The Leucine-rich Repeat Protein LRIG1 Is a Negative Regulator of ErbB Family Receptor Tyrosine Kinases

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function. The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function. The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases. Vol. 279 (2004) 47050–47056Journal of Biological ChemistryVol. 279Issue 50PreviewThis grant support statement was inadvertently omitted: This work was supported by California Breast Cancer Research Program Grant 7KB-0085 (to C. S.) and Department of Defense Breast Cancer Research Program Grant DAMD17-02-1-0322 (to K. L. C.). Full-Text PDF Open Access The four members of the ErbB family of receptor tyrosine kinases (epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; NRG1, neuregulin-1; LRR, leucine-rich repeat; Ig, immunoglobulin; Kek1, Kekkon-1; FCS, fetal calf serum; HA, hemagglutinin. receptor, ErbB2, ErbB3, and ErbB4) play key roles in mediating the development of a variety of tissues, and the aberrant activation of these receptors contributes to the growth and progression of numerous tumor types (1Holbro T. Civenni G. Hynes N.E. Exp. Cell Res. 2003; 284: 99-110Crossref PubMed Scopus (533) Google Scholar, 2Marmor M.D. Skaria K.B. Yarden Y. Int. J. Radiat. Oncol. Biol. Phys. 2004; 58: 903-913Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Binding of EGF-like family ligands to ErbB receptors stimulates receptor dimerization, kinase activation, autophosphorylation, and the engagement of multiple intracellular growth signaling pathways. Although considerable effort over the past two decades has gone into understanding mechanisms by which ErbB receptors are activated and signals are propagated, our understanding of the variety of molecular mechanisms underlying the suppression of growth factor receptor activity remains in its infancy. Growth factor-stimulated receptor down-regulation, involving receptor internalization and the cbl-mediated ubiquitination and trafficking of receptors to lysosomes (3Shtiegman K. Yarden Y. Semin. Cancer Biol. 2003; 13: 29-40Crossref PubMed Scopus (57) Google Scholar, 4Wiley H.S. Exp. Cell Res. 2003; 284: 78-88Crossref PubMed Scopus (300) Google Scholar), represents one mechanism for preventing hypersignaling by the ErbB receptors. However, whereas EGF receptor (ErbB1 or EGFR) efficiently couples to cbl following stimulation with its ligand EGF, the ErbB2, ErbB3, and ErbB4 receptors do not efficiently couple to cbl following stimulation with neuregulin-1 (NRG1) (5Levkowitz G. Klapper L.N. Tzahar E. Freywald A. Sela M. Yarden Y. Oncogene. 1996; 12: 1117-1125PubMed Google Scholar) and do not undergo efficient NRG1-stimulated down-regulation (6Baulida J. Kraus M.H. Alimandi M. Di Fiore P.P. Carpenter G. J. Biol. Chem. 1996; 271: 5251-5257Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 7Baulida J. Carpenter G. Exp. Cell Res. 1997; 232: 167-172Crossref PubMed Scopus (42) Google Scholar). Hence, other negative regulatory mechanisms may play major roles in suppressing ErbB receptor activity. Studies from the fruit fly Drosophila melanogaster point to the existence of several classes of proteins that negatively regulate EGF receptor activity in flies (8Sweeney C. Carraway III, K.L. Br. J. Cancer. 2004; 90: 289-293Crossref PubMed Scopus (49) Google Scholar). Argos is a secreted protein that bears some homology with EGF family growth factors and possibly acts as an antagonist of EGF receptor stimulation by activating ligands (9Vinos J. Freeman M. Oncogene. 2000; 19: 3560-3562Crossref PubMed Scopus (22) Google Scholar, 10Jin M.H. Sawamoto K. Ito M. Okano H. Mol. Cell. Biol. 2000; 20: 2098-2107Crossref PubMed Scopus (46) Google Scholar). Thus far, a mammalian functional homolog of Argos has not been identified. Kekkon-1 (Kek1) is a transmembrane protein whose extracellular region contains a domain of six leucine-rich repeats (LRRs) and an immunoglobulin (Ig) domain (11Musacchio M. Perrimon N. Dev. Biol. 1996; 178: 63-76Crossref PubMed Scopus (97) Google Scholar). Kek1 physically associates with Drosophila EGF receptor through its LRR domain to specifically suppress receptor signaling (12Ghiglione C. Carraway III, K.L. Amundadottir L.T. Boswell R.E. Perrimon N. Duffy J.B. Cell. 1999; 96: 847-856Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 13Ghiglione C. Amundadottir L. Andresdottir M. Bilder D. Diamonti J.A. Noselli S. Perrimon N. Carraway III, K.L. Development. 