RELT stains prominently in B-cell lymphomas and binds the hematopoietic transcription factor MDFIC

Abstract

Receptor Expressed in Lymphoid Tissues (RELT) is a human tumor necrosis factor receptor superfamily member (TNFRSF) that is expressed most prominently in cells and tissues of the hematopoietic system. RELL1 and RELL2 are two homologs that physically interact with RELT and co-localize with RELT at the plasma membrane. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members. The transcription factor MyoD family inhibitor domain-containing (MDFIC) was identified in a yeast two-hybrid genetic screen using RELL1 as bait. MDFIC co-localizes with RELT family members at the plasma membrane; this co-localization was most prominently observed with RELL1 and RELL2. In vitro co-immunoprecipitation (Co-IP) was utilized to demonstrate that MDFIC physically interacts with RELT, RELL1, and RELL2. Co-IP using deletion mutants of MDFIC and RELT identified regions important for physical association between MDFIC and RELT family members and a computational analysis revealed that RELT family members are highly disordered proteins. Immunohistochemistry of normal human lymph nodes revealed RELT staining that was most prominent in macrophages. Interestingly, the level of RELT staining significantly increased progressively in low and high-grade B-cell lymphomas versus normal lymph nodes. RELT co-staining with CD20 was observed in B-cell lymphomas, indicating that RELT is expressed in malignant B cells. Collectively, these results further our understanding of RELT-associated signaling pathways, the protein structure of RELT family members, and provide preliminary evidence indicating an association of RELT with B-cell lymphomas.

The RELT Family of Proteins: An Increasing Awareness of Their Importance for Cancer, the Immune System, and Development

“…Interestingly, the HPA predicts that RELT localizes to the nucleoplasm, based on information using an antibody raised against intracellular residues 204-275 as an epitope, suggesting that the ICD of RELT may be cleaved, resulting in the truncated carboxy terminus migrating to the nucleus. Expression of recombinant RELL1 and RELL2 individually was observed to localize at the plasma membrane in COS-7 [9] and 293 cells [23]. The HPA confirms that RELL1 is expressed at the plasma membrane, yet also indicates that it is associated with microtubules, whereas this database predicts that RELL2 localizes to intracellular vesicles.…”

“…All RELTfms bind each other as demonstrated by co-IP experiments, and co-expression of recombinant RELTfms resulted in co-localization of these proteins at the plasma membrane of COS-7 cells [9]. Although RELT co-localizes with either RELL1 or RELL2 at the plasma membrane, expression of recombinant RELT by itself resulted in predominant localization within cytosolic compartments in either COS-7 cells [9] and 293 cells [23,25]. Endogenous murine RELT (TNFRSF19L) was observed to be expressed at the plasma membrane in the human lung tumor cell line H226 [26].…”

“…The predicted transmembrane domain (TMD) spans from amino acids 156-191 and the HPA predicts at least two intracellular alpha helices. RELT is predicted to contain a highly disordered structure at its carboxy terminus, indicating the carboxy terminus may fluctuate and adopt different conformations dependent on either post-translational modifications or binding by other proteins [23]. Intracellular sequences of RELT proximal to the membrane are likely important for protein folding and stability, as deletion mutant constructs of RELT lacking amino acids 191-252 were very faintly detected by Western blotting in contrast to other deletion mutants, indicating that RELT constructs lacking amino acids 191-252 were likely degraded in the endoplasmic reticulum due to their inability to fold properly [21].…”

“…RELL1 and RELL2 are also Type I transmembrane proteins with short sequences in their ECD in comparison to other TNFRSF members, and neither RELL1 nor RELL2 contains extracellular Cys-rich domains used to bind TNFSF ligands (Figure 1). RELL1 encodes a 271 amino acid-long 29.3 kDa protein that appears to be subjected to significant post-translational modifications, as recombinant RELL1 migrates at a position larger than its predicted molecular weight, with multiple bands apparent [9,[23][24][25]. RELL2 encodes a 303 amino acid-long 32.4 kDa protein.…”

“…RELL2 encodes a 303 amino acid-long 32.4 kDa protein. Both RELL1 and RELL2 are predicted to contain disordered sequences in their carboxy-terminal tails, like RELT, suggesting that the carboxy-termini of RELTfms may adopt multiple conformations depending on either post-translational modifications or the binding of other proteins [23].…”