Cargando…
Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects
The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which thre...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602980/ https://www.ncbi.nlm.nih.gov/pubmed/33080786 http://dx.doi.org/10.3390/biom10101452 |
_version_ | 1783603813551177728 |
---|---|
author | Zheng, Li Chenavas, Sylvie Kieken, Fabien Trease, Andrew Brownell, Sarah Anbanandam, Asokan Sorgen, Paul L. Spagnol, Gaelle |
author_facet | Zheng, Li Chenavas, Sylvie Kieken, Fabien Trease, Andrew Brownell, Sarah Anbanandam, Asokan Sorgen, Paul L. Spagnol, Gaelle |
author_sort | Zheng, Li |
collection | PubMed |
description | The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects. |
format | Online Article Text |
id | pubmed-7602980 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-76029802020-11-01 Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects Zheng, Li Chenavas, Sylvie Kieken, Fabien Trease, Andrew Brownell, Sarah Anbanandam, Asokan Sorgen, Paul L. Spagnol, Gaelle Biomolecules Article The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects. MDPI 2020-10-17 /pmc/articles/PMC7602980/ /pubmed/33080786 http://dx.doi.org/10.3390/biom10101452 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Zheng, Li Chenavas, Sylvie Kieken, Fabien Trease, Andrew Brownell, Sarah Anbanandam, Asokan Sorgen, Paul L. Spagnol, Gaelle Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects |
title | Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects |
title_full | Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects |
title_fullStr | Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects |
title_full_unstemmed | Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects |
title_short | Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects |
title_sort | calmodulin directly interacts with the cx43 carboxyl-terminus and cytoplasmic loop containing three oddd-linked mutants (m147t, r148q, and t154a) that retain α-helical structure, but exhibit loss-of-function and cellular trafficking defects |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602980/ https://www.ncbi.nlm.nih.gov/pubmed/33080786 http://dx.doi.org/10.3390/biom10101452 |
work_keys_str_mv | AT zhengli calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects AT chenavassylvie calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects AT kiekenfabien calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects AT treaseandrew calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects AT brownellsarah calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects AT anbanandamasokan calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects AT sorgenpaull calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects AT spagnolgaelle calmodulindirectlyinteractswiththecx43carboxylterminusandcytoplasmicloopcontainingthreeodddlinkedmutantsm147tr148qandt154athatretainahelicalstructurebutexhibitlossoffunctionandcellulartraffickingdefects |