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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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Calreticulin-deficient mouse embryonic fibroblasts. (A) Western blot analysis of calreticulin in mouse embryonic fibroblasts. Wild-type (K41) and calreticulin-deficient (K42) cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anticalreticulin antibodies. Lane 1, wild-type K41 mouse embryonic fibroblasts; lane 2, calreticulin-deficient (crt−/−) K42 mouse embryonic fibroblasts; lane 3, calreticulin-deficient fibroblasts transfected with expression vector containing cDNA encoding calreticulin (K42CRT). (B) Phase–contrast analysis of K41 and K42 cells. (C) Immunostaining of K41, K42, and K42CRT cells with anti-CRT, anti-Grp94, and anti-PDI antibodies.
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fig1: Calreticulin-deficient mouse embryonic fibroblasts. (A) Western blot analysis of calreticulin in mouse embryonic fibroblasts. Wild-type (K41) and calreticulin-deficient (K42) cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anticalreticulin antibodies. Lane 1, wild-type K41 mouse embryonic fibroblasts; lane 2, calreticulin-deficient (crt−/−) K42 mouse embryonic fibroblasts; lane 3, calreticulin-deficient fibroblasts transfected with expression vector containing cDNA encoding calreticulin (K42CRT). (B) Phase–contrast analysis of K41 and K42 cells. (C) Immunostaining of K41, K42, and K42CRT cells with anti-CRT, anti-Grp94, and anti-PDI antibodies.

Mentions: To determine the effects of calreticulin deficiency on ER function, we isolated mouse embryonic fibroblasts from crt−/− and wild-type embryos. These cell lines were designated K41 for wild-type mouse embryonic fibroblasts and K42 for calreticulin-deficient mouse embryonic fibroblasts. Some of the calreticulin-deficient cells (K42) were also transfected with a calreticulin expression vector and were designated K42CRT. As expected, Western blot analysis revealed that the K41 and K42CRT cells contained immunoreactive calreticulin (Fig. 1 A, lanes 1 and 3), whereas the K42 crt−/− cells did not (Fig. 1 A, lane 2). The morphological appearance of the wild-type (K41) and calreticulin-deficient (K42) cells was indistinguishable and typical of fibroblasts (Fig. 1 B). The cell lines all attached firmly to plastic, and we detected no differences in the kinetics of their long-term (14 d) growth (unpublished data). Fig. 1 C shows that K41 and K42CRT cells both expressed calreticulin and that the protein was localized to an ER-like network. As expected, there was no expression of calreticulin in K42 cells. Morphologically, at a light microscope level the ER appeared intact in all cell lines as judged by staining with antibodies against protein disulfide isomerase (PDI) and Grp94 (Fig. 1 C). We observed typical nuclear morphology in all of the cell lines and the actin cytoskeleton visualized by labeling with fluorescent phalloidin also appeared normal (unpublished data).


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)

Calreticulin-deficient mouse embryonic fibroblasts. (A) Western blot analysis of calreticulin in mouse embryonic fibroblasts. Wild-type (K41) and calreticulin-deficient (K42) cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anticalreticulin antibodies. Lane 1, wild-type K41 mouse embryonic fibroblasts; lane 2, calreticulin-deficient (crt−/−) K42 mouse embryonic fibroblasts; lane 3, calreticulin-deficient fibroblasts transfected with expression vector containing cDNA encoding calreticulin (K42CRT). (B) Phase–contrast analysis of K41 and K42 cells. (C) Immunostaining of K41, K42, and K42CRT cells with anti-CRT, anti-Grp94, and anti-PDI antibodies.
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Related In: Results  -  Collection

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

fig1: Calreticulin-deficient mouse embryonic fibroblasts. (A) Western blot analysis of calreticulin in mouse embryonic fibroblasts. Wild-type (K41) and calreticulin-deficient (K42) cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anticalreticulin antibodies. Lane 1, wild-type K41 mouse embryonic fibroblasts; lane 2, calreticulin-deficient (crt−/−) K42 mouse embryonic fibroblasts; lane 3, calreticulin-deficient fibroblasts transfected with expression vector containing cDNA encoding calreticulin (K42CRT). (B) Phase–contrast analysis of K41 and K42 cells. (C) Immunostaining of K41, K42, and K42CRT cells with anti-CRT, anti-Grp94, and anti-PDI antibodies.
Mentions: To determine the effects of calreticulin deficiency on ER function, we isolated mouse embryonic fibroblasts from crt−/− and wild-type embryos. These cell lines were designated K41 for wild-type mouse embryonic fibroblasts and K42 for calreticulin-deficient mouse embryonic fibroblasts. Some of the calreticulin-deficient cells (K42) were also transfected with a calreticulin expression vector and were designated K42CRT. As expected, Western blot analysis revealed that the K41 and K42CRT cells contained immunoreactive calreticulin (Fig. 1 A, lanes 1 and 3), whereas the K42 crt−/− cells did not (Fig. 1 A, lane 2). The morphological appearance of the wild-type (K41) and calreticulin-deficient (K42) cells was indistinguishable and typical of fibroblasts (Fig. 1 B). The cell lines all attached firmly to plastic, and we detected no differences in the kinetics of their long-term (14 d) growth (unpublished data). Fig. 1 C shows that K41 and K42CRT cells both expressed calreticulin and that the protein was localized to an ER-like network. As expected, there was no expression of calreticulin in K42 cells. Morphologically, at a light microscope level the ER appeared intact in all cell lines as judged by staining with antibodies against protein disulfide isomerase (PDI) and Grp94 (Fig. 1 C). We observed typical nuclear morphology in all of the cell lines and the actin cytoskeleton visualized by labeling with fluorescent phalloidin also appeared normal (unpublished data).

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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