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Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

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Model depicting the functional consequences of the interaction between ERp57–CRT with SERCA 2b. At rest, the ER Ca2+ stores are full (high [Ca2+]ER), the N domain of CRT interacts with the COOH-terminal sequence of SERCA 2b, whereas the P domain recruits ERp57 to target L4 in SERCA 2b. At high [Ca2+]ER our data suggest that ERp57 promotes disulfide bond formation between thiol groups in the loop, reducing pump activity (i.e., decreasing the frequency of Ca2+ oscillations and t1/2). This activity of ERp57 favors disulfide bond formation because the prevailing conditions in the ER are relatively oxidizing. As [Ca2+]ER decreases below ∼50 μM, ERp57 dissociates from L4, allowing SERCA 2b in its reduced form (cysteine mutagenesis) to be more active. Thus, at low [Ca2+]ER SERCA 2b favors ER store refilling. The arrow indicates the point of divergence between SERCA 2a and SERCA 2b. NFS is the glycosylation consensus motif at the COOH-terminal sequence of SERCA 2b.
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fig10: Model depicting the functional consequences of the interaction between ERp57–CRT with SERCA 2b. At rest, the ER Ca2+ stores are full (high [Ca2+]ER), the N domain of CRT interacts with the COOH-terminal sequence of SERCA 2b, whereas the P domain recruits ERp57 to target L4 in SERCA 2b. At high [Ca2+]ER our data suggest that ERp57 promotes disulfide bond formation between thiol groups in the loop, reducing pump activity (i.e., decreasing the frequency of Ca2+ oscillations and t1/2). This activity of ERp57 favors disulfide bond formation because the prevailing conditions in the ER are relatively oxidizing. As [Ca2+]ER decreases below ∼50 μM, ERp57 dissociates from L4, allowing SERCA 2b in its reduced form (cysteine mutagenesis) to be more active. Thus, at low [Ca2+]ER SERCA 2b favors ER store refilling. The arrow indicates the point of divergence between SERCA 2a and SERCA 2b. NFS is the glycosylation consensus motif at the COOH-terminal sequence of SERCA 2b.

Mentions: To determine the specificity of these dominant negative constructs for SERCA 2b, we also coexpressed them with either SERCA 2a or SERCA 2b. Oocytes coexpressing L4, CRT-NC, CRT-N, or CRT-P with SERCA 2b exhibited significantly shorter t1/2 values than SERCA 2b overexpressing oocytes, consistent with our observations presented in the previous paragraph. Note that control oocytes coexpressing CRT with SERCA 2b exhibited the longest t1/2 values (lowest frequency of Ca2+ oscillations; Camacho and Lechleiter, 1995; Fig. 9, A and B). In agreement with the results reported in Fig. 7, we found that Ca2+ oscillations enhanced by SERCA 2a were not affected by coexpression with these constructs (Fig. 9 C). Together, these data support our model that CRT recruits ERp57 to the lumenal L4 in SERCA 2b, modulating pump activity to maintain ER Ca2+ homeostasis (Fig. 10).


Ca2+-dependent redox modulation of SERCA 2b by ERp57.

Li Y, Camacho P - J. Cell Biol. (2003)

Model depicting the functional consequences of the interaction between ERp57–CRT with SERCA 2b. At rest, the ER Ca2+ stores are full (high [Ca2+]ER), the N domain of CRT interacts with the COOH-terminal sequence of SERCA 2b, whereas the P domain recruits ERp57 to target L4 in SERCA 2b. At high [Ca2+]ER our data suggest that ERp57 promotes disulfide bond formation between thiol groups in the loop, reducing pump activity (i.e., decreasing the frequency of Ca2+ oscillations and t1/2). This activity of ERp57 favors disulfide bond formation because the prevailing conditions in the ER are relatively oxidizing. As [Ca2+]ER decreases below ∼50 μM, ERp57 dissociates from L4, allowing SERCA 2b in its reduced form (cysteine mutagenesis) to be more active. Thus, at low [Ca2+]ER SERCA 2b favors ER store refilling. The arrow indicates the point of divergence between SERCA 2a and SERCA 2b. NFS is the glycosylation consensus motif at the COOH-terminal sequence of SERCA 2b.
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fig10: Model depicting the functional consequences of the interaction between ERp57–CRT with SERCA 2b. At rest, the ER Ca2+ stores are full (high [Ca2+]ER), the N domain of CRT interacts with the COOH-terminal sequence of SERCA 2b, whereas the P domain recruits ERp57 to target L4 in SERCA 2b. At high [Ca2+]ER our data suggest that ERp57 promotes disulfide bond formation between thiol groups in the loop, reducing pump activity (i.e., decreasing the frequency of Ca2+ oscillations and t1/2). This activity of ERp57 favors disulfide bond formation because the prevailing conditions in the ER are relatively oxidizing. As [Ca2+]ER decreases below ∼50 μM, ERp57 dissociates from L4, allowing SERCA 2b in its reduced form (cysteine mutagenesis) to be more active. Thus, at low [Ca2+]ER SERCA 2b favors ER store refilling. The arrow indicates the point of divergence between SERCA 2a and SERCA 2b. NFS is the glycosylation consensus motif at the COOH-terminal sequence of SERCA 2b.
Mentions: To determine the specificity of these dominant negative constructs for SERCA 2b, we also coexpressed them with either SERCA 2a or SERCA 2b. Oocytes coexpressing L4, CRT-NC, CRT-N, or CRT-P with SERCA 2b exhibited significantly shorter t1/2 values than SERCA 2b overexpressing oocytes, consistent with our observations presented in the previous paragraph. Note that control oocytes coexpressing CRT with SERCA 2b exhibited the longest t1/2 values (lowest frequency of Ca2+ oscillations; Camacho and Lechleiter, 1995; Fig. 9, A and B). In agreement with the results reported in Fig. 7, we found that Ca2+ oscillations enhanced by SERCA 2a were not affected by coexpression with these constructs (Fig. 9 C). Together, these data support our model that CRT recruits ERp57 to the lumenal L4 in SERCA 2b, modulating pump activity to maintain ER Ca2+ homeostasis (Fig. 10).

Bottom Line: Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins.Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site.Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology, MSC 7756, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.

ABSTRACT
We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.

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