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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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Related in: MedlinePlus

InsP3 production and [3H]bradykinin binding in calreticulin-deficient cells. (A) InsP3 synthesis was measured in wild-type (K41) and calreticulin-deficient (K42) cells incubated in the absence (−) or presence (+) of bradykinin as described in Materials and methods. Data are means ± SD (n = 3). (B) [3H]bradykinin binding to wild-type (K41), calreticulin-deficient (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT).
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fig9: InsP3 production and [3H]bradykinin binding in calreticulin-deficient cells. (A) InsP3 synthesis was measured in wild-type (K41) and calreticulin-deficient (K42) cells incubated in the absence (−) or presence (+) of bradykinin as described in Materials and methods. Data are means ± SD (n = 3). (B) [3H]bradykinin binding to wild-type (K41), calreticulin-deficient (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT).

Mentions: Since InsP3-induced Ca2+ release in permeabilized crt−/− cells is normal, the impaired bradykinin-induced Ca2+ release in intact cells could result from a deficiency in InsP3 synthesis. To investigate this possibility, we incubated wild-type and calreticulin-deficient cells with 200 nM bradykinin and then measured InsP3 levels. Fig. 9 A shows that the incubation with bradykinin resulted in significant synthesis of InsP3 in the K41 cells. In contrast, when calreticulin-deficient K42 cells were treated with bradykinin there was no detectable synthesis of InsP3 (Fig. 9 A). We conclude that impairment of bradykinin-induced Ca2+ release in crt−/− K42 cells likely results from a failure to synthesize InsP3.


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)

InsP3 production and [3H]bradykinin binding in calreticulin-deficient cells. (A) InsP3 synthesis was measured in wild-type (K41) and calreticulin-deficient (K42) cells incubated in the absence (−) or presence (+) of bradykinin as described in Materials and methods. Data are means ± SD (n = 3). (B) [3H]bradykinin binding to wild-type (K41), calreticulin-deficient (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT).
© Copyright Policy
Related In: Results  -  Collection

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

fig9: InsP3 production and [3H]bradykinin binding in calreticulin-deficient cells. (A) InsP3 synthesis was measured in wild-type (K41) and calreticulin-deficient (K42) cells incubated in the absence (−) or presence (+) of bradykinin as described in Materials and methods. Data are means ± SD (n = 3). (B) [3H]bradykinin binding to wild-type (K41), calreticulin-deficient (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT).
Mentions: Since InsP3-induced Ca2+ release in permeabilized crt−/− cells is normal, the impaired bradykinin-induced Ca2+ release in intact cells could result from a deficiency in InsP3 synthesis. To investigate this possibility, we incubated wild-type and calreticulin-deficient cells with 200 nM bradykinin and then measured InsP3 levels. Fig. 9 A shows that the incubation with bradykinin resulted in significant synthesis of InsP3 in the K41 cells. In contrast, when calreticulin-deficient K42 cells were treated with bradykinin there was no detectable synthesis of InsP3 (Fig. 9 A). We conclude that impairment of bradykinin-induced Ca2+ release in crt−/− K42 cells likely results from a failure to synthesize InsP3.

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