EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer
- Xiaofeng Lin†1, 2, 10,
- Shuangshuang Zhong†1, 2,
- Xiaofeng Ye†1, 2, 11,
- Yueling Liao1, 2,
- Feng Yao3,
- Xiaohua Yang4,
- Beibei Sun4,
- Jie Zhang5,
- Qi Li6,
- Yong Gao7,
- Yifan Wang8,
- Jingyi Liu8,
- Baohui Han4,
- Y Eugene Chin4, 9,
- Binhua P Zhou8Email author and
- Jiong Deng1, 2, 4Email author
© Lin et al.; licensee BioMed Central Ltd. 2014
Received: 23 January 2014
Accepted: 7 October 2014
Published: 14 October 2014
GPRC5A is a retinoic acid inducible gene that is preferentially expressed in lung tissue. Gprc5a– knockout mice develop spontaneous lung cancer, indicating Gprc5a is a lung tumor suppressor gene. GPRC5A expression is frequently suppressed in majority of non-small cell lung cancers (NSCLCs), however, elevated GPRC5A is still observed in a small portion of NSCLC cell lines and tumors, suggesting that the tumor suppressive function of GPRC5A is inhibited in these tumors by an unknown mechanism.
In this study, we examined EGF receptor (EGFR)-mediated interaction and tyrosine phosphorylation of GPRC5A by immunoprecipitation (IP)-Westernblot. Tyrosine phosphorylation of GPRC5A by EGFR was systematically identified by site-directed mutagenesis. Cell proliferation, migration, and anchorage-independent growth of NSCLC cell lines stably transfected with wild-type GPRC5A and mutants defective in tyrosine phosphorylation were assayed. Immunohistochemical (IHC) staining analysis with specific antibodies was performed to measure the total and phosphorylated GPRC5A in both normal lung and lung tumor tissues.
We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y350, at the C-terminal tail of GPRC5A. EGF induced phosphorylation of GPRC5A, which disrupted GPRC5A-mediated suppression on anchorage-independent growth of NSCLC cells. On contrary, GPRC5A-4 F, in which the four tyrosine residues have been replaced with phenylalanine, was resistant to EGF-induced phosphorylation and maintained tumor suppressive activities. Importantly, IHC analysis with anti-Y317/Y320-P sites showed that GPRC5A was non-phosphorylated in normal lung tissue whereas it was highly tyrosine-phosphorylated in NSCLC tissues.
GPRC5A can be inactivated by receptor tyrosine kinase via tyrosine phosphorylation. Thus, targeting EGFR can restore the tumor suppressive functions of GPRC5A in lung cancer.
The retinoic acid inducible G protein-coupled receptor family C group 5 member A (GPRC5A) is expressed predominantly in normal lung tissue. Several lines of evidences suggest that it function as a lung-specific tumor suppressor: a) Gprc5a-/- mice develop spontaneous lung tumors; b) loss of heterozygosity of chromosome 12p, where GPRC5A gene (12p12.3-p13) resides, is common in NSCLCs[3, 4]; and c), GPRC5A expression is suppressed in many lung cancer cell lines and tumor tissues compared to adjacent normal lung tissues[2, 5]. In addition, we showed previously that lung tissues from Gprc5a -/- mouse have increased NF-κB activation compared with that of wild-type mice. This suggests that Gprc5a negatively regulates a subset of genes involved in inflammation, proliferation, and survival. However, there are several controversy reports showing that elevated GPRC5A expression was found in some tumor cell lines and tumor samples and this expression correlated with increased cell growth and colony formation[7–9]. One possible explanation for these opposite observations is that the tumor-suppressive function of GPRC5A can be inactivated under certain conditions. Understanding the underlying mechanisms of GPRC5A functions will yield new insights into lung tumorigenesis and permit development of novel therapeutic invention for restoring the tumor suppressive functions of GPRC5A in lung cancer.
