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• W2049969188 abstract "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 sacrifice and was homogenized in 3 ml of co-immunoprecipitation buffer. The homogenate was cleared by centrifugation at 14,000 × g. 1 ml of supernatant was immunoprecipitated with 1.5 μg of rabbit anti-IgM or rabbit anti-LRIG1 and blotted with anti-receptor and anti-LRIG1 antibodies. Detection was carried out using horseradish peroxidase-conjugated secondary antibodies (Zymed Laboratories Inc.). Images were captured using an Alpha Innotech imaging station and FluorChem software. Biotinylation Experiments—293T cells in 100-mm dishes were transfected with cDNAs encoding ErbB receptors or LRIG1, and proteins were allowed to express for 30 h. Cells were rinsed twice with ice-cold phosphate-buffered saline and treated with 0.5 mg/ml sulfo-NHS-biotin (Pierce) in phosphate-buffered saline for 1 h at 4 °C. Cells were then rinsed twice with phosphate-buffered saline, lysed in co-immunoprecipitation buffer, and precipitated with appropriate anti-Myc or anti-receptor antibodies. Precipitates were resolved by 6% SDS-PAGE and blotted with horseradish peroxidase-conjugated anti-biotin antibodies (Cell Signaling Technologies). Ubiquitination Experiments—293T cells were transfected with HA-tagged ubiquitin and EGF receptor or ErbB4, without or with LRIG1. Immunoprecipitations were carried out as described above except that cells were lysed in radioimmune precipitation assay 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 5 mmN-ethyl maleimide and sample buffer containing 8 m urea. Ubiquitinated receptors in 293T cells were detected by blotting with anti-HA antibodies. Ubiquitinated receptors in PC3-LRIG1 cells were detected by blotting with anti-ubiquitin monoclonal antibody (Covance). Immunofluorescence—COS7 cells were seeded onto cover slips, transiently transfected with cDNAs encoding LRIG1 and ErbB2 for 24 h, and then fixed with 4% paraformaldehyde for 20 min at room temperature. Cells on cover slips were permeabilized and blocked 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 incubated with 1/500 dilutions of anti-Myc IgG1 and anti-ErbB2 3E8 IgG2a in blocking buffer. Proteins were detected using secondary antibodies AlexaFluor 488 goat anti-mouse IgG2a and AlexaFluor 647 goat anti-mouse IgG1. Fluorescence microscopy was carried out using an Olympus BX61 fluorescence microscope to capture 12 z-plane images at 0.25-μm intervals; Slidebook software was used to deconvolve images. Soft Agar Assays—NIH3T3 cells in 60-mm plates were transfected with combinations of vector alone, EGF receptor, H-Ras, or LRIG1. After 24 h, cells were trypsinized and counted. 2 × 103 cells were seeded in triplicate into 1.5 ml of plating agar (Dulbecco's modified Eagle's medium with 10% FCS and 33% agar, with or without 3 nm EGF) and plated on top of 60-mm Petri dishes precoated with 2 ml of underlay agar (Dulbecco's modified Eagle's medium with 10% FCS and 50% agar, with or without 3 nm EGF). Cells were fed once a week for 3.5 weeks and then stained with thiazolyl blue (Sigma). Colonies on plates were counted using an Alpha Innotech imaging station and FluorChem software. Cell Cycle Analysis—PC3-LRIG1 cells were simultaneously serum starved and treated with and without tetracycline for 12 h and then treated with 1 mm hydroxyurea for an additional 12 h. Cells were then treated with 10% serum, 50 nm NRG1β, or 33 nm EGF for various times, collected, and fixed in 70% ethanol/phosphate-buffered saline for a minimum of 2 h at 4 °C. They were then stained with 0.1 mg/ml propidium iodide staining solution (0.1 mg/ml propidium iodide, 2 mm EDTA, 0.2% Triton X-100, 50 mg/ml RNase A) for 1 h at 4 °C. The fluorescence intensities of the samples were measured by quantitative flow cytometry using a BD PharMingen FACScan scanner. Analysis was based on 20,000 cells and was linearly scaled. To determine whether LRIG1 might influence the properties of human ErbB receptors, we first examined the association of the proteins by co-immunoprecipitation. In the experiment shown in Fig. 1A, 293T cells were transiently transfected with human ErbB receptor tyrosine kinases in the absence or presence of human LRIG1 tagged in the extracellular domain with the