ARTICLE Received 8 Mar 2016 | Accepted 28 Apr 2016 | Published 8 Jun 2016 DOI: 10.1038/ncomms11795 OPEN GPRC5A suppresses protein synthesis at the endoplasmic reticulum to prevent radiation-induced lung tumorigenesis Jian Wang1, Alton B. Farris2, Kaiming Xu1, Ping Wang1, Xiangming Zhang1, Duc M. Duong3, Hong Yi4, Hui-Kuo Shu1, Shi-Yong Sun5 & Ya Wang1 GPRC5A functions as a lung tumour suppressor to prevent spontaneous and environmentally induced lung carcinogenesis; however, the underlying mechanism remains unclear. Here we reveal that GPRC5A at the endoplasmic reticulum (ER) membrane suppresses synthesis of the secreted or membrane-bound proteins including a number of oncogenes, the most important one being Egfr. The ER-located GPRC5A disturbs the assembly of the eIF4F-mediated translation initiation complex on the mRNA cap through directly binding to the eIF4F complex with its two middle extracellular loops. Particularly, suppression of EGFR by GPRC5A contributes significantly to preventing ionizing radiation (IR)-induced lung tumorigenesis. Thus, GPRC5A deletion enhances IR-promoted EGFR expression through an increased translation rate, thereby significantly increasing lung tumour incidence in Gprc5a À / À mice. Our findings indicate that under-expressed GPRC5A during lung tumor- igenesis enhances any transcriptional stimulation through an active translational status, which can be used to control oncogene expression and potentially the resulting related disease. 1 Department of Radiation Oncology, Emory University School of Medicine and the Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, USA. 2 Department of Pathology, Emory University School of Medicine and the Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, USA. 3 Emory Integrated Proteomics Core and Biochemistry Department, Atlanta, Georgia 30322, USA. 4 Robert P. Apkaran Integrated Electron Microscope Core, Emory University, Atlanta, Georgia 30322, USA. 5 Department of Hemotology and Medical Oncology, Emory University School of Medicine and the Winship Cancer Institute, Atlanta, Georgia 30322, USA. Correspondence and requests for materials should be addressed to Y.W. (email: [email protected]). NATURE COMMUNICATIONS | 7:11795 | DOI: 10.1038/ncomms11795 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11795 ung tumours are the second most common tumours and the multiple levels in cells, we used a quantitative global proteomics leading cause of cancer death in both men and women approach by mTRAQ labelling (Fig. 1a) to identify the differen- Laround the world, and adenocarcinoma is one of the most tially expressed proteins between wild-type (Gprc5a þ / þ ) common lung tumours. On the basis of a published analysis of and Gprc5a À / À mouse lung bronchial epithelial (LBE) cells. human data, G-protein-coupled receptor family C group 5 type Interestingly, the quantitative analysis revealed a substantial A (GPRC5A) was significantly repressed in lung tumours, perturbation of the cellular proteome, showing a marked particularly in non-small-cell lung cancers (NSCLC)1. The distribution shift of quantified proteins relative to representative GPRC5A gene locus is 12p13 and loss of heterozygosity of normal distribution (Fig. 1b, top panel and Supplementary chromosome 12p was frequently found in NSCLC2,3. In addition, Data 1), suggesting a structural change in the protein expression B10% of Gprc5a knockout mice spontaneously developed lung profile caused by GPRC5A deletion. Since there is no evidence of adenocarcinoma and lung cancer patients showed a significantly a decrease in the protein expression level in the center of the lower level of GPRC5A (ref. 1), indicating that GPRC5A is a distribution curve (such as PCNA, XRCC5, SEC23a, XRCC1, lung tumour suppressor. However, the mechanism underlying actin and so on) by western blot analysis in Gprc5a À / À LBE cells how GPRC5A prevents lung tumorigenesis remains unclear. compared with equal cell numbers of wild-type LBE cells, we Investigation of GPRC5A-regulated gene expression will facilitate excluded the possibility of a global downregulation of proteins by a better understanding of the role GPRC5A plays in preventing GPRC5A deletion. Thereby, we believe that GPRC5A deletion lung tumorigenesis. might cause a dramatic increase in the expression level of a Epidermal growth factor receptor (EGFR) is a key oncogene in specific group of proteins, resulting in a reduction on the lung adenocarcinoma4. EGFR is a transmembrane protein located proportion of other proteins in total proteins. Although the in the cell surface membrane as well as in the nucleus5, which quantitative global proteomics data only includes a small portion involves transcriptional regulation6,7, DNA replication