Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins

2015 ◽  
Vol 465 (3) ◽  
pp. 489-501 ◽  
Author(s):  
Moshe Giladi ◽  
Su Youn Lee ◽  
Reuben Hiller ◽  
Ka Young Chung ◽  
Daniel Khananshvili
Keyword(s):  
Calcium Binding ◽  
Splice Variant ◽  
Main Chain ◽  
Splice Variants ◽  
Binding Domain ◽  
Dependent Manner ◽  
Regulatory Response ◽  
Structure Dynamic ◽  
Calcium Binding Domain

Ca2+ binding to CBD1 (calcium-binding domain 1) and CBD2 regulates Na+/Ca2+ exchangers (NCX). In the present study, we demonstrate that Ca2+ binding rigidifies the main chain of CBD2, but not of CBD1, in a splice variant-dependent manner. The dynamic differences account for variant-dependent responses to Ca2+.

scholarly journals FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus

2020 ◽  
Vol 22 (1) ◽  
pp. 111
Author(s):  
Oksana M. Subach ◽  
Natalia V. Barykina ◽  
Elizaveta S. Chefanova ◽  
Anna V. Vlaskina ◽  
Vladimir P. Sotskov ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Hela Cells ◽  
Calcium Binding ◽  
Dynamic Range ◽  
Spectral Characteristics ◽  
Binding Domain ◽  
Calcium Transients ◽  
Neuronal Cultures ◽  
Decay Kinetics ◽  
Calcium Binding Domain

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.

scholarly journals Mutagenesis of the fourth calcium-binding domain of yeast calmodulin

1993 ◽  
Vol 268 (18) ◽  
pp. 13267-13273
Author(s):  
I. Matsuura ◽  
E. Kimura ◽  
K. Tai ◽  
M. Yazawa
Keyword(s):  
Calcium Binding ◽  
Binding Domain ◽  
Calcium Binding Domain

A novel GSN variant outside the G2 calcium‐binding domain associated with Amyloidosis of the Finnish type

2021 ◽  
Author(s):  
Sean Mullany ◽  
Emmanuelle Souzeau ◽  
Sonja Klebe ◽  
Tiger Zhou ◽  
Lachlan S. W. Knight ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Domain ◽  
Finnish Type ◽  
Calcium Binding Domain

scholarly journals Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-35
Author(s):  
Sara L. Seegers ◽  
Amanda Lance ◽  
Lawrence J Druhan ◽  
Belinda R Avalos
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Splice Variant ◽  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Human Cells ◽  
Dependent Manner ◽  
Epitope Tag ◽  
Proliferative Signaling ◽  
Primary Human Cells

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

Calsyntenin-1, a Proteolytically Processed Postsynaptic Membrane Protein with a Cytoplasmic Calcium-Binding Domain

2001 ◽  
Vol 17 (1) ◽  
pp. 151-166 ◽  
Author(s):  
Lorenz Vogt ◽  
Sabine P. Schrimpf ◽  
Virginia Meskenaite ◽  
Renato Frischknecht ◽  
Jochen Kinter ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Calcium Binding ◽  
Postsynaptic Membrane ◽  
Binding Domain ◽  
Cytoplasmic Calcium ◽  
Calcium Binding Domain

scholarly journals An Alternative Splice Variant of HIPK2 with Intron Retention Contributes to Cytokinesis

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 484 ◽  
Author(s):  
Veronica Gatti ◽  
Manuela Ferrara ◽  
Ilaria Virdia ◽  
Silvia Matteoni ◽  
Laura Monteonofrio ◽  
...  
Keyword(s):  
Cell Division ◽  
Stress Response ◽  
Alternative Splice ◽  
Functional Activity ◽  
Splice Variant ◽  
Splice Variants ◽  
Stop Codon ◽  
Cellular Stress Response ◽  
Dependent Manner ◽  
Western Blots

HIPK2 is a DYRK-like kinase involved in cellular stress response pathways, development, and cell division. Two alternative splice variants of HIPK2, HIPK2-FL and HIPK2-Δe8, have been previously identified as having different protein stability but similar functional activity in the stress response. Here, we describe one additional HIPK2 splice variant with a distinct subcellular distribution and functional activity in cytokinesis. This novel splice variant lacks the last two exons and retains intron13 with a stop codon after 89 bp of the intron, generating a short isoform, HIPK2-S, that is detectable by 2D Western blots. RT-PCR analyses of tissue arrays and tumor samples show that HIPK2-FL and HIPK2-S are expressed in normal human tissues in a tissue-dependent manner and differentially expressed in human colorectal and pancreatic cancers. Gain- and loss-of-function experiments showed that in contrast to HIPK2-FL, HIPK2-S has a diffuse, non-speckled distribution and is not involved in the DNA damage response. Rather, we found that HIPK2-S, but not HIPK2-FL, localizes at the intercellular bridge, where it phosphorylates histone H2B and spastin, both required for faithful cell division. Altogether, these data show that distinct human HIPK2 splice variants are involved in distinct HIPK2-regulated functions like stress response and cytokinesis.

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