The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling
A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3? as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3? protein dire...
| Main Authors: | , , , , , , |
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| Format: | Journal Article |
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2012
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| Online Access: | http://hdl.handle.net/20.500.11937/31034 |
| _version_ | 1848753262358953984 |
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| author | Arulpragasam, Ajanthy Magno, A. Ingley, E. Brown, S. Conigrave, A. Ratajczak, T. Ward, B. |
| author_facet | Arulpragasam, Ajanthy Magno, A. Ingley, E. Brown, S. Conigrave, A. Ratajczak, T. Ward, B. |
| author_sort | Arulpragasam, Ajanthy |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3? as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3? protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3? when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3? did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3? in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3? overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3? in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3?, exhibited similar behaviour to that of 14-3-3? with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3?, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR. © The Authors Journal compilation © 2012 Biochemical Society. |
| first_indexed | 2025-11-14T08:21:43Z |
| format | Journal Article |
| id | curtin-20.500.11937-31034 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T08:21:43Z |
| publishDate | 2012 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-310342017-09-13T15:14:33Z The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling Arulpragasam, Ajanthy Magno, A. Ingley, E. Brown, S. Conigrave, A. Ratajczak, T. Ward, B. A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3? as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3? protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3? when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3? did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3? in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3? overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3? in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3?, exhibited similar behaviour to that of 14-3-3? with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3?, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR. © The Authors Journal compilation © 2012 Biochemical Society. 2012 Journal Article http://hdl.handle.net/20.500.11937/31034 10.1042/BJ20111277 unknown |
| spellingShingle | Arulpragasam, Ajanthy Magno, A. Ingley, E. Brown, S. Conigrave, A. Ratajczak, T. Ward, B. The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling |
| title | The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling |
| title_full | The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling |
| title_fullStr | The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling |
| title_full_unstemmed | The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling |
| title_short | The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling |
| title_sort | adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated rho kinase signalling |
| url | http://hdl.handle.net/20.500.11937/31034 |