and DNA of cell membrane proteins due to limitations in the approach, repair8,9. Direct stimulation of EGFR by binding to a ligand, such some cell membrane proteins in Gprc5a À / À LBE cells, such as as EGF, to the receptor’s extracellular domain leads to EGFR, Tmem43, Cdh1, and Itgb4 and so on showed an extremely dimerization and subsequent autophosphorylation of two high expression, implying that GPRC5A might be involved in receptor molecules, thereby creating phosphotyrosine docking suppressing the cell membrane protein expression. Notably, sites that activate intracellular signalling cascades. It is well the previously released mRNA microarray data of wild-type and known, based on mine workers and atomic bomb survivors10,11, Gprc5a À / À LBE cells (GSE21309) did not show a distribution that ionizing radiation (IR) promotes lung tumorigenesis and curve shift (Fig. 1b, bottom panel) and the genes with significant abnormal EGFR is involved in radiation-stimulated lung protein level changes in the quantitative global proteomics cancers12; however, the whole picture needs to be elucidated. IR data did not show corresponding mRNA level changes can stimulate the EGFR transcription, whereas only a moderate (Supplementary Data 1). In addition, the changes in the change in the protein level is induced by IR13, suggesting a steady state of the mRNA levels versus corresponding proteins strict control of EGFR expression aside from transcriptional between wild-type and Gprc5a À / À LBE cells were plotted control. Previous studies have shown a significant increase in in Supplementary Fig. 1a. This analysis does not exhibit a EGFR expression when normal bronchial mucosa transforms correlation between changes in the steady state of the mRNA epithelial hyperplasia and cancer14,15, suggesting that increasing and protein levels in GPRC5A depleted versus control cells, EGFR expression may contribute to lung tumorigenesis in suggesting that GPRC5A depletion affects the proteome in the Gprc5a À / À mice. absence of the major effect on the steady state of the mRNA Recently, it was reported that hypoxia/HIF2 activation could levels. Altogether, these results suggest a post-transcriptional upregulate EGFR overexpression through increasing EGFR regulation of membrane proteins by GPRC5A. synthesis16, suggesting that the translation machinery plays an EGFR is one of the most significantly increased proteins by the important role in EGFR regulation. In mammals, mRNA- proteomic quantitative analysis in Gprc5a À / À LBE cells but independent translational regulation relies mainly on a direct without mRNA changes (Supplementary Data 1). The western modification of the translation initiation factors. The 43S pre- blot of mouse LBE cells verified a much higher EGFR protein initiation complex binds to the messenger RNA (mRNA), which level in the Gprc5a À / À LBE cells (Fig. 1c), and mRNA levels is thought to involve bridging interactions between eIF3 and the measured by semi-quantitative PCR with reverse transcription cap-binding eukaryotic initiation factor 4F (eIF4F) complex that (RT–PCR) or quantitative real-time PCR did not show a is associated with the 50-cap structure of the mRNA17. Alternated significant difference between wild-type and Gprc5a À / À LBE regulation of the eIF4F complex has been recently reported to cells (Fig. 1d). In addition, the deletion of GPRC5A did not cause play an essential role in carcinogenesis18,19. The eIF4F complex any changes in Egfr mRNA turnover rates (Supplementary contains several proteins: eIF4E (it physically binds to the Fig. 1b). Consistent with the data from LBE cells, a stronger m7GpppN cap structure), eIF4A (a dead-box RNA helicase to EGFR staining was observed in the lung tissue of Gprc5a À / À unwind secondary structures in the 50-UTR so that the 43S mice compared with that of their wild-type counterparts (Fig. 1e), complex can bind and scan the mRNA20) and eIF4G that where the GPRC5A expression was examined by immuno- functions as a scaffold protein by interacting with eIF4E, eIF4A histochemistry (IHC) and Laz staining1 (Supplementary Fig. 1c, d). and eIF3 (ref. 21). Also, the Egfr mRNA level did not show any difference between In this study, our data reveal a new regulation for EGFR by wild-type and Gprc5a À / À mouse lung tissue (Fig. 1f). Next, GPRC5A through translational suppression by directly binding complementary expression of GPRC5A in Gprc5a À / À mice LBE to the eIF4F complex. Deletion of Gprc5a significantly enhances cells markedly decreased the EGFR expression (Fig. 1g), but did IR-stimulated EGFR expression due to loss of translational not affect the Egfr mRNA level (Fig. 1h). Taken together, these suppression, thereby causing an increase in IR-induced