G protein-coupled receptors (GPCRs) can be modified by glycosylation, phosphorylation and palmitoylation, which alter protein conformation, protein association, subcellular localization, and/or biological functions[10, 11]. For example, GPCRs are desensitized via phosphorylation following agonist stimulation. This phosphorylation is directed to serine/threonine residues in the cytoplasmic tail and third cytoplasmic loop but rarely on tyrosine residues. The Ser/Thr phosphorylation by GPCR kinases (GRKs) leads to the internalization of GPCRs[10, 11] and hampers GPCR signaling. GPCRs can also undergo Tyr-phosphorylation on residues located in the cytoplasmic domain. It has been suggested that tyrosine phosphorylation of GPCR is required for Src recruitment and subsequent Ser/Thr phosphorylation by GRK. In some GPCRs, a tyrosine containing motif in the cytoplasmic tail has been linked to the internalization of GPCRs. For example, cytokine-induced tyrosine phosphorylation of CXCR4, which reduces the level of functional CXCR4 on cell surface, contributes to GRK3 and β-arrestin2-mediated sequestration of this receptor in the cytoplasm. It remains elusive whether GPRC5A is subjected to phosphorylation, leading to altered activities in lung cells.
EGFR and its family members are the major groups of receptor tyrosine kinases that are aberrantly activated in many NSCLCs. EGFR is over-expressed in more than 60% of NSCLC cases. In addition, oncogenically-activated mutant forms of EGFR and HER2 have been found in lung cancer, and contribute to the development of this disease. Moreover, EGF and TGF-α, ligands of EGFR, are frequently expressed in NSCLCs, which provides an autocrine mechanism to sustain the hyper-activation of these receptor tyrosine kinases (RTKs). In an un-biased whole cell phospho-peptide analysis, GPRC5A was identified as one of the tyrosine phosphorylated protein in HER2-overexpressing HMEC cells after EGF or heregulin (HRG) treatment[20, 21]. This suggests a potential cross-regulation between EGFR and GPRC5A. In this study, we showed that EGFR interacts with and phosphorylates GPRC5A in two highly conserved double-tyrosine modules (Y317/Y320 and Y347/Y350) at the C-terminal domain of GPRC5A. EGF treatment inhibited GPRC5A-mediated repression of anchorage-independent growth via phosphorylation of these tyrsoine sites since the same treatment failed to do so on GPRC5A-4 F mutant, in which tyrosine residues were replaced with phenylalanine (F). IHC analysis with specific antibody to Y317/Y320-P site showed that GPRC5A in NSCLC tissues is mostly phosphorylated, whereas GPRC5A in adjacent tumor tissues is mostly non-phosphorylated. Thus, EGFR-mediated tyrosine phosphorylation represents a newly identified mechanism by which the tumor suppressive function of GPRC5A is inactivated in lung cancer.
EGFR interacts with and phosphorylates GPRC5A
Y317 and Y320 at the C-terminal tail of GPRC5A are phosphorylated by EGFR
GPRC5A contains seven transmembrane regions and a C-terminal cytoplasmic tail. When the primary sequences of GPRC5A from difference species were aligned, we found that there are two potential double-tyrosine modules, including Y317/Y320 and Y347/Y350, located at the C-terminal tail of GPRC5A. These two double-tyrosine modules are highly conserved among all species (Figure 2A). To determine whether the predicted four tyrosine residues are responsible for EGF-induced tyrosine phosphorylation of GPRC5A, we constructed a GPRC5A-4 F mutant, in which four tyrosine residues (Y317, Y320, Y347 and Y350 site) were replaced by phenylalanine (F), that is unable to be phosphorylated (Figure 2B). When EGFR was co-expressed with GPRC5A-WT (wild type), we found that EGF induced tyrosine-phosphorylation of GPRC5A-WT (Figure 2C). However, when EGFR was co-expressed with 4 F mutant, no tyrosine- phosphorylation was induced by EGFR in GPRC5A-4 F (Figure 2D). Thus, it is likely that Y317, Y320, Y347 and Y350 are the major residues involved in the phosphorylation of GPRC5A mediated by EGFR.
