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Differential modulation of SERCA2 isoforms by calreticulin.

Authors: John LM, Lechleiter JD, Camacho P
Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA
J Cell Biol 1998;142:963-73

Presenter: A.K.Grover

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Presenter's Summary:

Calcium waves:
Release of Ca2+-sequestered in the endoplasmic reticulum (ER) by IP3 may initiate an intracellular Ca2+-wave. Unique Ca2+-wave patterns may represent complex intracellular signals? Neurons exhibit complex Ca2+-waves and their interactions can generate even more complex patterns which have the potential to explain the operational basis of neural networks. Determining how Ca2+-wave patterns are generated and how they get translated into neural networks is a challenge that is likely to form a central challenge in neuroscience for the next decade.

SERCA pumps:

Ca2+-Mg2+-ATPases termed SERCA pumps sequester Ca2+ into the sarco/endoplasmic reticulum. An appropriate stimulus, such as IP3, cADP ribose or Ca2+, can then release the sequestered Ca2+. There are several SERCA protein isoforms encoded by three genes: SERCA1, 2 and 3. SERCA2 gene is expressed most ubiquitously and its splices encode two proteins SERCA2a and SERCA2b. Cardiac and slow twitch skeletal muscles express SERCA2a in great abundance. Most tissues express SERCA2b but at much lower levels. SERCA2a and b proteins have the same 999 N-terminal residues but differ in the C-terminus. SERCA2a has an additional 4 amino acid sequence and SERCA2b has a different 46 amino acids. SERCA2b may have a slightly higher Ca2+ affinity than SERCA2a.

What is the importance of Ca2+-affinity of SERCA pumps versus their proximity to IP3 receptors in initiating and propagating Ca2+-waves?

Previous related work of the authors:

The authors use Xenopus oocyte model to study Ca2+ waves. Activating IP3-receptors in the oocytes releases Ca2+ from intracellular stores in very low amplitude propagating waves. Expressing SERCA1 mRNA in the oocytes increases the amplitude and the frequency of the IP3 induced Ca2+ waves and narrows the width of individual Ca2+ waves showing that accumulation of Ca2+ into ER can alter the Ca2+ waveforms generated.

Postulate:

Patterns of Ca2+-waves depend on whether the Ca2+-pump isoform SERCA2a isoform is expressed or SERCA2b because SERCA2b protein may be chaperoned to unique sites within the ER.
Evidence provided in this paper:
1. Injecting IP3 into oocytes expressing SERCA isoforms SERCA2a or SERCA2b generates high amplitude Ca2+ waves.
2. Ca2+ waves in oocytes expressing SERCA2a have shorter decay times and higher frequencies than in those with SERCA2b.
3. Co-expressing the ER Ca2+-binding protein calreticulin with SERCA2b results in fewer oocytes showing Ca2+ waves.
4. Co-expressing calreticulin does not affect the waves generated in cells expressing SERCA2a.
5. The effect of calreticulin is not related to its large Ca2+-buffering capacity. Calreticulin has two types of Ca2+ binding sites - low affinity (high micromolar) large number of sites (20-50 per calreticulin molecule) and 1 high affinity site per calreticulin. Co-expressing a deletion mutant missing the large number of the low affinity binding sites Ca2+- binding capacity produces the same effect as the native calreticulin.
6. Fusion proteins of SERCA2a and b into the C-terminus of the green fluorescence protein. are still functional in generating the expected waveforms. When examined by confocal microscopy, the distribution of SERCA2a and SERCA2b-green fluorescence fusion proteins does not differ from each other. Calreticulin does not alter this distribution. This result is confirmed by immunocytochemistry.
7. SERCA2b contains a potential glycosylation site (N1036) near its C-terminus which is missing in SERCA2a. Glucosidase inhibition prevents the effect of calreticulin on SERCA2b arguing that glycosylation may be involved in the SERCA2b-calreticulin interaction.
8. Expressing a SERCA2b mutant with A replacing N at 1036 gives waveforms similar to those of SERCA2a and no effects of calreticulin. There is no difference in targeting of this mutant as observed using confocal microscopy.

Conclusions:

Different isoforms of SERCA may be localized to different sites within the ER, thereby being able to generate unique Ca2+ waves and that calreticulin may chaperone SERCA2b to form a unique macromolecular complex.

Why is this paper important?
Ca2+-waves have the potential to explain complex intracellular Ca2+ signaling and hence it is important to understand what properties of Ca2+-channels and pumps determine the amplitudes and patterns of the Ca2+-waves. This paper shows that the isoform of the Ca2+-pump is an important factor.
Once one identifies the Ca2+-pump activity as a factor in generating the Ca2+-wave patterns, one can begin to ask questions on the effects of regulation of the pump which can change affinity or maximum activity of the Ca2+- pump.
The authors show that even if one sees identical reticular like distributions of pumps and channels by confocal microscopy, there may be differences between them.
Calcium binding protein calreticulin which is present in the ER/SR of many tissues can play a chaperoning role. Ability to chaperone SERCA2b to form a unique complex or a lack of it would add another dimension to the diversity of wave patterns.
The C-terminal domain of the SERCA3b isoform also has a similar potential glycosylation site as SERCA2b but there are no reports on its role.
There may well be other chaperones involved in the formation of such complexes?
Could PDZ domain containing proteins also play similar roles to modulate Ca2+-wave patterns a cell can generate?
What is the role of distribution of channels vs. pumps in generating different wave patterns?

Authors' 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 sarco/endoplasmic 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.