EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer.
EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer.
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EGFR 磷酸化并抑制肺癌中的肺肿瘤抑制因子 GPRC5A
DOI：
10.1186/1476-4598-13-233
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发表时间：
2014-10-14
期刊：
影响因子：
37.3
通讯作者：
Deng J
中科院分区：
文献类型：
--
作者：
Lin X;Zhong S;Ye X;Liao Y;Yao F;Yang X;Sun B;Zhang J;Li Q;Gao Y;Wang Y;Liu J;Han B;Chin YE;Zhou BP;Deng J
GPRC5A是维甲酸诱导基因，在肺组织中优先表达。Gprc5a敲除小鼠发生自发性肺癌，提示Gprc5a是肺肿瘤抑制基因。GPRC5A在大多数非小细胞肺癌（non-small cell lung cancer， NSCLC）中表达经常受到抑制，然而，在一小部分NSCLC细胞系和肿瘤中仍观察到GPRC5A的表达升高，提示GPRC5A的抑瘤功能在这些肿瘤中受到未知机制的抑制。在这项研究中，我们通过免疫沉淀(IP)-Westernblot检测了EGF受体（EGFR）介导的相互作用和GPRC5A的酪氨酸磷酸化。通过定点诱变系统地鉴定了EGFR对GPRC5A酪氨酸的磷酸化作用。研究了稳定转染野生型GPRC5A和酪氨酸磷酸化缺陷突变体的NSCLC细胞系的细胞增殖、迁移和不依赖锚定生长。采用特异性抗体免疫组化（IHC）染色法测定正常肺组织和肺肿瘤组织中GPRC5A的总量和磷酸化水平。我们发现EGFR与GPRC5A相互作用，并在GPRC5A的c端尾部的Y317/Y320和Y347/ Y350两个保守的双酪氨酸基序中磷酸化GPRC5A。EGF诱导GPRC5A磷酸化，破坏了GPRC5A介导的对非锚定依赖性生长的抑制。相反，gprc5a - 4f中四个酪氨酸残基被苯丙氨酸取代，能够抵抗egf诱导的磷酸化并保持肿瘤抑制活性。重要的是，抗y317 /Y320-P位点的免疫组化分析显示，GPRC5A在正常肺组织中不磷酸化，而在非小细胞肺癌组织中则高度酪氨酸磷酸化。受体酪氨酸激酶可通过酪氨酸磷酸化使GPRC5A失活。因此，靶向EGFR可以恢复GPRC5A在肺癌中的抑瘤功能。
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.
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