Endogenous EGFR phosphorylates endogenous GPRC5A in NSCLCs
Next, we investigated whether endogenous EGFR can phosphorylate endogenous GPRC5A in NSCLC cell lines. We examined 11 NSCLC cell lines for expression of GPRC5A, EGFR and HER2, and found that 4 out of 11 NSCLC cell lines (H292G, Calu-1, H322, and H226b) expressed elevated levels of GPRC5A (Figure 3A). Notably, all of these four cell lines also have relatively high levels of EGFR. This suggests that the endogenous RTKs of EGFR family members are available in these cells for providing potential tyrosine-phosphorylation on GPRC5A. After serum starvation for 24 hours, we treated H292G cells with EGF (100 ng/ml) in serum-free medium, then immunoprecipitated endogenous GPRC5A for immunobloting. The IP-Western assay showed that GPRC5A was indeed tyrosine-phosphorylated after EGF exposure (Figure 3B), indicating that endogenous EGFR can interact and phosphorylate GPRC5A.
To expand the characterization of the tyrosine-phosphorylation of GPRC5A, we developed GPRC5A-WT (5A) and GPRC5A-4 F (4 F) stable transfectants in H1792. NSCLC cell line H1792 was selected for two reasons: 1) H1792 cells express a low level of endogenous GPRC5A (Figure 3A), thus expression of exogenous GPRC5A-WT and GPRC5A-4 F could be detected and evaluated; and 2) this cell line expresses endogenous EGFR (Figure 3A), thus EGFR activity can be induced by EGF. To determine the effects of endogenous EGFR on GPRC5A phosphorylation, we treated GPRC5A-WT stable H1792 transfectants (H1792-GPRC5A) with medium (C), EGF (E), or EGF plus pretreatment with EGFR inhibitor AG1478 (30 ng/ml for 6 hours) (E + A). We found that, GPRC5A was tyrosine-phosphorylated after EGF treatment, and this phosphorylation was blocked when cells were treated with AG1478 (Figure 3C).
Tyrosine phosphorylation inhibits the tumor suppressive activities of GPRC5A
To determine the biological effects of tyrosine phosphorylation on GPRC5A, we examined cell growth, migration, and anchorage-independent growth in H1792 cells stably expressing GPRC5A-WT (H1792-5A) or GPRC5A-4 F (H1792-4 F). We found no significant difference in cell proliferation among H1792-5A and H1792-4 F cells both before and after EGF treatment (Figure 4A). Noticeably, EGF treatment significantly increased the number of migrated H1792-V cells compared to the untreated cells (Figure 4B). We found that EGFR-mediated cell migration was inhibited by overexpression of GPRC5A-WT and 4 F (Figure 4B). However, the differences between the number of migratory cells in EGF-treated GPRC5A-WT and -4 F stable transfectants in H1792 cells were not statistically significant (P > .005; two-sided z test) (Figure 4B). This result suggests that EGF-mediated tyrosine phosphorylation of GPRC5A does not affect the suppressive effect of GPRC5A on cell migration.
GPRC5A exists as a non-phosphorylated form in normal lung tissue and tyrosine-phosphorylated form in NSCLC tissues
To determine the phosphorylated status of endogenous GPRC5A in normal lung and lung tumor tissues in vivo, we developed specific antibody against the double-tyrosine phosphorylation (Y317/Y320) sites of GPRC5A. Using co-transfection and immunoblot assay, we found that this antibody detected EGFR-mediated tyrosine phosphorylation of GPRC5A and Y347/350-F, but not Y317/320-F and 4 F (Figure 5A). Thus, the antibody is specifically for Y317/Y320 phosphorylated sites of GPRC5A (Figure 5A).
To determine the phosphorylation status of GPRC5A in normal lung and lung tumor tissues, we performed immunohistochemical (IHC) staining analysis by using antibodies to either GPRC5A or tyrosine-phosphorylated-GPRC5A (Y317/Y320-P) in 129 or 150 paired adjacent normal lung tissues and NSCLC tissues, respectively. The results of IHC staining show that total GPRC5A expression was significantly higher in adjacent normal lung tissues than NSCLC ones (Figure 5C-D), which is consistent with the previous report ([2, 5]), supporting GPRC5A is a lung tumor suppressor. Interestingly, the tyrosine-phosphorylated GPRC5A, although the total level was low, was significantly higher in NSCLC tissues than normal lung tissues (Figure 5E-F). These results suggest that, in normal lung tissue, GPRC5A was non-phosphorylated; whereas in lung tumor tissues, GPRC5A became highly tyrosine-phosphorylated, supporting that GPRC5A in lung tumor tissues are the function-defective ones.