2003; 130: 4483-4493Crossref PubMed Scopus (46) Google Scholar). Interestingly Drosophila Kek1 can physically associate with each of the four mammalian ErbB family members and potently suppresses the growth rate of mammary tumor cells whose growth is dependent on ErbB signaling (13Ghiglione C. Amundadottir L. Andresdottir M. Bilder D. Diamonti J.A. Noselli S. Perrimon N. Carraway III, K.L. Development. 2003; 130: 4483-4493Crossref PubMed Scopus (46) Google Scholar). These observations strongly suggest that human proteins containing extracellular LRR domains are candidate suppressors of ErbB activity. The human genome encodes dozens of transmembrane proteins from different families that contain extracellular LRR domains. The functions of the vast majority of these remain to be elucidated. Interestingly, it has been demonstrated that mice lacking LRIG1, a protein possessing a domain of 15 LRRs and 3 Ig domains in its extracellular region (14Suzuki Y. Sato N. Tohyama M. Wanaka A. Takagi T. J. Biol. Chem. 1996; 271: 22522-22527Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 15Nilsson J. Vallbo C. Guo D. Golovleva I. Hallberg B. Henriksson R. Hedman H. Biochem. Biophys. Res. Commun. 2001; 284: 1155-1161Crossref PubMed Scopus (120) Google Scholar), develop psoriatic-like skin lesions (16Suzuki Y. Miura H. Tanemura A. Kobayashi K. Kondoh G. Sano S. Ozawa K. Inui S. Nakata A. Takagi T. Tohyama M. Yoshikawa K. Itami S. FEBS Lett. 2002; 521: 67-71Crossref PubMed Scopus (89) Google Scholar). Hyperactivation of ErbB receptor signaling is commonly observed in keratinocytes of psoriatic lesions, and expression of EGF-like growth factors in keratinocytes of transgenic mice results in a psoriatic phenotype (17Piepkorn M. Pittelkow M.R. Cook P.W. J. Investig. Dermatol. 1998; 111: 715-721Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). These observations point to the possibility that LRIG1 might act as a negative regulator of ErbB receptor signaling. Here we have examined whether LRIG1 can suppress ErbB-mediated growth regulation. Materials and Cell Culture—EGF was purchased from Upstate Biotechnology, and glutathione S-transferase-fused NRG1β EGF-like domain was produced in bacteria and purified by glutathione affinity. Plasmids expressing human ErbB receptors have been previously described (18Diamonti A.J. Guy P.M. Ivanof C. Wong K. Sweeney C. Carraway III, K.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2866-2871Crossref PubMed Scopus (104) Google Scholar). Antibodies to Myc and HA tags were from Oncogene Sciences, and receptor antibodies from NeoMarkers or Santa Cruz Biotechnology were used as described previously (19Sweeney C. Fambrough D. Huard C. Diamonti A.J. Lander E.S. Cantley L.C. Carraway III, K.L. J. Biol. Chem. 2001; 276: 22685-22698Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Anti-ErbB2 antibody 3E8 used in immunofluorescence was from Genentech. Antibodies to LRIG1 were raised in rabbit to peptide acetyl-QTRKKSEEYSVTNTDETC-amide located in the intracellular domain. Antibodies were affinity purified prior to use in immunoprecipitation and blotting experiments. FuGENE 6 was from Roche Applied Science. All cell lines were from ATCC. NIH3T3, 293T, SKBR3, MDA-MB-453, MDA-MB-361, and BT474 cells were cultured in Dulbecco's modified Eagle's medium/10% fetal calf serum (FCS). All prostate tumor cell lines and T47D cells were cultured in RPMI/10% FCS. The PC3-LRIG1 cell line was created by the simultaneous transfection of tTA plasmid (Clontech) and LRIG1 subcloned into pTRE2-Hyg plasmid and selection with 0.2 mg/ml hygromycin B and 0.4 mg/ml G418 in the presence of 2 μg/ml tetracycline. Individual colonies were screened for tetracycline-suppressed LRIG1 expression. Cloning of Human LRIG1 cDNA—A partial clone encompassing the carboxyl-terminal half of human LRIG1 cDNA was obtained from ATCC (IMAGE:4310439). The amino-terminal half was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA from human MDA-MB-453 breast cancer cells. The parts were joined using the unique internal restriction site SphI. Sequencing revealed that the cloned fragment represents a splice variant in which 1 leucine-rich repeat encoded by a 72-nucleotide exon is inserted after the first leucine-rich repeat of the published sequence (14Suzuki Y. Sato N. Tohyama M. Wanaka A. Takagi T. J. Biol. Chem. 1996; 271: 22522-22527Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 15Nilsson J. Vallbo C. Guo D. Golovleva I. Hallberg B. Henriksson R. Hedman H. Biochem. Biophys. Res. Commun. 