Limits...
Differential modulation of SERCA2 isoforms by calreticulin.

John LM, Lechleiter JD, Camacho P - J. Cell Biol. (1998)

Bottom Line: We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences.Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding.In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

Show MeSH
ΔC inhibits Ca2+ oscillations when coexpressed with  SERCA2b, but not when coexpressed with SERCA2a. (a and b)  Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity  in a SERCA2b-overexpressing oocyte with the Ca2+ wave activity of a SERCA2b + ΔC-coexpressing oocyte. (c and d) Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity in a  SERCA2a-overexpressing oocyte with the Ca2+ wave activity of  a SERCA2a + ΔC-coexpressing oocyte. Individual confocal images (745 μm × 745 μm) of Ca2+ wave activity were taken at the  indicated times. The bottom trace in each panel represents the  change in fluorescence (ΔF/F) shown as a function of time for a  5 × 5 pixel area (white square in the first panel of each image).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132884&req=5

Figure 4: ΔC inhibits Ca2+ oscillations when coexpressed with SERCA2b, but not when coexpressed with SERCA2a. (a and b) Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity in a SERCA2b-overexpressing oocyte with the Ca2+ wave activity of a SERCA2b + ΔC-coexpressing oocyte. (c and d) Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity in a SERCA2a-overexpressing oocyte with the Ca2+ wave activity of a SERCA2a + ΔC-coexpressing oocyte. Individual confocal images (745 μm × 745 μm) of Ca2+ wave activity were taken at the indicated times. The bottom trace in each panel represents the change in fluorescence (ΔF/F) shown as a function of time for a 5 × 5 pixel area (white square in the first panel of each image).

Mentions: We have previously demonstrated that injecting IP3 in Xenopus laevis oocytes coexpressing calreticulin and SERCA2b results in a sustained release of Ca2+ without repetitive Ca2+ oscillations (Camacho and Lechleiter, 1995). This effect of calreticulin survives deletion of the high-capacity Ca2+- binding domain (ΔC mutant), and therefore it is not due to the high Ca2+ storage capacity of calreticulin in the ER stores. The inhibition of Ca2+ oscillations by calreticulin (or ΔC) overexpression is consistent with a modulation of SERCA activity to slow Ca2+ uptake (Camacho and Lechleiter, 1995). Because ΔC contains the proline-rich P-domain of calreticulin shared by the other members of this class of ER chaperones (Ohsako et al., 1994; Tjoelker et al., 1994; Watanabe et al., 1994), and because lectin activity resides in the P-domain (Krause and Michalak, 1997), we tested whether ΔC modulation of SERCA2b was responsible for the decreased rate of Ca2+ transport characteristic of this pump (Lytton et al., 1992). Furthermore, since SERCA2a does not have a glycosylation motif facing the lumen of the ER, we also decided to test whether the functional differences between the two isoforms are due to a lack of interaction of calreticulin with SERCA2a. To this end, we coexpressed ΔC with SERCA2b or with SERCA2a (Fig. 4). A high percentage of SERCA2b-overexpressing oocytes exhibited robust high-frequency IP3-mediated Ca2+ oscillations (94%; n = 32; Fig. 4 a). The number of oocytes showing repetitive Ca2+ activity was significantly reduced when SERCA2b was coexpressed with ΔC (66%, n = 32, P < 0.01). In the remaining oocytes (34%), injections of IP3 caused sustained release of Ca2+ without repetitive Ca2+ waves (Fig. 4 b). Detailed analysis of Ca2+ waves revealed that even in those SERCA2b + ΔC-coexpressing oocytes that had repetitive Ca2+ waves, the interwave periods were significantly longer (P < 0.005), and wave widths were significantly broader (P < 0.005) than those oocytes overexpressing SERCA2b alone (Fig. 5 a). In the majority of SERCA2a-overexpressing oocytes (95%, n = 20), injections of IP3 caused high-frequency Ca2+ oscillations such as those observed in the oocyte shown in Fig. 4 a. In contrast to the inhibition of Ca2+ oscillations seen in oocytes coexpressing SERCA2b + ΔC, all SERCA2a + ΔC–overexpressing oocytes tested (100%, n=20) exhibited high-frequency Ca2+ oscillations (Fig. 4 d). Detailed analysis revealed that there were no statistically significant differences in Ca2+ wave properties between SERCA2a and SERCA2a + ΔC– overexpressing oocytes (Fig. 5 a). Western blot analysis revealed that the ΔC mutant was coexpressed with either SERCA2a or SERCA2b in this set of experiments, suggesting that the lack of modulation of SERCA2a by ΔC cannot be attributed to a lack of ΔC expression (Fig. 5 b). Correct targeting of ΔC to the ER was confirmed by confocal immunofluorescence performed on oocyte slices probed with an anti-calreticulin Ab as previously described (Camacho and Lechleiter, 1995; data not shown). Together these results indicate that the differential Ca2+ transport by the SERCA2 isoforms must be due to the presence of the luminal-facing COOH terminus extension of SERCA2b, conferring susceptibility of SERCA2b to modulation by calreticulin.