In this study, we showed that EGFR interacts with and phosphorylates GPRC5A, leading to inactivation of the tumor suppressive function of GPRC5A in lung cancer cells. In this study, EGFR and GPRC5A were found to form a complex together. It remains unclear whether this interaction is directly or indirectly and whether other adaptor molecules, such as Grb-2, are involved in the interaction of EGFR-GPRC5A complex. Further investigation will reveal detailed mechanistic insight for this interaction.
Using systematic site-directed mutagenesis analysis, we identified that Y317, Y320, Y347 and Y350 are involved in the tyrosine phosphorylation of GPRC5A. In a whole-cell proteomic phospho-peptide analysis, Y317, Y320, Y347 and Y350 of GPRC5A were found to be phosphorylated in cells overexpressing EGFR and Src by mass Spectrometry[22, 23]. These results support our finding that the two double-tyrosine modules in GPRC5A are the major residues responsible for tyrosine phosphorylation. Interestingly, Y317F exhibited much reduced tyrosine phosphorylation than Y320F. One possible explanation is that Y317 is a primary/initial site for tyrosine phosphorylation, whereas Y320 is phosphorylated subsequently. This interesting phenomenon is analogous to GSK-3β-mediated β-catenin phosphorylation, in which CK1 induced-phosphorylation on Ser45 of β-catenin facilitates/primes GSK-3β-mediated sequential phosphorylation on Thr41, Ser37, and Ser33. This reverse order of phosphorylation results in the degradation of phosphorylated β-catenin by ubiquitin–proteasome system. It is likely that phosphorylation of one residue leads to conformation change of the protein, which in turn results in exposure of other target residue for subsequent phosphorylation. In addition, different phosphorylation motif may have different biochemical or biological changes. For example, we previously demonstrated that two phosphorylation motifs on Snail regulate two different functional properties of Snail, one controls the subcellular localization whereas the other controls proteins ubiquitination and degradation of snail. In this study, we found that the major phosphorylation sites are in the first double-tyrosine module (Y317 and Y320) in the C-terminal domain of GPRC5A. It is unclear whether other tyrosine kinases are responsible for the phosphorylation of the second double-tyrosine module (Y347 and Y350) of GPRC5A.
Several reports suggest that GPRC5A could be tyrosine phosphorylated in the C-terminal domain, and different stimuli in different cell lines or tissues may induce different tyrosine phosphorylation patterns. For example, HRG-treated 184A1 HMECs cells, which overexpress HER-230-fold, showed 332 tyrosine-phosphorylation sites in 175 proteins. Interestingly, Y347 on GPRC5A (RAI3) was among these peptides. These discrepancies between theirs and our studies may be due to different tissues or experiment conditions. It is also possible that Y347/Y350 might be preferentially phosphorylated by other tyrosine kinases in different cellular context.
The biological activities of GPRC5A appeared to be regulated differently by tyrosine phosphorylation. First, GPRC5A did not affect cell proliferation, regardless of tyrosine phosphorylation status. Second, GPRC5A-mediated inhibition of cell migration is independent of tyrosine phosphorylation. These results are consistent with Kumar’s finding, in which tyrosine phosphorylation of GPRC5A showed no effect on cell proliferation and migration ability. And third, tyrosine phosphorylation did decrease or abolish GPRC5A-mediated inhibition on EGF-enhanced anchorage-independent growth of H1792 cells. Taken together, these observations strongly support out hypothesis, that EGFR-mediated tyrosine phosphorylation of GPRC5A inactivates some of the tumor suppressor activities of GPRC5A. Thus, targeting EGFR by RTK inhibitors will restore the tumor suppressor functions of GPRC5A in lung cancer cells.