2001; 284: 1155-1161Crossref PubMed Scopus (120) Google Scholar), and the 14th leucine-rich repeat encoded by a 72-nucleotide exon is deleted (GenBank™ accession number AY730707). LRIG1 was epitope tagged immediately carboxyl-terminal to its leader sequence by cutting the cDNA with NaeI and ligating in annealed oligonucleotides encoding the Myc epitope. Northern blotting of 20 μg of total RNA from human breast and prostate cancer cell lines was carried out using previously established procedures (20Yen L. Benlimame N. Nie Z.R. Xiao D. Wang T. Al Moustafa A.E. Esumi H. Milanini J. Hynes N.E. Pages G. Alaoui-Jamali M.A. Mol. Biol. Cell. 2002; 13: 4029-4044Crossref PubMed Scopus (121) Google Scholar). Co-immunoprecipitation and Immunoblotting—293T cells in 100-mm dishes were transfected with each ErbB receptor without or with LRIG1 using FuGENE 6 according to procedures recommended by the manufacturer. Cells were serum starved in Dulbecco's modified Eagle's medium/0.1% FCS overnight prior to growth factor treatment. Dishes were treated with nothing, 50 nm NRG1β, or 33 nm EGF as indicated in the figure legends. Cells were lysed in co-immunoprecipitation buffer (19Sweeney C. Fambrough D. Huard C. Diamonti A.J. Lander E.S. Cantley L.C. Carraway III, K.L. J. Biol. Chem. 2001; 276: 22685-22698Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and cleared lysates were immunoprecipitated with 1.5 μg of anti-Myc or anti-receptor antibodies. Proteins in lysates were blotted with Myc, HA, or receptor antibodies. For immunoprecipitation experiments from mouse brain lysates, a single mouse brain was obtained immediately after and was in 3 of co-immunoprecipitation The was cleared by 1 of was immunoprecipitated with 1.5 μg of rabbit or rabbit and blotted with anti-receptor and antibodies. was carried out using antibodies were using an and cells in 100-mm dishes were transfected with encoding ErbB receptors or LRIG1, and proteins were to for Cells were with and treated with mg/ml in for 1 Cells were with lysed in co-immunoprecipitation and with anti-Myc or anti-receptor antibodies. were by and blotted with antibodies cells were transfected with and EGF receptor or without or with were carried out as described that cells were lysed in buffer (19Sweeney C. Fambrough D. Huard C. Diamonti A.J. Lander E.S. Cantley L.C. Carraway III, K.L. J. Biol. Chem. 2001; 276: 22685-22698Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) containing and buffer containing receptors in 293T cells were by blotting with antibodies. receptors in PC3-LRIG1 cells were by blotting with antibody cells were transfected with encoding LRIG1 and for and with for 20 Cells on were and as previously described (18Diamonti A.J. Guy P.M. Ivanof C. Wong K. Sweeney C. Carraway III, K.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2866-2871Crossref PubMed Scopus (104) Google Scholar) and with of anti-Myc and 3E8 in Proteins were using antibodies and was carried out using an to was used to cells in were transfected with of EGF receptor, or cells were and 2 cells were in into 1.5 of modified Eagle's with FCS and with or without 3 nm and on of dishes with 2 of modified Eagle's with FCS and with or without 3 nm Cells were a for and with on were using an and Cell cells were serum starved and treated with and without for and treated with 1 for an Cells were treated with 50 nm NRG1β, or 33 nm EGF for and in for a of 2 were with mg/ml mg/ml 2 50 mg/ml for 1 The of the were by using a was on cells and was whether LRIG1 might the of human ErbB we first examined the of the proteins by the in 293T cells were transfected with human ErbB receptor tyrosine kinases in the or presence of human LRIG1 tagged in the extracellular domain with the Myc epitope. with growth lysates from cells were immunoprecipitated with Myc and were examined for the presence of receptors by We observed that EGFR, ErbB2, ErbB3, and ErbB4 each with the LRIG1, co-immunoprecipitation of receptor was not These