EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer

Xiaofeng Lin; Shuangshuang Zhong; Xiaofeng Ye; Yueling Liao; Feng Yao; Xiaohua Yang; Beibei Sun; Jie Zhang; Qi Li; Yong Gao; Yifan Wang; Jingyi Liu; Baohui Han; Y. Baohui Chin; Binhua P. Zhou; Jiong Deng

doi:10.1186/1476-4598-13-233

EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer

Xiaofeng Lin
, Shuangshuang Zhong
, Xiaofeng Ye
, Yueling Liao
, Feng Yao
, Xiaohua Yang
, Beibei Sun
, Jie Zhang
, Qi Li
, Yong Gao
, Yifan Wang
, Jingyi Liu
, Baohui Han
, Y. Baohui Chin
, Binhua P. Zhou
, Jiong Deng

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

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. Result: We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y1, 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. Conclusion: 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.

Original language	English
Article number	233
Journal	Molecular Cancer
Volume	13
Issue number	1
DOIs	https://doi.org/10.1186/1476-4598-13-233
State	Published - Oct 14 2014

Bibliographical note

Publisher Copyright:
© 2014 Lin et al.; licensee BioMed Central Ltd.

Funding

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).

Funders	Funder number
National Institutes of Health (NIH)	2RO1CA125454
Mary Kay Ash Foundation
Susan G Komen Foundation	KG081310
National Natural Science Foundation of China (NSFC)	81071923, 81272714, 81201840, 91129303
Science and Technology Commission of Shanghai Municipality	10140902100
Ministry of Science and Technology, Taiwan	2011CB504300, 2013CB910900
National Key Research and Development Program of China	2013CB910901

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

EGFR
GPRC5A
Lung cancer
Post-translation modification
Tyrosine kinase

ASJC Scopus subject areas

Molecular Medicine
Oncology
Cancer Research

Access to Document

10.1186/1476-4598-13-233

Cite this

@article{d30995ba64f74e2aaaa20afa7618b1e0,

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. Result: We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y1, 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. Conclusion: 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.",

keywords = "EGFR, GPRC5A, Lung cancer, Post-translation modification, Tyrosine kinase",

author = "Xiaofeng Lin and Shuangshuang Zhong and Xiaofeng Ye and Yueling Liao and Feng Yao and Xiaohua Yang and Beibei Sun and Jie Zhang and Qi Li and Yong Gao and Yifan Wang and Jingyi Liu and Baohui Han and Chin, \{Y. Baohui\} and Zhou, \{Binhua P.\} and Jiong Deng",

note = "Publisher Copyright: {\textcopyright} 2014 Lin et al.; licensee BioMed Central Ltd.",

year = "2014",

month = oct,

day = "14",

doi = "10.1186/1476-4598-13-233",

language = "English",

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TY - JOUR

T1 - EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer

AU - Lin, Xiaofeng

AU - Zhong, Shuangshuang

AU - Ye, Xiaofeng

AU - Liao, Yueling

AU - Yao, Feng

AU - Yang, Xiaohua

AU - Sun, Beibei

AU - Zhang, Jie

AU - Li, Qi

AU - Gao, Yong

AU - Wang, Yifan

AU - Liu, Jingyi

AU - Han, Baohui

AU - Chin, Y. Baohui

AU - Zhou, Binhua P.

AU - Deng, Jiong

PY - 2014/10/14

Y1 - 2014/10/14

N2 - 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. Result: We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y1, 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. Conclusion: 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.

AB - 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. Result: We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y1, 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. Conclusion: 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.

KW - EGFR

KW - GPRC5A

KW - Lung cancer

KW - Post-translation modification

KW - Tyrosine kinase

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M3 - Article

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AN - SCOPUS:84935000709

SN - 1476-4598

VL - 13

JO - Molecular Cancer

JF - Molecular Cancer

IS - 1

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ER -

EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer

Abstract

Bibliographical note

Funding

UN SDGs

Keywords

ASJC Scopus subject areas

Access to Document

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