Eagle, Robert A. and Flack, Gillian and Warford, Anthony and Martínez-Borra, Jesús and Jafferji, Insiya and Traherne, James A. and Ohashi, Maki and Boyle, Louise H. and Barrow, Alexander D. and Caillat-Zucman, Sophie and Young, Neil T. and Trowsdale, John (2009) Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G. PLoS ONE, 4 (2). e4503. ISSN 1932-6203 http://resolver.caltech.edu/CaltechAUTHORS:20090529-104311699
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Background: The 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 Findings: We 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/Significance: We 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.
|Additional Information:||© 2009 Eagle et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received September 23, 2008; Accepted November 20, 2008; Published February 18, 2009. This work is funded by grants from Cancer Research UK and The Isaac Newton Trust to RAE, The Leukaemia Research Fund to NTY, and The Wellcome Trust to JT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Cherie Blenkiron, Carlos Caldas, and Paul Lehner for kind gifts of reagents. Thanks also to Aradhna Tripati for helpful comments and support. Please see supplementary file 1 for author contribution statement. Author Contributions: Conceived and designed the experiments: RE. Performed the experiments: RE GF AW JMB IJ JAT MO LHB ADB SCZ NY. Analyzed the data: RE AW SCZ NY. Wrote the paper: RE. Performed cDNA cloning: RAE JMB. Antibody testing by western blot and confocal microscopy: RAE. Produced cDNA for RT-PCR: RAE. Produced stable transfectants: RAE. Produced recombinant protein: RAE. Performed FACS and western blot experiments: RAE. Performed and analyzed tissue microarrays experiments: GF AW. Performed western blot of RAET1G2: JMB. Performed antibody internalization experiments: IJ. Assisted in primer design and RT-PCR experiments: JAT. Assisted in stable cell line production and maintenance: MO. Performed pulse chase experiments: LHB. ADB Assisted in the production and refolding of recombinant proteins: ADB. Performed and analyzed celiac disease tissue staining: SCZ. Performed soluble RAET1G co-culture experiments with NK cells, and carried out FACS analysis of NKG2D expression: NTY. Proofread the manuscript: JT. Competing interests: RAE and JT hold a patent on RAET1G.|
|Usage Policy:||This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.|
|Deposited By:||Jason Perez|
|Deposited On:||25 Aug 2009 23:36|
|Last Modified:||26 Dec 2012 11:02|
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