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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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ER Ca2+ content in calreticulin-deficient cells. Calreticulin-deficient cells (K42) were transfected with expression vectors encoding calreticulin or calreticulin domains followed by measurement of a total cellular Ca2+. (A) A total cellular Ca2+ content was determined using equilibrium incubation with 45Ca2+ followed by addition of thapsigargin (estimates Ca2+ pool in thapsigargin-sensitive Ca2+ stores) or ionomycin (estimates Ca2+ pool in thapsigargin-insensitive Ca2+ stores) (Mery et al., 1996). Ca2+ content was measured in wild-type (K41), crt−/− (K42), and K42 cells transfected with expression vectors encoding either calreticulin (K42CRT), the N + P domain of calreticulin (K42N+P), or the P + C domain of the protein (K42P+C). Data shown are means ± SE (n = 3). (B) Western blot analysis with anti-HA tag antibodies of K42 calreticulin-deficient cells expressing calreticulin domains. Lane 1, calreticulin-deficient cells (K42); lane 2, K42 cells transfected with expression vectors encoding the N + P domain of calreticulin (K42N+P); lane 3, K42 transfected with expression vector encoding the P + C domain of the protein (K42P+C). Similar levels of both, the 38-kD N + P domain and the 36-kD P + C domain were expressed in transfected cells.
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fig2: ER Ca2+ content in calreticulin-deficient cells. Calreticulin-deficient cells (K42) were transfected with expression vectors encoding calreticulin or calreticulin domains followed by measurement of a total cellular Ca2+. (A) A total cellular Ca2+ content was determined using equilibrium incubation with 45Ca2+ followed by addition of thapsigargin (estimates Ca2+ pool in thapsigargin-sensitive Ca2+ stores) or ionomycin (estimates Ca2+ pool in thapsigargin-insensitive Ca2+ stores) (Mery et al., 1996). Ca2+ content was measured in wild-type (K41), crt−/− (K42), and K42 cells transfected with expression vectors encoding either calreticulin (K42CRT), the N + P domain of calreticulin (K42N+P), or the P + C domain of the protein (K42P+C). Data shown are means ± SE (n = 3). (B) Western blot analysis with anti-HA tag antibodies of K42 calreticulin-deficient cells expressing calreticulin domains. Lane 1, calreticulin-deficient cells (K42); lane 2, K42 cells transfected with expression vectors encoding the N + P domain of calreticulin (K42N+P); lane 3, K42 transfected with expression vector encoding the P + C domain of the protein (K42P+C). Similar levels of both, the 38-kD N + P domain and the 36-kD P + C domain were expressed in transfected cells.

Mentions: Next, we used thapsigargin, an inhibitor of the Ca2+-ATPase, to measure the amount of Ca2+ associated with rapidly exchangeable stores. To measure the residual amounts of Ca2+ contained within thapsigargin-insensitive stores, we added the Ca2+ ionophore ionomycin. In these experiments, cells were equilibrium loaded with 45Ca2+ and then resuspended in a nonradioactive Ca2+-free medium (Mery et al., 1996). Fig. 2 shows that in response to thapsigargin the wild-type cells (K41) released almost twice as much 45Ca2+ as the calreticulin-deficient cells (K42). When the remaining 45Ca2+ was released with ionomycin, the amount released from K41 cells was greater than from K42 cells (Fig. 2 A). Fig. 2 A also shows that there was an increase in Ca2+ release in the presence of ionomycin compared with thapsigargin alone. This is likely due to ionomycin-dependent Ca2+ release from intracellular compartments other than ER including mitochondria. To confirm that the absence of calreticulin caused the decreased Ca2+ content of the K42 cells, some K42 cells were stably transfected with a calreticulin expression vector, creating the K42CRT cell line (Fig. 1). Fig. 2 A shows that the Ca2+ content of thapsigargin-sensitive stores was indistinguishable in K42CRT and wild-type (K41) cells. We conclude that the absence of calreticulin in K42 cells causes a significant decrease in the Ca2+ content of thapsigargin-sensitive stores.


