scholarly journals The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3

2004 ◽  
Vol 377 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Tomohiro NAKAYAMA ◽  
Mitsuharu HATTORI ◽  
Keiko UCHIDA ◽  
Takeshi NAKAMURA ◽  
Yoko TATEISHI ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Physiological Significance ◽  
Regulatory Domain ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
In Cells

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is an intracellular Ca2+ channel protein that plays crucial roles in generating complex Ca2+ signalling patterns. IP3R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP3R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP3R1 or IP3R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells’ responsiveness to an IP3-generating agonist ATP and Ca2+ leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP–IP3R1-casp) or the channel domain alone (GFP–IP3R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca2+ concentration. Interestingly, store-operated (−like) Ca2+ entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca2+ stores of cells expressing GFP–IP3R1-casp or GFP–IP3R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca2+. We therefore propose that the channel domain of IP3R1 tends to remain open and that the large regulatory domain of IP3R1 is necessary to keep the channel domain closed. Thus cleavage of IP3R1 by caspase 3 may contribute to the increased cytosolic Ca2+ concentration often observed in cells undergoing apoptosis. Finally, GFP–IP3R1-casp or GFP–IP3R1-ES can be used as a novel tool to deplete intracellular Ca2+ stores.

The complex regulatory function of the ligand-binding domain of the inositol 1,4,5-trisphosphate receptor

Cell Calcium ◽  
2008 ◽  
Vol 43 (1) ◽  
pp. 17-27 ◽  
Author(s):  
Benoit Devogelaere ◽  
Leen Verbert ◽  
Jan B. Parys ◽  
Ludwig Missiaen ◽  
Humbert De Smedt
Keyword(s):  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Regulatory Function ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

scholarly journals Structure/function analysis of the integrin beta 1 subunit by epitope mapping

1993 ◽  
Vol 122 (6) ◽  
pp. 1361-1371 ◽  
Author(s):  
DT Shih ◽  
JM Edelman ◽  
AF Horwitz ◽  
GB Grunwald ◽  
CA Buck
Keyword(s):  
Ligand Binding ◽  
Epitope Mapping ◽  
Transmembrane Domain ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Alpha Subunit ◽  
Cell Surface Expression ◽  
Regulatory Domain ◽  
Ligand Specificity ◽  
Amino Terminal

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti-chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.

Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination

ChemBioChem ◽  
2016 ◽  
Vol 17 (16) ◽  
pp. 1509-1512 ◽  
Author(s):  
Tai Oura ◽  
Kaori Murata ◽  
Takao Morita ◽  
Akihiro Nezu ◽  
Mitsuhiro Arisawa ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Domain Combination ◽  
Highly Sensitive ◽  
Inositol 1,4,5 Trisphosphate ◽  
Fluorescent Ligand

scholarly journals Inactivation of the Human Vitamin D Receptor by Caspase-3

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 679-686 ◽  
Author(s):  
Peter J. Malloy ◽  
David Feldman
Keyword(s):  
Vitamin D ◽  
Ligand Binding ◽  
Cancer Cell ◽  
Vitamin D Receptor ◽  
Cleavage Site ◽  
Caspase 3 ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Lncap Cells

Calcitriol actions are mediated by the vitamin D receptor (VDR), a nuclear transcription factor of the steroid-retinoid-thyroid nuclear receptor gene superfamily. Calcitriol inhibits the growth of many cells including cancer cells by inducing cell cycle arrest. In some cancer cell lines, calcitriol also induces apoptosis. In the LNCaP prostate cancer cell line, induction of apoptosis and caspase-3/7 activities by staurosporine (STS) abolished [3H]1,25-dihydroxy vitamin D3 binding and VDR protein, suggesting that the VDR may be targeted for inactivation by caspases during apoptosis. A potential caspase-3 site (D195MMD198S) was identified in the human VDR ligand-binding domain. Mutations D195A, D198A, and S199A were generated in the putative capase-3 cleavage site. In transfected COS-7 cells, STS treatment resulted in the cleavage of the wild-type (WT) VDR and S199A mutant VDR but not the D195A or D198A mutants. In in vitro assays, the WT VDR and S199A mutant VDR were cleaved by caspase-3, although the D195A and D198A mutants were resistant to caspase-3. In vitro, the WT VDR was also cleaved by caspase-6 and caspase-7 and in extracts of STS-treated LNCaP cells. In STS-treated LNCaP cells and human skin fibroblasts, the proteasome inhibitor MG-132 protected the VDR caspase cleavage fragment from further degradation by the 26S proteasome. The rat VDR that does not contain the caspase-3 cleavage site was not cleaved in STS-treated COS-7 cells. In conclusion, our results demonstrate that the human VDR is a target of caspase-3 and suggest that activation of caspase-3 may limit VDR activity. The vitamin D receptor contains a caspase-3 cleavage site in the ligand-binding domain that can be cleaved by caspase-3 in vitro and in intact cells.

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