B) Basal [Ca2+]c in wild-type (white bar), calreticulin- deficient (black bar), and calreticulin-deficient cells transfected with calreticulin expression vector (hatched bar). The absolute values of [Ca2+]c were: 100 ± 12 nM (K41), 148 ± 10 nM (crt−/− K42), and 95 ± 8 nM (crt−/− + CRT, K42CRT). (C) Ca2+ released by bradykinin was significantly different in cell lines investigated. K41, wild-type mouse embryonic fibroblasts; K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector. Data are mean ± SE (n = 3).
Mentions: We next performed experiments with the Ca2+-sensitive fluorescent dye fura-2. Fig. 4 shows that in wild-type (K41) cells, the resting free cytoplasmic Ca2+ concentration ([Ca2+]c) was ∼100 ± 12 nM (mean ± SD; n = 3). In calreticulin-deficient fibroblasts (K42), the resting free [Ca2+]c was significantly increased (148 ± 10 nM; mean ± SD; n = 3). We also performed fura-2 analysis of [Ca2+]c in calreticulin-deficient mouse embryonic fibroblasts, which had been transfected with a calreticulin expression vector (K42CRT cells). These cells, which express recombinant calreticulin, had 108 ± 13 nM (mean ± SD; n = 3) basal [Ca2+]c, indicating that reintroduction of calreticulin to calreticulin-deficient cells lowered the basal [Ca2+]c to the levels observed in the wild-type (K41) cells (Fig. 4, A and B).

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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