Myc epitope. Following treatment with appropriate growth factor, lysates from cells were immunoprecipitated with Myc antibodies, and precipitates were examined for the presence of receptors by immunoblotting. We observed that EGFR, ErbB2, ErbB3, and ErbB4 each co-immunoprecipitated with the 145-kDa LRIG1, but co-immunoprecipitation of endogenous insulin receptor was not observed. These observations indicate that LRIG1 is capable of forming a specific complex with each of the receptors of the ErbB family. Because previous studies have demonstrated that LRIG1 is prominently expressed in glial 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 assay to examine the association of LRIG1 and EGFR in mouse brain lysates. We observed that EGFR could be specifically co-immunoprecipitated with LRIG1 (Fig. 1B), indicating that the two proteins are associated in a complex in vivo. Interestingly, when lysates from co-transfection experiments were blotted with anti-receptor antibodies, a loss of EGFR, ErbB2, ErbB3, and ErbB4 was observed in the presence of LRIG1, but no loss of insulin receptor was observed despite very efficient transfection of these cells (Fig. 1C, compare lanes 1 and 3 in all panels). These observations suggest that a functional consequence of LRIG1 interaction with receptor tyrosine kinases may be suppression of receptor levels to lower the efficiency of signaling. As a transmembrane protein, LRIG1 could be present in the plasma membrane and interact with receptors at the cell surface, or it could be located within membranous intracellular compartments and interact with receptors during their transit to or from the cell surface. To determine whether LRIG1 is present at the cell surface, we first examined its reactivity with sulfo-biotin-X-NHS, an amine-reactive, membrane-impermeant form of biotin commonly employed to biotinylate cell surface proteins. In the experiment depicted in Fig. 2A, 293T cells were transfected with LRIG1 or each of the ErbB receptors, cells were biotinylated, and expressed proteins were immunoprecipitated with their respective antibodies and blotted with anti-biotin. We observed that each of the proteins could be biotinylated, indicating that some fraction of each is present at the cell surface. To determine the subcellular location of LRIG1 and possible sites of LRIG1-receptor interaction, we examined the localization of receptors and LRIG1 in co-transfected COS7 cells by three-dimensional indirect immunofluorescence followed by deconvolution of the resulting z-plane images. As an example, Fig. 2B illustrates the localization of LRIG1 and ErbB2 in co-transfected cells. LRIG1 is found largely in perinuclear intracellular compartments and is particularly concentrated in a region to one side of the nucleus. A fraction of LRIG1 is also found at the cell periphery. ErbB2 exhibited a similar distribution, although its presence in intracellular structures was less pronounced. Merging of the images demonstrated that LRIG1 and ErbB2 signals overlap at both peripheral and intracellular locations, suggesting that these are sites of LRIG1-ErbB2 interaction. Similar co-localization patterns were observed with the other ErbB receptors (not shown). Previous studies have shown that the attachment of a single ubiquitin moiety to the EGF receptor is sufficient to decrease its stability and to suppress cellular receptor levels (21Haglund K. Sigismund S. Polo S. Szymkiewicz I. Di Fiore P.P. Dikic I. Nat. Cell Biol. 2003; 5: 461-466Crossref PubMed Scopus (666) Google Scholar, 22Mosesson Y. Shtiegman K. Katz M. Zwang Y. Vereb G. Szollosi J. Yarden Y. J. Biol. Chem. 2003; 278: 21323-21326Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Thus, one mechanism that could account for the observed LRIG1-induced loss of receptors is promotion of receptor ubiquitination and degradation by LRIG1. To test this, we examined the basal and ligand-stimulated ubiquitination of EGFR and ErbB4. In the experiment illustrated in Fig. 3, 293T cells were co-transfected with HA-tagged ubiquitin and either EGFR or ErbB4. Cells were treated without and with their respective growth factors for 5 min, receptors were immunoprecipitated, and precipitates were blotted for the presence of HA. The ratio of HA immunoreactivity to receptor immunoreactivity