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Research Article
Nader Rahimi
Boston University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6745-1725
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The cell adhesion molecule immunoglobulin and proline-rich receptor-1 (IGPR-1) regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy. However, the molecular mechanisms governing IGPR-1 cell surface expression levels remains unknown. In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Similarly, knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Likewise, deletion of 57 amino acids encompassing polyproline rich (PPR) motifs on the C-terminus of IGPR-1 nullified the binding of NEDD4 with IGPR-1. Furthermore, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulated lysosomal-dependent degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine and ammonium chloride increased IGPR-1 levels in the HEK-293 cells ectopically expressing IGPR-1 and in multiple human skin melanoma cell lines. Hence, these findings suggest that ubiquitin E3 ligase NEDD4 is a key regulator of IGPR-1 with a significant implication in the therapeutic targeting of IGPR-1.
Keywords
NEDD4, IGPR-1, TMIGD2, CD28H, UbcH6, ubiquitination, lysosomal degradation
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This is a preprint. It has not completed peer review.
Research Article
Nader Rahimi
Boston University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6745-1725
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 22 Feb, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The cell adhesion molecule immunoglobulin and proline-rich receptor-1 (IGPR-1) regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy. However, the molecular mechanisms governing IGPR-1 cell surface expression levels remains unknown. In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Similarly, knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Likewise, deletion of 57 amino acids encompassing polyproline rich (PPR) motifs on the C-terminus of IGPR-1 nullified the binding of NEDD4 with IGPR-1. Furthermore, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulated lysosomal-dependent degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine and ammonium chloride increased IGPR-1 levels in the HEK-293 cells ectopically expressing IGPR-1 and in multiple human skin melanoma cell lines. Hence, these findings suggest that ubiquitin E3 ligase NEDD4 is a key regulator of IGPR-1 with a significant implication in the therapeutic targeting of IGPR-1.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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