Importantly, IHC analysis showed that GPRC5A in adjacent normal lung tissues is non-tyrosine- phosphorylated, whereas it is tyrosine-phosphorylated in NSCLCs. This observation strongly supports the model that tyrosine phosphorylation inhibited the tumor suppressive activities of GPRC5A. Because GPRC5A in H292G cells was tyrosine-phosphorylated either with or without EGF in vitro, we assume that GPRC5A could be either tyrosine-phosphorylated by EGFR or other receptor tyrosine kinases (RTKs). Thus, we proposed that the tyrosine-phosphorylated GPRC5A could be used as a prognostic marker for tumor progression. Thus, targeting receptor tyrosine kinases will be an effective in preventing and treating lung cancer by restoring the tumor suppressor function of GPRC5A. Our results provide a novel mechanism in supporting this strategy.
Materials and methods
Cell culture and reagents
Human NSCLC cell lines (H460, A549, H226, H157, Calu-1, H226B, H322, H292G, H1792, H358 and Sk-Mes-1) were obtained from Adi Gazdar and John Minna (UT Southwestern, Dallas, TX). Cell culture was performed as described previously. EGF was obtained from R&D system (Minneapolis, MN, USA). AG1478 was purchased from Merck Millipore (Billerica, MA, USA). EGFR mouse mAb (#2256, IP), EGFR Rabbit mAb (#4267), were from Cell Signaling Technology. Antibody to P-Tyr (PY99) was from Santa Cruz Biotechnology. Antibodies against myc and actin were from Sigma-Aldrich. HRP-conjugated secondary antibodies were from eBioScience (San Diego, CA). Rabbit polyclonal antibody to human GPRC5A was as described previously. Mouse monoclonal antibody (clone 1 L23) to the C-terminus of GPRC5A (PYKDYEVKKE) was developed and purified in Abmart (Shanghai, China). Polyclonal rabbit anti-GPRC5A-Y317/Y320-P antibody was developed against DTLYpAPYpSTH from Abmart (Shanghai, China).
Plasmids and transfection assay
HEK293T cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen, Grand Island, NY). Transfected plasmids include: pcDNA-EGFR, pcDNA-GPRC5A-myc, and GPRC5A mutants. Cells were starved in serum-free medium for 24 hours, then treated with EGF (100 ng/ml) for various time periods as indicated.
Construction of wild-type and mutant GPRC5A plasmids
Plasmid pcDNA3.1 (+)-GPRC5A-Myc was as described. GPRC5A mutants were constructed with the following primers: Primer Y317/320 F-F: (5′-GGT TTT GAA GAG ACC GGT GAC ACG CTC TTT GCC CCC TTT TCC ACA CAT TTT C-3′) and primer Y317/320 F-R (5′-GAA AAT GTG TGG AAA AGG GGG CAA AGA GCG TGT CAC CGG TCT CTT CAA AAC C-3′). Primer Y347/350 F-F: (5′-CCA CGC TTG GCC GAG CCC TTT TAA AGA CTT TGA AGT AAA GAA AGA GG-3′) and primer Y347/350 F-R: (5′-CCT CTT TCT TTA CTT CAA AGT CTT TAA AAG GGC TCG GCC AAG CGT GG-3′). 4 F mutant (GPRC5A-Y317/320/347/350 F-myc) of GPRC5A was generated by ligation of GPRC5A-Y317/320 F-myc and GPRC5A-Y347/350 F-myc after digestion with KpnI and EcoRI separately. All sequences were verified by DNA sequencing.
Transient and stable transfection of cells
Transfection of H1792 cells with plasmids was performed by electrophoresis: Briefly, the cells were harvested, and about 3 × 105 cells were mixed with 100 μl of electroporation transfection solution (Solution V, Dharmacon, Lafayette, CO), plus plasmids, and the mixtures were transferred to electroporation cuvettes and subjected to electroporation (Amaxa Biosystems, Cologne, Germany) according to the manufacturer’s pro-grams and instructions.
Immunoprecipitation (IP) and western blotting
IP was performed with 2 μg of antibodies against myc or EGFR or normal IgG (N IgG) (as a negative control) in 1.0 mg whole cell lysate. Immunoblot[25, 28] and cell fractionation was performed as described.
