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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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SERCA2 and InsP3R expression in calreticulin-deficient cells. (A) Wild-type (K41) and calreticulin-deficient (K42) mouse embryonic fibroblasts were lysed, and an increasing amount of protein (from 4 to 30 μg) was separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-SERCA2. (B) Microsomal vesicles were isolated from wild-type and calreticulin-deficient cells followed by protein separation in SDS-PAGE. InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) proteins were identified by Western blot analysis. Lane 1, K41 cells; lane 2, K42 cells; lane 3, RBL-2H3 control cells. The lower molecular mass protein band in the InsP3R2 panel is an immunoreactive protein not related to InsP3R2, and it was not included in quantitative analysis. 50 μg protein/lane was loaded except for the InsP3R2 samples (100 μg protein/lane). The position of InsP3R is indicated by the arrow.
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fig6: SERCA2 and InsP3R expression in calreticulin-deficient cells. (A) Wild-type (K41) and calreticulin-deficient (K42) mouse embryonic fibroblasts were lysed, and an increasing amount of protein (from 4 to 30 μg) was separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-SERCA2. (B) Microsomal vesicles were isolated from wild-type and calreticulin-deficient cells followed by protein separation in SDS-PAGE. InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) proteins were identified by Western blot analysis. Lane 1, K41 cells; lane 2, K42 cells; lane 3, RBL-2H3 control cells. The lower molecular mass protein band in the InsP3R2 panel is an immunoreactive protein not related to InsP3R2, and it was not included in quantitative analysis. 50 μg protein/lane was loaded except for the InsP3R2 samples (100 μg protein/lane). The position of InsP3R is indicated by the arrow.

Mentions: There are several potential explanations for the observation that bradykinin-induced Ca2+ release is impaired in calreticulin-deficient cells. For example, in crt−/− (K42) cells there could be changes in the expression and/or function of Ca2+ transport proteins in the ER or in the bradykinin receptor in the plasma membrane. To investigate these possibilities, we first compared expression of SERCA2 and the InsP3R in wild-type and calreticulin-deficient cells. Fig. 6 A shows that the expression of SERCA2 was not altered in calreticulin-deficient cells. Further, the level of mRNA for SERCA was the same in wild-type (K41) and calreticulin-deficient (K42) cells (unpublished data). We also used Western blot analysis (Fig. 6 B) and reverse transcriptase PCR (Table I) to compare expression of the three isoforms of the InsP3R (type 1, 2, and 3) in K41 and K42 cells. All three isoforms of the InsP3R were expressed in wild-type and crt−/− cells (Fig. 6 B and Table I). However, we found a 30–40% reduction in mRNA for the InsP3R in calreticulin-deficient cells compared with the wild-type cells (Table I). Western blot analysis of types 1, 2, and 3 of the InsP3R revealed that these cells contain all three types of InsP3R at a ratio of 10:70:20 (Fig. 6). A significant decrease of ∼20% was found in the calreticulin-deficient K42 cells for InsP3R type 1 (3.1 ± 0.4 versus 2.6 ± 0.4; n = 4; P < 0.01) and for InsP3R type 3 (9.4 ± 0.4 versus 7.5 ± 0.6; n = 4; P < 0.01). The relative level of InsP3R type 2 was approximately equal in both cell types (0.2 ± 0.1; n = 4).


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)

SERCA2 and InsP3R expression in calreticulin-deficient cells. (A) Wild-type (K41) and calreticulin-deficient (K42) mouse embryonic fibroblasts were lysed, and an increasing amount of protein (from 4 to 30 μg) was separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-SERCA2. (B) Microsomal vesicles were isolated from wild-type and calreticulin-deficient cells followed by protein separation in SDS-PAGE. InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) proteins were identified by Western blot analysis. Lane 1, K41 cells; lane 2, K42 cells; lane 3, RBL-2H3 control cells. The lower molecular mass protein band in the InsP3R2 panel is an immunoreactive protein not related to InsP3R2, and it was not included in quantitative analysis. 50 μg protein/lane was loaded except for the InsP3R2 samples (100 μg protein/lane). The position of InsP3R is indicated by the arrow.
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Related In: Results  -  Collection

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fig6: SERCA2 and InsP3R expression in calreticulin-deficient cells. (A) Wild-type (K41) and calreticulin-deficient (K42) mouse embryonic fibroblasts were lysed, and an increasing amount of protein (from 4 to 30 μg) was separated in SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-SERCA2. (B) Microsomal vesicles were isolated from wild-type and calreticulin-deficient cells followed by protein separation in SDS-PAGE. InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) proteins were identified by Western blot analysis. Lane 1, K41 cells; lane 2, K42 cells; lane 3, RBL-2H3 control cells. The lower molecular mass protein band in the InsP3R2 panel is an immunoreactive protein not related to InsP3R2, and it was not included in quantitative analysis. 50 μg protein/lane was loaded except for the InsP3R2 samples (100 μg protein/lane). The position of InsP3R is indicated by the arrow.
Mentions: There are several potential explanations for the observation that bradykinin-induced Ca2+ release is impaired in calreticulin-deficient cells. For example, in crt−/− (K42) cells there could be changes in the expression and/or function of Ca2+ transport proteins in the ER or in the bradykinin receptor in the plasma membrane. To investigate these possibilities, we first compared expression of SERCA2 and the InsP3R in wild-type and calreticulin-deficient cells. Fig. 6 A shows that the expression of SERCA2 was not altered in calreticulin-deficient cells. Further, the level of mRNA for SERCA was the same in wild-type (K41) and calreticulin-deficient (K42) cells (unpublished data). We also used Western blot analysis (Fig. 6 B) and reverse transcriptase PCR (Table I) to compare expression of the three isoforms of the InsP3R (type 1, 2, and 3) in K41 and K42 cells. All three isoforms of the InsP3R were expressed in wild-type and crt−/− cells (Fig. 6 B and Table I). However, we found a 30–40% reduction in mRNA for the InsP3R in calreticulin-deficient cells compared with the wild-type cells (Table I). Western blot analysis of types 1, 2, and 3 of the InsP3R revealed that these cells contain all three types of InsP3R at a ratio of 10:70:20 (Fig. 6). A significant decrease of ∼20% was found in the calreticulin-deficient K42 cells for InsP3R type 1 (3.1 ± 0.4 versus 2.6 ± 0.4; n = 4; P < 0.01) and for InsP3R type 3 (9.4 ± 0.4 versus 7.5 ± 0.6; n = 4; P < 0.01). The relative level of InsP3R type 2 was approximately equal in both cell types (0.2 ± 0.1; n = 4).

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