observations indicate that LRIG1 is of a complex with each of the receptors of the ErbB studies have demonstrated that LRIG1 is in cells of mouse brain (14Suzuki Y. Sato N. Tohyama M. Wanaka A. Takagi T. J. Biol. Chem. 1996; 271: 22522-22527Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), we used the co-immunoprecipitation to the of LRIG1 and in mouse brain We observed that be specifically with LRIG1 that the two proteins are in a complex in Interestingly, lysates from experiments were blotted with anti-receptor a of EGFR, ErbB2, ErbB3, and ErbB4 was observed in the presence of LRIG1, of receptor was observed efficient transfection of these cells 1 and 3 in These observations suggest that a functional of LRIG1 with receptor tyrosine kinases may be suppression of receptor to the of signaling. a transmembrane LRIG1 be in the and with receptors the cell or it be located intracellular and with receptors to or from the cell whether LRIG1 is the cell we first examined its with an of commonly to cell the in 293T cells were transfected with LRIG1 or each of the ErbB cells were and proteins were immunoprecipitated with antibodies and blotted with We observed that each of the proteins be that some of each is the cell the of LRIG1 and of we examined the of receptors and LRIG1 in co-transfected cells by immunofluorescence by of the an the of LRIG1 and in co-transfected cells. LRIG1 is largely in intracellular and is in a region to one of the of LRIG1 is the cell a its presence in intracellular was of the demonstrated that LRIG1 and signals and intracellular that these are of were observed with the other ErbB receptors Previous studies have that the of a single to the EGF receptor is to its and to suppress cellular receptor K. S. S. I. Di Fiore P.P. I. Cell Biol. 2003; PubMed Scopus Google Scholar, Y. K. M. Y. G. J. Yarden Y. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). one mechanism that for the observed of receptors is of receptor ubiquitination and degradation by we examined the and ligand-stimulated ubiquitination of and ErbB4. the in 293T cells were co-transfected with and or ErbB4. Cells were treated without and with growth factors for receptors were and were blotted for the presence of The of HA to receptor was after the We observed that for receptors in the of LRIG1 the of receptor to total with we observed that growth factor the ubiquitination of receptors. Interestingly, for receptors LRIG1 the of receptor to total receptor following growth factor The observations in 3 suggest that the functional of LRIG1 with ErbB receptors may be to receptor ubiquitination in the presence and of growth The of receptors in the presence of LRIG1 and in the of ligand be of the of the LRIG1-mediated receptor degradation in the of ligand may through a mechanism independent of receptor ubiquitination. For LRIG1 is are the ErbB receptors the of receptors with LRIG1 undergo in receptor the other LRIG1 EGF-stimulated receptor that LRIG1 receptor with the ubiquitination the of LRIG1 in receptor degradation and to the of LRIG1 expression on ErbB-mediated cellular growth we established an inducible expression using a cell line that not of We first a of breast and prostate cancer cell lines for LRIG1 expression. We observed that of six breast cancer cell lines and one of four prostate cancer cell lines LRIG1 by Northern blotting We these cell lines for inducible LRIG1 expression studies cells the EGF receptor, ErbB2, and D. Oncogene. 1997; PubMed Scopus Google Scholar) and of the cells may be by EGF receptor ligands J. C. Cancer Res. Google Scholar). it has been that PC3 cells of LRIG1 with prostate as by H. J. Guo D. Henriksson R. Oncol. 2002; PubMed Scopus Google Scholar). clone of PC3 cells was established using the to LRIG1 expression. from these cells in efficient of expression of LRIG1 of LRIG1 expression in these cells in the of of and ErbB2, with the of receptors observed in 293T cell The of receptor in these cells to 293T may from LRIG1 expression whether LRIG1 expression the ubiquitination and degradation rate of ErbB receptors in the inducible expression we on the of LRIG1 expression on the of in the PC3-LRIG1 cells. to the results with transfected 293T cells we observed that the of LRIG1 expression the ubiquitination of in to EGF This of ligand-stimulated ubiquitination