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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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Expression of bradykinin receptor in calreticulin-deficient cells. Mouse embryonic fibroblasts were harvested, lysed, and proteins were separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibradykinin receptor antibodies (A). The positions of molecular markers are indicated. The open arrowhead indicates the position of bradykinin receptor. (B) Localization of bradykinin receptor in wild-type (K41), calreticulin-deficient cells (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT). K42 mock-transfected control cells are also shown. In all cell lines, bradykinin receptor localizes to cell surface. (C) Bradykinin receptor expression on cell surface. Flow cytometry analysis of mouse embryonic fibroblasts was carried out with antibradykinin receptor antibodies. Results are presented as the relative mean fluorescence intensity after subtracting fluorescent values for the secondary antibodies alone. Results shown are representative of five experiments. K41, wild type cells; K42, calreticulin-deficient cells; BK, bradykinin receptor.
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fig7: Expression of bradykinin receptor in calreticulin-deficient cells. Mouse embryonic fibroblasts were harvested, lysed, and proteins were separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibradykinin receptor antibodies (A). The positions of molecular markers are indicated. The open arrowhead indicates the position of bradykinin receptor. (B) Localization of bradykinin receptor in wild-type (K41), calreticulin-deficient cells (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT). K42 mock-transfected control cells are also shown. In all cell lines, bradykinin receptor localizes to cell surface. (C) Bradykinin receptor expression on cell surface. Flow cytometry analysis of mouse embryonic fibroblasts was carried out with antibradykinin receptor antibodies. Results are presented as the relative mean fluorescence intensity after subtracting fluorescent values for the secondary antibodies alone. Results shown are representative of five experiments. K41, wild type cells; K42, calreticulin-deficient cells; BK, bradykinin receptor.

Mentions: Next, we compared expression of the bradykinin receptor and its targeting to the plasma membrane in wild-type (K41) and calreticulin-deficient (K42) cells. Fig. 7 A shows that calreticulin-deficient cells had decreased level of bradykinin receptor protein. Quantitative analysis of Western blots indicated that there was ∼50% less bradykinin receptor in calreticulin-deficient cells compared with K41 wild-type cells. These results indicate that calreticulin deficiency had some effect on expression and/or turnover of the bradykinin receptor. Using confocal microscopy and antibodies against the bradykinin receptor, we found that the receptor was distributed in a “dotty” pattern across the cell surface in K41, K42, and K42CRT cells (Fig. 7 B). We noticed no difference between the cell lines in distribution of the receptor (Fig. 7 B). Cell surface localization of the receptor was further confirmed by flow cytometry assay using antibradykinin receptor antibodies. We observed no significant difference in the antibradykinin antibodies surface labeling of K41 and K42 cells (Fig. 7 C), indicating the similar number of immunoreactive receptor protein molecules was present on cell surface. We concluded that in calreticulin-deficient cells the bradykinin receptor was properly targeted to and localized in the plasma membrane.


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)

Expression of bradykinin receptor in calreticulin-deficient cells. Mouse embryonic fibroblasts were harvested, lysed, and proteins were separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibradykinin receptor antibodies (A). The positions of molecular markers are indicated. The open arrowhead indicates the position of bradykinin receptor. (B) Localization of bradykinin receptor in wild-type (K41), calreticulin-deficient cells (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT). K42 mock-transfected control cells are also shown. In all cell lines, bradykinin receptor localizes to cell surface. (C) Bradykinin receptor expression on cell surface. Flow cytometry analysis of mouse embryonic fibroblasts was carried out with antibradykinin receptor antibodies. Results are presented as the relative mean fluorescence intensity after subtracting fluorescent values for the secondary antibodies alone. Results shown are representative of five experiments. K41, wild type cells; K42, calreticulin-deficient cells; BK, bradykinin receptor.
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Related In: Results  -  Collection

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fig7: Expression of bradykinin receptor in calreticulin-deficient cells. Mouse embryonic fibroblasts were harvested, lysed, and proteins were separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibradykinin receptor antibodies (A). The positions of molecular markers are indicated. The open arrowhead indicates the position of bradykinin receptor. (B) Localization of bradykinin receptor in wild-type (K41), calreticulin-deficient cells (K42), and calreticulin-deficient cells transfected with calreticulin expression vector (K42CRT). K42 mock-transfected control cells are also shown. In all cell lines, bradykinin receptor localizes to cell surface. (C) Bradykinin receptor expression on cell surface. Flow cytometry analysis of mouse embryonic fibroblasts was carried out with antibradykinin receptor antibodies. Results are presented as the relative mean fluorescence intensity after subtracting fluorescent values for the secondary antibodies alone. Results shown are representative of five experiments. K41, wild type cells; K42, calreticulin-deficient cells; BK, bradykinin receptor.
Mentions: Next, we compared expression of the bradykinin receptor and its targeting to the plasma membrane in wild-type (K41) and calreticulin-deficient (K42) cells. Fig. 7 A shows that calreticulin-deficient cells had decreased level of bradykinin receptor protein. Quantitative analysis of Western blots indicated that there was ∼50% less bradykinin receptor in calreticulin-deficient cells compared with K41 wild-type cells. These results indicate that calreticulin deficiency had some effect on expression and/or turnover of the bradykinin receptor. Using confocal microscopy and antibodies against the bradykinin receptor, we found that the receptor was distributed in a “dotty” pattern across the cell surface in K41, K42, and K42CRT cells (Fig. 7 B). We noticed no difference between the cell lines in distribution of the receptor (Fig. 7 B). Cell surface localization of the receptor was further confirmed by flow cytometry assay using antibradykinin receptor antibodies. We observed no significant difference in the antibradykinin antibodies surface labeling of K41 and K42 cells (Fig. 7 C), indicating the similar number of immunoreactive receptor protein molecules was present on cell surface. We concluded that in calreticulin-deficient cells the bradykinin receptor was properly targeted to and localized in the plasma membrane.

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