Cell migration assay
The trans-well cell migration system consisted of cell culture inserts with an 8.0 μM pore size in 24-well plate (BD BioCoat #354578, San Jose, CA). Cells were resuspended with fetal bovine serum (FBS)-free DMEM, and seeded onto the insert (4 × 104 cells). DMEM (10% FBS) with or without EGF (100 ng/ml) was loaded into the lower chamber and incubated overnight. Migrated cells, which attached to the lower side of the filter were fixed with 96% ethanol for 30 minutes and stained with 1.5% crystal violet. Migrated cells were counted in five different microscopic fields by 10 × magnifications using a Nikon fluorescence microscope.
Anchorage-independent colony formation in soft agar assay
Soft-agarose assay was performed as described previously. Aggregates of 50 or more cells were considered to be a colony. Colonies were counted in four different fields under a microscope at 4× magnification and photographed. The means and 95% confidence intervals (CIs) of the number of colonies in four microscopic fields were calculated. Two independent experiments in triplicates were performed.
We obtained archival, formalin-fixed and paraffin-embedded (FFPE) material from surgically resected lung cancer specimens containing tumor and paired adjacent non-malignant epithelium tissue from the Shanghai Chest Hospital from 2008 to 2013 (Shanghai, CHINA). The adjacent non-malignant epithelia were collected at sites at least 2 cm away from the edge of tumor mass, with best efforts of avoiding contamination by the tumor cells. In total, 150 primary NSCLC patients without prior radiotherapy or chemotherapy were enrolled in this study. All NSCLC samples were confirmed histologically, and tumor samples were rechecked to ensure that tumor tissue was present in more than 80% of the specimens.
The samples of lung tumor and adjacent normal lung tissues were fixed with formalin buffer and embedded in paraffin. Immunohistochemical (IHC) staining was performed on 3-μm sections of paraffin-embedded specimens with the use of mouse monoclonal antibody (clone 1 L23) to the C-terminus of GPRC5A (PYKDYEVKKE) or rabbit anti-GPRC5A-Y317/Y320-P. These antibodies were developed and purified in Abmart (Shanghai, China). The process of IHC was performed as previously described.
All analyses were performed in triplicates, and the significance of differences between groups was calculated using the Student’s t test. P values <0.05 were considered to be statistically significant.
We would like to thank Dr. Reuben Lotan (M.D. Anderson, Houston, TX) for his generosity providing NSCLC cell lines and his support. We thank Cathy Anthony for critical reading and editing of this manuscript.
This work was supported by grants from, Ministry of Science and Technology No. 2011CB504300 (J.Deng) and 2013CB910900 (J.Deng and E.Y.Chin), National Nature Science Foundation of China 91129303 (J.Deng), 81071923 (J.Deng), 81201840 (F.Yao), 81272714 (Q.Li), National Key Basic Research 973 Program of China (2013CB910901) (J.Deng), and Science and Technology Commission of Shanghai (10140902100) (J.Deng). This work was also supported by grants from NIH (2RO1CA125454), Susan G Komen Foundation (KG081310), Mary Kay Ash Foundation (to B.P. Zhou).