Differential modulation of SERCA2 isoforms by calreticulin.

John LM, Lechleiter JD, Camacho P - J. Cell Biol. (1998)

ΔC inhibits Ca2+ oscillations when coexpressed with  SERCA2b, but not when coexpressed with SERCA2a. (a and b)  Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity  in a SERCA2b-overexpressing oocyte with the Ca2+ wave activity of a SERCA2b + ΔC-coexpressing oocyte. (c and d) Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity in a  SERCA2a-overexpressing oocyte with the Ca2+ wave activity of  a SERCA2a + ΔC-coexpressing oocyte. Individual confocal images (745 μm × 745 μm) of Ca2+ wave activity were taken at the  indicated times. The bottom trace in each panel represents the  change in fluorescence (ΔF/F) shown as a function of time for a  5 × 5 pixel area (white square in the first panel of each image).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132884&req=5

Figure 4: ΔC inhibits Ca2+ oscillations when coexpressed with SERCA2b, but not when coexpressed with SERCA2a. (a and b) Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity in a SERCA2b-overexpressing oocyte with the Ca2+ wave activity of a SERCA2b + ΔC-coexpressing oocyte. (c and d) Comparison of the IP3 (∼300 nM)-induced Ca2+ wave activity in a SERCA2a-overexpressing oocyte with the Ca2+ wave activity of a SERCA2a + ΔC-coexpressing oocyte. Individual confocal images (745 μm × 745 μm) of Ca2+ wave activity were taken at the indicated times. The bottom trace in each panel represents the change in fluorescence (ΔF/F) shown as a function of time for a 5 × 5 pixel area (white square in the first panel of each image).
Mentions: We have previously demonstrated that injecting IP3 in Xenopus laevis oocytes coexpressing calreticulin and SERCA2b results in a sustained release of Ca2+ without repetitive Ca2+ oscillations (Camacho and Lechleiter, 1995). This effect of calreticulin survives deletion of the high-capacity Ca2+- binding domain (ΔC mutant), and therefore it is not due to the high Ca2+ storage capacity of calreticulin in the ER stores. The inhibition of Ca2+ oscillations by calreticulin (or ΔC) overexpression is consistent with a modulation of SERCA activity to slow Ca2+ uptake (Camacho and Lechleiter, 1995). Because ΔC contains the proline-rich P-domain of calreticulin shared by the other members of this class of ER chaperones (Ohsako et al., 1994; Tjoelker et al., 1994; Watanabe et al., 1994), and because lectin activity resides in the P-domain (Krause and Michalak, 1997), we tested whether ΔC modulation of SERCA2b was responsible for the decreased rate of Ca2+ transport characteristic of this pump (Lytton et al., 1992). Furthermore, since SERCA2a does not have a glycosylation motif facing the lumen of the ER, we also decided to test whether the functional differences between the two isoforms are due to a lack of interaction of calreticulin with SERCA2a. To this end, we coexpressed ΔC with SERCA2b or with SERCA2a (Fig. 4). A high percentage of SERCA2b-overexpressing oocytes exhibited robust high-frequency IP3-mediated Ca2+ oscillations (94%; n = 32; Fig. 4 a). The number of oocytes showing repetitive Ca2+ activity was significantly reduced when SERCA2b was coexpressed with ΔC (66%, n = 32, P < 0.01). In the remaining oocytes (34%), injections of IP3 caused sustained release of Ca2+ without repetitive Ca2+ waves (Fig. 4 b). Detailed analysis of Ca2+ waves revealed that even in those SERCA2b + ΔC-coexpressing oocytes that had repetitive Ca2+ waves, the interwave periods were significantly longer (P < 0.005), and wave widths were significantly broader (P < 0.005) than those oocytes overexpressing SERCA2b alone (Fig. 5 a). In the majority of SERCA2a-overexpressing oocytes (95%, n = 20), injections of IP3 caused high-frequency Ca2+ oscillations such as those observed in the oocyte shown in Fig. 4 a. In contrast to the inhibition of Ca2+ oscillations seen in oocytes coexpressing SERCA2b + ΔC, all SERCA2a + ΔC–overexpressing oocytes tested (100%, n=20) exhibited high-frequency Ca2+ oscillations (Fig. 4 d). Detailed analysis revealed that there were no statistically significant differences in Ca2+ wave properties between SERCA2a and SERCA2a + ΔC– overexpressing oocytes (Fig. 5 a). Western blot analysis revealed that the ΔC mutant was coexpressed with either SERCA2a or SERCA2b in this set of experiments, suggesting that the lack of modulation of SERCA2a by ΔC cannot be attributed to a lack of ΔC expression (Fig. 5 b). Correct targeting of ΔC to the ER was confirmed by confocal immunofluorescence performed on oocyte slices probed with an anti-calreticulin Ab as previously described (Camacho and Lechleiter, 1995; data not shown). Together these results indicate that the differential Ca2+ transport by the SERCA2 isoforms must be due to the presence of the luminal-facing COOH terminus extension of SERCA2b, conferring susceptibility of SERCA2b to modulation by calreticulin.

Bottom Line: We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences.Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding.In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

Show MeSH