Post-translational Modification of the NKG2D Ligand RAET1G Leads to Cell Surface Expression of a Glycosylphosphatidylinositol-linked Isoform
- Creators
- Ohashi, Maki
- Eagle, Robert A.
- Trowsdale, John
Abstract
NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform.
Additional Information
© 2010 American Society for Biochemistry and Molecular Biology. Received October 23, 2009. Revision received February 19, 2010. First Published on March 19, 2010. This work was supported by a grant from Cancer Research United Kingdom. We thank Dr. N. Kanzawa and Prof. T. Kinoshita for cell lines, Prof. P. Lehner for plasmid, Dr. A. P. Kelly for helpful advice, and Dr. L. H. Boyle for help with the pulse-chase experiment.Attached Files
Published - Ohashi2010p10335Journal_of_Biological_Chemistry.pdf
Supplemental Material - jbc.M109.077636-1.pdf
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Additional details
- PMCID
- PMC2878053
- Eprint ID
- 18691
- Resolver ID
- CaltechAUTHORS:20100615-134555139
- Cancer Research United Kingdom
- Created
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2010-07-15Created from EPrint's datestamp field
- Updated
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2021-11-08Created from EPrint's last_modified field