was then determined after quantifying the respective bands. We observed that for both receptors in the absence of added ligand, LRIG1 reduced the ratio of ubiquitinated receptor to total receptor. Consistent with previous reports, we observed that growth factor treatment promoted the rapid ubiquitination of receptors. Interestingly, for both receptors LRIG1 markedly potentiated the ratio of ubiquitinated receptor to total receptor following growth factor stimulation. The observations illustrated in Fig. 3 suggest that the functional consequence of LRIG1 interaction with ErbB receptors may be to promote receptor ubiquitination both in the presence and absence of growth factor. The apparent loss of ubiquitinated receptors in the presence of LRIG1 and in the absence of added ligand could be because of the relatively rapid loss of the ubiquitinated population. Alternatively, LRIG1-mediated receptor degradation in the absence of ligand may occur through a mechanism independent of receptor ubiquitination. For example, because LRIG1 is more rapidly degraded than are the ErbB receptors (not shown) the population of receptors associated with LRIG1 would undergo accelerated degradation, resulting in lower basal receptor levels. On the other hand, LRIG1 clearly augments EGF-stimulated receptor ubiquitination, suggesting that LRIG1 also facilitates receptor interaction with the ubiquitination machinery. To examine the role of LRIG1 in receptor degradation and to determine the impact of LRIG1 expression on ErbB-mediated cellular growth properties, we established an inducible expression system using a cell line that does not express significant endogenous levels of LRIG1. We first surveyed a series of breast and prostate cancer cell lines for LRIG1 expression. We observed that three of six breast cancer cell lines and one of four prostate cancer cell lines detectably express LRIG1 by Northern blotting (Fig. 4A). We chose these cell lines for inducible LRIG1 expression studies because cells express the EGF receptor, ErbB2, and ErbB3 (23Grasso A.W. Wen D. Miller C.M. Rhim J.S. Pretlow T.G. Kung H.J. Oncogene. 1997; 15: 2705-2716Crossref PubMed Scopus (128) Google Scholar) and proliferation of the cells may be stimulated by EGF receptor ligands (24Hofer D.R. Sherwood E.R. Bromberg W.D. Mendelsohn J. Lee C. Kozlowski J.M. Cancer Res. 1991; 51: 2780-2785PubMed Google Scholar). In addition, it has been reported that PC3 cells express extremely low levels of LRIG1 when compared with normal prostate tissue as assessed by real-time RT-PCR (25Hedman H. Nilsson J. Guo D. Henriksson R. Acta Oncol. 2002; 41: 352-354Crossref PubMed Scopus (80) Google Scholar). A clone of PC3 cells (PC3-LRIG1) was established using the Tet-Off system to repress LRIG1 expression. Tetracycline withdrawal from these cells resulted in efficient induction of expression of Myc-tagged LRIG1 (Fig. 4B). Induction of LRIG1 expression in these cells resulted in the ligand-independent loss of 30–60% of endogenous EGFR and ErbB2, consistent with the LRIG1-induced loss of receptors observed in 293T cell co-transfectants (Fig. 1B). The lower efficiency of receptor loss in these cells relative to 293T co-transfectants may result from lower LRIG1 expression levels. To determine whether LRIG1 expression influences the ubiquitination and degradation rate of ErbB receptors in the inducible expression system, we focused on the effect of LRIG1 expression on the properties of EGFR in the PC3-LRIG1 cells. Similar to the results with transfected 293T cells (Fig. 3), we observed that the induction of LRIG1 expression markedly augmented the ubiquitination of EGFR in response to EGF treatment (Fig. 5A). This potentiation of ligand-stimulated EGFR ubiquitination correlated with a suppression of EGFR tyrosine phosphorylation and receptor levels after growth factor treatment. In the experiment illustrated in Fig. 5B, PC3-LRIG1 cells were treated with and without tetracycline to induce LRIG1 expression, and a time course of EGF stimulation of the cells was carried out. Lysates were immunoblotted with anti-phosphotyrosine and anti-EGFR antibodies. In the presence of LRIG1, EGFR responded less efficiently to EGF as determined by blotting autophosphorylated receptors with anti-phosphotyrosine. Interestingly, LRIG1 