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)

ER Ca2+ content in calreticulin-deficient cells. Calreticulin-deficient cells (K42) were transfected with expression vectors encoding calreticulin or calreticulin domains followed by measurement of a total cellular Ca2+. (A) A total cellular Ca2+ content was determined using equilibrium incubation with 45Ca2+ followed by addition of thapsigargin (estimates Ca2+ pool in thapsigargin-sensitive Ca2+ stores) or ionomycin (estimates Ca2+ pool in thapsigargin-insensitive Ca2+ stores) (Mery et al., 1996). Ca2+ content was measured in wild-type (K41), crt−/− (K42), and K42 cells transfected with expression vectors encoding either calreticulin (K42CRT), the N + P domain of calreticulin (K42N+P), or the P + C domain of the protein (K42P+C). Data shown are means ± SE (n = 3). (B) Western blot analysis with anti-HA tag antibodies of K42 calreticulin-deficient cells expressing calreticulin domains. Lane 1, calreticulin-deficient cells (K42); lane 2, K42 cells transfected with expression vectors encoding the N + P domain of calreticulin (K42N+P); lane 3, K42 transfected with expression vector encoding the P + C domain of the protein (K42P+C). Similar levels of both, the 38-kD N + P domain and the 36-kD P + C domain were expressed in transfected cells.
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Related In: Results  -  Collection

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fig2: ER Ca2+ content in calreticulin-deficient cells. Calreticulin-deficient cells (K42) were transfected with expression vectors encoding calreticulin or calreticulin domains followed by measurement of a total cellular Ca2+. (A) A total cellular Ca2+ content was determined using equilibrium incubation with 45Ca2+ followed by addition of thapsigargin (estimates Ca2+ pool in thapsigargin-sensitive Ca2+ stores) or ionomycin (estimates Ca2+ pool in thapsigargin-insensitive Ca2+ stores) (Mery et al., 1996). Ca2+ content was measured in wild-type (K41), crt−/− (K42), and K42 cells transfected with expression vectors encoding either calreticulin (K42CRT), the N + P domain of calreticulin (K42N+P), or the P + C domain of the protein (K42P+C). Data shown are means ± SE (n = 3). (B) Western blot analysis with anti-HA tag antibodies of K42 calreticulin-deficient cells expressing calreticulin domains. Lane 1, calreticulin-deficient cells (K42); lane 2, K42 cells transfected with expression vectors encoding the N + P domain of calreticulin (K42N+P); lane 3, K42 transfected with expression vector encoding the P + C domain of the protein (K42P+C). Similar levels of both, the 38-kD N + P domain and the 36-kD P + C domain were expressed in transfected cells.
Mentions: Next, we used thapsigargin, an inhibitor of the Ca2+-ATPase, to measure the amount of Ca2+ associated with rapidly exchangeable stores. To measure the residual amounts of Ca2+ contained within thapsigargin-insensitive stores, we added the Ca2+ ionophore ionomycin. In these experiments, cells were equilibrium loaded with 45Ca2+ and then resuspended in a nonradioactive Ca2+-free medium (Mery et al., 1996). Fig. 2 shows that in response to thapsigargin the wild-type cells (K41) released almost twice as much 45Ca2+ as the calreticulin-deficient cells (K42). When the remaining 45Ca2+ was released with ionomycin, the amount released from K41 cells was greater than from K42 cells (Fig. 2 A). Fig. 2 A also shows that there was an increase in Ca2+ release in the presence of ionomycin compared with thapsigargin alone. This is likely due to ionomycin-dependent Ca2+ release from intracellular compartments other than ER including mitochondria. To confirm that the absence of calreticulin caused the decreased Ca2+ content of the K42 cells, some K42 cells were stably transfected with a calreticulin expression vector, creating the K42CRT cell line (Fig. 1). Fig. 2 A shows that the Ca2+ content of thapsigargin-sensitive stores was indistinguishable in K42CRT and wild-type (K41) cells. We conclude that the absence of calreticulin in K42 cells causes a significant decrease in the Ca2+ content of thapsigargin-sensitive stores.

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