Cellular Expression, Trafficking, and Function of Two Isoforms of Human ULBP5/RAET1G

Abstract

BackgroundThe activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised.Methodology/Principal FindingsWe pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy.Conclusions/SignificanceWe demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

Secretory pathways generating immunosuppressive NKG2D ligands: New targets for therapeutic intervention

“…However, the mechanisms related to the shedding of ULBP1 remain unknown. Alternative splicing of ULBP4 and ULBP5 produce soluble forms of these ligands, but these molecules have not been detected in primary tumors 42 , 43 …”

ULBP6/RAET1L is an additional human NKG2D ligand

“…For example, NKG2D ligand expression was shown on activated T cells in both mouse and human, possibly representing a mechanism by which NK cells restrict excessive T-cell immune responses [25][26][27]. NKG2D ligand expression has also been reported in cell layers that interface with the environment, such as the gut mucosal epithelium [28,29], airway epithelial cells [30], and skin [12].Although all bind the same receptor, NKG2D ligands are very variable in their amino acid sequence, domain structure, and expression profile. This suggests that they may not be functionally equivalent.…”

Down‐regulation of HLA Class I and NKG2D ligands through a concerted action of MAPK and DNA methyltransferases in colorectal cancer cells

“…negative signals finally decides about the effector functions. The activating receptor NKG2D is expressed by virtually all NK cells, most CD8 + CTL, some CD4 + T cells, and recognizes 8 ligands, MICA, MICB, ULBP1‐5 and isoforms 21, 22. Although several mechanisms like infection, DNA damage and stress have been described for induction or up‐regulation of NKG2D ligands, only limited information is available about mechanisms that can lead to down‐regulation in malignant cells and the possible consequences for recognition and elimination of tumor cells by T and NK cells.…”