title: EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lin
authors.forename: Shuangshuang
authors.initials: S
authors.lastname: Zhong
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Ye
authors.forename: Yueling
authors.initials: Y
authors.lastname: Liao
authors.forename: Feng
authors.initials: F
authors.lastname: Yao
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Yang
authors.forename: Beibei
authors.initials: B
authors.lastname: Sun
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.forename: Yong
authors.initials: Y
authors.lastname: Gao
authors.forename: Yifan
authors.initials: Y
authors.lastname: Wang
authors.forename: Jingyi
authors.initials: J
authors.lastname: Liu
authors.forename: Baohui
authors.initials: B
authors.lastname: Han
authors.forename: Y Eugene
authors.initials: YE
authors.lastname: Chin
authors.affiliation: Department of Pathophysiology, Key laboratory of Cell Differentiation and Apoptosis of Minister of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. peter.zhou@uky.edu.
authors.forename: Binhua P
authors.initials: BP
authors.lastname: Zhou
authors.forename: Jiong
authors.initials: J
authors.lastname: Deng
mesh_terms: D000595:Amino Acid Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000378:metabolism*; D016147:Genes, Tumor Suppressor; D057809:HEK293 Cells; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*
chemical_list: C116760:GPRC5A protein, human; D043562:Receptors, G-Protein-Coupled; D019000:Phosphotyrosine; D066246:ErbB Receptors