with a suppression of tyrosine and receptor after growth factor treatment. the in PC3-LRIG1 cells were treated with and without to LRIG1 and a of EGF stimulation of the cells was carried were with and antibodies. the presence of LRIG1, efficiently to EGF as by blotting receptors with Interestingly, LRIG1 expression in an of revealed that in the of LRIG1 expression an of receptor after of by a However, stimulation of cells expressing LRIG1 in a of receptor after with that the presence of LRIG1 the degradation of after ligand The of ErbB receptors in the of growth factor that its presence may receptor degradation the of LRIG1 expression on EGF receptor PC3-LRIG1 cells were treated with and without to LRIG1 expression and treated with to protein The of EGF receptor following of protein were by and for cells with and without LRIG1 expression. in the presence of LRIG1 the rate which EGF receptor was by cells. the results in suggest that LRIG1 negatively ErbB receptors by receptor ubiquitination and the and ligand-stimulated of receptor The suppression of ErbB receptor by LRIG1 the as to whether LRIG1 might suppress ErbB-mediated cellular growth we first examined the of LRIG1 expression on EGF transformation of NIH3T3 mouse the in NIH3T3 cells were transfected with EGF receptor, or EGF receptor with Cells were treated without or with EGF, and colonies were after of with numerous we observed that EGF in This was LRIG1 was LRIG1 expression was not to suppress transformation by whether LRIG1 the of ErbB receptors to cell cycle EGF- and NRG1-stimulated progression of PC3-LRIG1 cells the to was examined in the presence or of these cells were first treated with or without to LRIG1 expression and treated with to cells to the of the cell cell cycle was and growth factors were Cells were and cell cycle by cell We observed that of LRIG1 expression in the of growth factors a on the of cells the in the of the vast majority of cells with EGF or NRG1β in a of cells into in the of LRIG1 expression. However, the presence of LRIG1 these LRIG1 expression on cell cycle progression These observations indicate that LRIG1 acts as a negative regulator of ErbB-mediated cellular growth regulation. Our observations point to the following for the of LRIG1 in signaling through receptor tyrosine kinases. LRIG1 forms a complex with each of the mammalian ErbB receptors and receptor to a of and ligand-stimulated receptor and a suppression of receptor to with studies have an in the expression of LRIG1 and EGF receptor in human cell M. Hedman H. Guo D. B. Henriksson R. Br. J. Cancer. 2003; PubMed Scopus Google Scholar), the possibility that LRIG1 might act as a of tumor cell growth H. J. Guo D. Henriksson R. Oncol. 2002; PubMed Scopus Google Scholar) by the of receptor be of to the of the to which LRIG1 is in human and to whether LRIG1 enhances the rate of tumor and progression in cultured cell and mouse of human is that the mechanisms by which Kek1 and LRIG1 act may The of Kek1 with mammalian ErbB receptors with ligand and may with receptor or activation (13Ghiglione C. Amundadottir L. Andresdottir M. Bilder D. Diamonti J.A. Noselli S. Perrimon N. Carraway III, K.L. Development. 2003; 130: 4483-4493Crossref PubMed Scopus (46) Google Scholar). the other LRIG1 to suppress ErbB signaling by receptor to receptor and Interestingly, other LRR proteins have been demonstrated to with receptor tyrosine kinases. a secreted containing has been to with the EGF receptor and to M. Wong A.J. J. Investig. 1998; PubMed Scopus Google Scholar, M. I. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). with receptors has been to receptor down-regulation and the of cellular growth through expression of A. M. A. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). a transmembrane protein containing LRRs and a domain 1999; PubMed Scopus Google Scholar), has been demonstrated to with growth factor receptors to receptor signaling specifically through the in N. H. C. Cell Biol. 2004; PubMed Scopus Google Scholar). These observations suggest that some LRR proteins may as of receptor tyrosine kinase to suppress activity or to regulate of signaling pathways. was an observations and was published by G. C. M. I. A. J. N. Henriksson R. G. Hedman H. R. Yarden Y. J. 2004; PubMed Scopus Google Scholar). We for the for and for mouse