- Cheng Y, Lotan R: Molecular cloning and characterization of a novel retinoic acid-inducible gene that encodes a putative G protein-coupled receptor. J Biol Chem. 1998, 273: 35008-35015. 10.1074/jbc.273.52.35008View ArticlePubMedGoogle Scholar
- Tao Q, Fujimoto J, Men T, Ye X, Deng J, Lacroix L, Clifford JL, Mao L, Van Pelt CS, Lee JJ, Lotan D, Lotan R: Identification of the retinoic acid-inducible Gprc5a as a new lung tumor suppressor gene. J Natl Cancer Inst. 2007, 99: 1668-1682. 10.1093/jnci/djm208View ArticlePubMedGoogle Scholar
- Takeuchi S, Mori N, Koike M, Slater J, Park S, Miller CW, Miyoshi I, Koeffler HP: Frequent loss of heterozygosity in region of the KIP1 locus in non-small cell lung cancer: evidence for a new tumor suppressor gene on the short arm of chromosome 12. Cancer Res. 1996, 56: 738-740.PubMedGoogle Scholar
- Grepmeier U, Dietmaier W, Merk J, Wild PJ, Obermann EC, Pfeifer M, Hofstaedter F, Hartmann A, Woenckhaus M: Deletions at chromosome 2q and 12p are early and frequent molecular alterations in bronchial epithelium and NSCLC of long-term smokers. Int J Oncol. 2005, 27: 481-488.PubMedGoogle Scholar
- Fujimoto J, Kadara H, Garcia MM, Kabbout M, Behrens C, Liu DD, Lee JJ, Solis LM, Kim ES, Kalhor N, Moran C, Sharafkhaneh A, Lotan R, Wistuba : G-protein coupled receptor family C, group 5, member A (GPRC5A) expression is decreased in the adjacent field and normal bronchial epithelia of patients with chronic obstructive pulmonary disease and non-small-cell lung cancer. J Thorac Oncol. 2012, 7: 1747-1754. 10.1097/JTO.0b013e31826bb1ffPubMed CentralView ArticlePubMedGoogle Scholar
- Deng J, Fujimoto J, Ye XF, Men TY, Van Pelt CS, Chen YL, Lin XF, Kadara H, Tao Q, Lotan D, Lotan R: Knockout of the tumor suppressor gene Gprc5a in mice leads to NF-kappaB activation in airway epithelium and promotes lung inflammation and tumorigenesis. Cancer Prev Res (Phila). 2010, 3: 424-437. 10.1158/1940-6207.CAPR-10-0032View ArticleGoogle Scholar
- Wu Q, Ding W, Mirza A, Van Arsdale T, Wei I, Bishop WR, Basso A, McClanahan T, Luo L, Kirschmeier P, Gustafson E, Hernandez M, Liu S: Integrative genomics revealed RAI3 is a cell growth-promoting gene and a novel P53 transcriptional target. J Biol Chem. 2005, 280: 12935-12943. 10.1074/jbc.M409901200View ArticlePubMedGoogle Scholar
- Hirano M, Zang L, Oka T, Ito Y, Shimada Y, Nishimura Y, Tanaka T: Novel reciprocal regulation of cAMP signaling and apoptosis by orphan G-protein-coupled receptor GPRC5A gene expression. Biochem Biophys Res Commun. 2006, 351: 185-191. 10.1016/j.bbrc.2006.10.016View ArticlePubMedGoogle Scholar
- Nagahata T, Sato T, Tomura A, Onda M, Nishikawa K, Emi M: Identification of RAI3 as a therapeutic target for breast cancer. Endocr Relat Cancer. 2005, 12: 65-73. 10.1677/erc.1.00890View ArticlePubMedGoogle Scholar
- Marchese A, Paing MM, Temple BR, Trejo J: G protein-coupled receptor sorting to endosomes and lysosomes. Annu Rev Pharmacol Toxicol. 2008, 48: 601-629. 10.1146/annurev.pharmtox.48.113006.094646PubMed CentralView ArticlePubMedGoogle Scholar
- Ulloa-Aguirre A, Conn PM: Targeting of G protein-coupled receptors to the plasma membrane in health and disease. Front Biosci (Landmark Ed). 2009, 14: 973-994.View ArticleGoogle Scholar
- Premont RT, Gainetdinov RR: Physiological roles of G protein-coupled receptor kinases and arrestins. Annu Rev Physiol. 2007, 69: 511-534. 10.1146/annurev.physiol.69.022405.154731View ArticlePubMedGoogle Scholar
- Tobin AB, Butcher AJ, Kong KC: Location, location, location…site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling. Trends Pharmacol Sci. 2008, 29: 413-420. 10.1016/j.tips.2008.05.006PubMed CentralView ArticlePubMedGoogle Scholar
