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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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Role of the N + P domain of calreticulin in bradykinin-induced Ca2+ release. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. A shows a basal [Ca2+]c, and B shows the amount of Ca2+ released by 200 nM bradykinin. K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector; K42N+P, K42 cells transfected with N + P domain expression vector; K42P+C, K42 cells transfected with P + C domain expression vector. Data are means ± SD (n = 3).
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fig10: Role of the N + P domain of calreticulin in bradykinin-induced Ca2+ release. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. A shows a basal [Ca2+]c, and B shows the amount of Ca2+ released by 200 nM bradykinin. K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector; K42N+P, K42 cells transfected with N + P domain expression vector; K42P+C, K42 cells transfected with P + C domain expression vector. Data are means ± SD (n = 3).

Mentions: The data presented in Figs. 4 and 9 indicate that the impairment of bradykinin-induced Ca2+ release in calreticulin-deficient cells results from the failure of bradykinin to bind to its receptor. This indicates that the bradykinin receptor may be misfolded and therefore unable to bind bradykinin. In preliminary experiments, we showed that calreticulin and bradykinin receptor form complexes, which can be immunoprecipitated (unpublished data), indicating that calreticulin may play a role in folding of the bradykinin receptor. This presented us with a unique opportunity to investigate the role of calreticulin's different domains in its function as a chaperone. We used bradykinin-induced changes in [Ca2+]c as a measure of the function of the bradykinin receptor. Cells were loaded with fura-2, stimulated with 200 nM bradykinin, and changes in [Ca2+]c were monitored. As shown in Fig. 10 , in K42 crt−/− cells expressing calreticulin (K42CRT), bradykinin-induced Ca2+ release was restored to the levels seen in wild-type cells. This indicates that full-length calreticulin is required for normal ligand binding to the receptor. Bradykinin-induced Ca2+ release was also reestablished in K42 cells after transfection with the N + P domain of calreticulin (Fig. 10 B, K42N+P). However, it was not reestablished in cells expressing the P + C domain of the protein (Fig. 10 B, K42P+C). This indicates that the N and P domain of calreticulin may play a role in peptide binding and/or folding of the bradykinin receptor in mouse embryonic fibroblasts.


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)

Role of the N + P domain of calreticulin in bradykinin-induced Ca2+ release. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. A shows a basal [Ca2+]c, and B shows the amount of Ca2+ released by 200 nM bradykinin. K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector; K42N+P, K42 cells transfected with N + P domain expression vector; K42P+C, K42 cells transfected with P + C domain expression vector. Data are means ± SD (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Role of the N + P domain of calreticulin in bradykinin-induced Ca2+ release. Cells were loaded with the fluorescent Ca2+ indicator fura-2 and stimulated with 200 nM bradykinin. A shows a basal [Ca2+]c, and B shows the amount of Ca2+ released by 200 nM bradykinin. K42, calreticulin-deficient cells; K42CRT, K42 cells transfected with calreticulin expression vector; K42N+P, K42 cells transfected with N + P domain expression vector; K42P+C, K42 cells transfected with P + C domain expression vector. Data are means ± SD (n = 3).
Mentions: The data presented in Figs. 4 and 9 indicate that the impairment of bradykinin-induced Ca2+ release in calreticulin-deficient cells results from the failure of bradykinin to bind to its receptor. This indicates that the bradykinin receptor may be misfolded and therefore unable to bind bradykinin. In preliminary experiments, we showed that calreticulin and bradykinin receptor form complexes, which can be immunoprecipitated (unpublished data), indicating that calreticulin may play a role in folding of the bradykinin receptor. This presented us with a unique opportunity to investigate the role of calreticulin's different domains in its function as a chaperone. We used bradykinin-induced changes in [Ca2+]c as a measure of the function of the bradykinin receptor. Cells were loaded with fura-2, stimulated with 200 nM bradykinin, and changes in [Ca2+]c were monitored. As shown in Fig. 10 , in K42 crt−/− cells expressing calreticulin (K42CRT), bradykinin-induced Ca2+ release was restored to the levels seen in wild-type cells. This indicates that full-length calreticulin is required for normal ligand binding to the receptor. Bradykinin-induced Ca2+ release was also reestablished in K42 cells after transfection with the N + P domain of calreticulin (Fig. 10 B, K42N+P). However, it was not reestablished in cells expressing the P + C domain of the protein (Fig. 10 B, K42P+C). This indicates that the N and P domain of calreticulin may play a role in peptide binding and/or folding of the bradykinin receptor in mouse embryonic fibroblasts.

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