NEDD4 regulates ubiquitination and stability of the cell adhesion molecule IGPR-1 via lysosomal pathway

J Biomed Sci. 2021 May 7;28(1):35. doi: 10.1186/s12929-021-00731-9.


Background: The cell adhesion molecule IGPR-1 regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy and plays important roles in angiogenesis and tumor growth; however, the molecular mechanism governing the cell surface levels of IGPR-1 remains unknown.

Results: 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. Knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Deletion of 57 amino acids encompassing the polyproline rich (PPR) motifs on the C-terminus of IGPR-1 nullified its binding with NEDD4. Furthermore, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulates lysosomal-dependent degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine or ammonium chloride increased IGPR-1 levels ectopically expressed in HEK-293 cells and in multiple endogenously IGPR-1 expressing human skin melanoma cell lines.

Conclusions: NEDD4 ubiquitin E3 ligase binds to and mediates polyubiquitination of IGPR-1 leading to its lysosomal-dependent degradation. NEDD4 is a key regulator of IGPR-1 expression with implication in the therapeutic targeting of IGPR-1 in human cancers.

Keywords: CD28H; IGPR-1; K48-linked ubiquitination; K63-linked ubiquitination; Lysosomal degradation; Melanoma; NEDD4; TMIGD2; UbcH6; Ubiquitination.

MeSH terms

  • CD28 Antigens / chemistry*
  • Cell Membrane / metabolism*
  • HEK293 Cells
  • Humans
  • Lysosomes / metabolism*
  • Nedd4 Ubiquitin Protein Ligases / genetics*
  • Nedd4 Ubiquitin Protein Ligases / metabolism
  • Protein Stability
  • Ubiquitination


  • CD28 Antigens
  • TMIGD2 protein, human
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, human