- Wang J, Guan E, Roderiquez G, Calvert V, Alvarez R, Norcross MA: Role of tyrosine phosphorylation in ligand-independent sequestration of CXCR4 in human primary monocytes-macrophages. J Biol Chem. 2001, 276: 49236-49243. 10.1074/jbc.M108523200View ArticlePubMedGoogle Scholar
- Brambilla C, Spiro S: Highlights in lung cancer. Eur Respir J. 2001, 18: 617-618. 10.1183/09031936.01.00238801View ArticlePubMedGoogle Scholar
- Hirsch FR, Varella-Garcia M, Bunn PA, Di Maria MV, Veve R, Bremmes RM, Baron AE, Zeng C, Franklin WA: Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol. 2003, 21: 3798-3807. 10.1200/JCO.2003.11.069View ArticlePubMedGoogle Scholar
- Stephens P, Hunter C, Bignell G, Edkins S, Davies H, Teague J, Stevens C, O’Meara S, Smith R, Parker A, Barthorpe A, Blow M, Brackenbury L, Butler A, Clarke O, Cole J, Dicks E, Dike A, Drozd A, Edwards K, Forbes S, Foster R, Gray K, Greenman C, Halliday K, Hills K, Kosmidou V, Lugg R, Menzies A, Perry J: Lung cancer: intragenic ERBB2 kinase mutations in tumours. Nature. 2004, 431: 525-526.View ArticlePubMedGoogle Scholar
- Riese DJ, Gallo RM, Settleman J: Mutational activation of ErbB family receptor tyrosine kinases: insights into mechanisms of signal transduction and tumorigenesis. Bioessays. 2007, 29: 558-565. 10.1002/bies.20582PubMed CentralView ArticlePubMedGoogle Scholar
- Sharma SV, Bell DW, Settleman J, Haber DA: Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007, 7: 169-181. 10.1038/nrc2088View ArticlePubMedGoogle Scholar
- Okamoto Y, Ninomiya H, Masaki T: Posttranslational modifications of endothelin receptor type B. Trends Cardiovasc Med. 1998, 8: 327-329. 10.1016/S1050-1738(98)00027-9View ArticlePubMedGoogle Scholar
- Qanbar R, Bouvier M: Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function. Pharmacol Ther. 2003, 97: 1-33. 10.1016/S0163-7258(02)00300-5View ArticlePubMedGoogle Scholar
- Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, Hu Y, Tan Z, Stokes M, Sullivan L, Mitchell J, Wetzel R, Macneill J, Ren JM, Yuan J, Bakalarski CE, Villen J, Kornhauser JM, Smith B, Li D, Zhou X, Gygi SP, Gu TL, Polakiewicz RD, Rush J, Comb MJ: Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 2007, 131: 1190-1203. 10.1016/j.cell.2007.11.025View ArticlePubMedGoogle Scholar
- Wolf-Yadlin A, Kumar N, Zhang Y, Hautaniemi S, Zaman M, Kim HD, Grantcharova V, Lauffenburger DA, White FM: Effects of HER2 overexpression on cell signaling networks governing proliferation and migration. Mol Syst Biol. 2006, 2: 54-PubMed CentralView ArticlePubMedGoogle Scholar
- Kikuchi A, Kishida S, Yamamoto H: Regulation of Wnt signaling by protein-protein interaction and post-translational modifications. Exp Mol Med. 2006, 38: 1-10. 10.1038/emm.2006.1View ArticlePubMedGoogle Scholar
- Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M, Hung MC: Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol. 2004, 6: 931-940. 10.1038/ncb1173View ArticlePubMedGoogle Scholar
- Kumar N, Zaman MH, Kim HD, Lauffenburger DA: A high-throughput migration assay reveals HER2-mediated cell migration arising from increased directional persistence. Biophys J. 2006, 91: L32-L34. 10.1529/biophysj.106.088898PubMed CentralView ArticlePubMedGoogle Scholar
- Liu SL, Zhong SS, Ye DX, Chen WT, Zhang ZY, Deng J: Repression of G protein-coupled receptor family C group 5 member A is associated with pathologic differentiation grade of oral squamous cell carcinoma. J Oral Pathol Med. 2013, 42: 761-768. 10.1111/jop.12077View ArticlePubMedGoogle Scholar
- Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP: Stabilization of snail by NF-kappaB is required for inflammation-induced cell migration and invasion. Cancer Cell. 2009, 15: 416-428. 10.1016/j.ccr.2009.03.016PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC: Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol. 2001, 3: 245-252. 10.1038/35060032View ArticlePubMedGoogle Scholar
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