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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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Overexpression of L4, NC, N, or P domains of CRT increase Ca2+ oscillation frequency. Representative confocal images of Ca2+ oscillations in oocytes as labeled: H2O (n = 46); L4 (n = 31); CRT (n = 22); CRT-NP (n = 28); CRT-NC (n = 28); CRT-N domain (n = 45); and CRT-P domain (n = 49). Two independent experiments with 11–25 oocytes per group were performed. Histogram plots t1/2 of individual waves normalized to the H2O control group. Asterisks indicate statistical significance (P < 0.05, t test) between L4, CRT, CRT-NP, CRT-NC, CRT-N, CRT-P, and the control group. Bar, 100 μm.
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fig8: Overexpression of L4, NC, N, or P domains of CRT increase Ca2+ oscillation frequency. Representative confocal images of Ca2+ oscillations in oocytes as labeled: H2O (n = 46); L4 (n = 31); CRT (n = 22); CRT-NP (n = 28); CRT-NC (n = 28); CRT-N domain (n = 45); and CRT-P domain (n = 49). Two independent experiments with 11–25 oocytes per group were performed. Histogram plots t1/2 of individual waves normalized to the H2O control group. Asterisks indicate statistical significance (P < 0.05, t test) between L4, CRT, CRT-NP, CRT-NC, CRT-N, CRT-P, and the control group. Bar, 100 μm.

Mentions: The globular domain of CRT comprises the N and C domains and interacts with substrate glycoproteins, whereas the tip of the P domain binds to ERp57 and promotes disulfide bond formation (Ellgaard et al., 2001, 2002; Schrag et al., 2001; Frickel et al., 2002; Leach et al., 2002). Previous observations from our group demonstrated that CRT-NP (i.e., CRT lacking the C domain) modulated SERCA 2b just like wild-type CRT, indicating that in this case the C domain might not be necessary for the interaction (Camacho and Lechleiter, 1995; John et al., 1998). Thus, CRT most likely targets the COOH-terminal sequence of SERCA 2b via its N domain, whereas the P domain recruits ERp57 to interact with the L4. To test this hypothesis, we overexpressed four dominant negative constructs (L4, CRT-NC, CRT-N, or CRT-P) to determine whether they would interrupt the interaction between endogenous SERCA 2b with the ERp57–CRT complex. We found that oocytes overexpressing these four constructs exhibited shorter t1/2 values (faster SERCA 2b activity) than control oocytes. In contrast, oocytes overexpressing CRT or CRT-NP exhibited longer t1/2 values, consistent with previous findings (Camacho and Lechleiter, 1995; John et al., 1998; Fig. 8).


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

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

Overexpression of L4, NC, N, or P domains of CRT increase Ca2+ oscillation frequency. Representative confocal images of Ca2+ oscillations in oocytes as labeled: H2O (n = 46); L4 (n = 31); CRT (n = 22); CRT-NP (n = 28); CRT-NC (n = 28); CRT-N domain (n = 45); and CRT-P domain (n = 49). Two independent experiments with 11–25 oocytes per group were performed. Histogram plots t1/2 of individual waves normalized to the H2O control group. Asterisks indicate statistical significance (P < 0.05, t test) between L4, CRT, CRT-NP, CRT-NC, CRT-N, CRT-P, and the control group. Bar, 100 μm.
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fig8: Overexpression of L4, NC, N, or P domains of CRT increase Ca2+ oscillation frequency. Representative confocal images of Ca2+ oscillations in oocytes as labeled: H2O (n = 46); L4 (n = 31); CRT (n = 22); CRT-NP (n = 28); CRT-NC (n = 28); CRT-N domain (n = 45); and CRT-P domain (n = 49). Two independent experiments with 11–25 oocytes per group were performed. Histogram plots t1/2 of individual waves normalized to the H2O control group. Asterisks indicate statistical significance (P < 0.05, t test) between L4, CRT, CRT-NP, CRT-NC, CRT-N, CRT-P, and the control group. Bar, 100 μm.
Mentions: The globular domain of CRT comprises the N and C domains and interacts with substrate glycoproteins, whereas the tip of the P domain binds to ERp57 and promotes disulfide bond formation (Ellgaard et al., 2001, 2002; Schrag et al., 2001; Frickel et al., 2002; Leach et al., 2002). Previous observations from our group demonstrated that CRT-NP (i.e., CRT lacking the C domain) modulated SERCA 2b just like wild-type CRT, indicating that in this case the C domain might not be necessary for the interaction (Camacho and Lechleiter, 1995; John et al., 1998). Thus, CRT most likely targets the COOH-terminal sequence of SERCA 2b via its N domain, whereas the P domain recruits ERp57 to interact with the L4. To test this hypothesis, we overexpressed four dominant negative constructs (L4, CRT-NC, CRT-N, or CRT-P) to determine whether they would interrupt the interaction between endogenous SERCA 2b with the ERp57–CRT complex. We found that oocytes overexpressing these four constructs exhibited shorter t1/2 values (faster SERCA 2b activity) than control oocytes. In contrast, oocytes overexpressing CRT or CRT-NP exhibited longer t1/2 values, consistent with previous findings (Camacho and Lechleiter, 1995; John et al., 1998; Fig. 8).

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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