expression also resulted in an accelerated loss of EGFR. Anti-EGFR blots revealed that in the absence of LRIG1 expression an immediate loss of receptor occurred after 3–15 min of stimulation, followed by a recovery at 30–60 min. However, stimulation of cells expressing LRIG1 also resulted in a loss of receptor after 3–15 min but with little if any recovery, suggesting that the presence of LRIG1 accelerates the degradation of EGFR after ligand stimulation. The LRIG1-induced loss of ErbB receptors in the absence of growth factor suggests that its presence may also affect basal receptor degradation rate. To examine the impact of LRIG1 expression on basal EGF receptor stability, PC3-LRIG1 cells were treated with and without tetracycline to induce LRIG1 expression and then treated with cycloheximide to inhibit protein synthesis. The levels of EGF receptor remaining at various times following inhibition of protein synthesis were then determined by immunoblotting and quantitatively compared for cells with and without LRIG1 expression. As shown in Fig. 5C, the presence of LRIG1 accelerated the rate at which EGF receptor was degraded by cells. Taken together, the results depicted in Fig. 5 suggest that LRIG1 negatively regulates ErbB receptors by promoting receptor ubiquitination and increasing the basal and ligand-stimulated rates of receptor degradation. The suppression of ErbB receptor levels by LRIG1 raises the question as to whether LRIG1 might also suppress ErbB-mediated cellular growth properties. To test this, we first examined the impact of LRIG1 expression on ligand-dependent EGF receptor-mediated transformation of NIH3T3 mouse fibroblasts. In the experiment depicted in Fig. 6A, NIH3T3 cells were transfected with control vector alone, EGF receptor, or EGF receptor together with LRIG1. Cells were treated without or with EGF, and colonies were counted after 3.5 weeks of growth. Consistent with numerous previous reports, we observed that EGF stimulated colony formation in this assay. This effect was completely blocked when LRIG1 was co-expressed, but LRIG1 expression was not able to suppress transformation induced by H-Ras (not shown). To determine whether LRIG1 modulates the ability of ErbB receptors to promote cell cycle progression, EGF- and NRG1-stimulated progression of PC3-LRIG1 cells synchronized at the G1/S boundary to S-phase was examined in the presence or absence of LRIG1. In these experiments, cells were first treated with or without tetracycline to induce LRIG1 expression and then treated with hydroxyurea to synchronize cells to the G1/S boundary of the cell cycle. At time 0, cell cycle block was removed and growth factors were added. Cells were then stopped at various time points and their cell cycle distribution determined by fluorescence-activated cell sorting. We observed that induction of LRIG1 expression in the absence of growth factors had a negligible impact on the synchronization of cells at the G1/S boundary, reflected in the accumulation of the vast majority of cells at G0/G1 under both conditions (Fig. 3B). Stimulation with EGF or NRG1β resulted in a dramatic shift of cells into S-phase in the absence of LRIG1 expression. However, the presence of LRIG1 markedly suppressed these effects. In contrast, LRIG1 expression had no effect on serum-stimulated cell cycle progression (not shown). These observations indicate that LRIG1 acts as a negative regulator of ErbB-mediated cellular growth regulation. Our observations point to the following model for the role of LRIG1 in modulating signaling through receptor tyrosine kinases. LRIG1 forms a complex with each of the mammalian ErbB receptors and promotes receptor ubiquitination, leading to a loss of steady state and ligand-stimulated receptor levels and a suppression of receptor ability to mediate mitogenic responses. Consistent with this, previous studies have reported an inverse link in the expression levels of LRIG1 and EGF receptor in human renal cell carcinomas (26Thomasson M. Hedman H. Guo D. Ljungberg B. Henriksson R. Br. J. Cancer. 2003; 89: 1285-1289Crossref PubMed Scopus (67) Google Scholar), raising the possibility that LRIG1 might act as a suppressor of tumor cell growth (25Hedman H. Nilsson J. Guo D. Henriksson R. Acta Oncol. 2002; 41: 352-354Crossref PubMed Scopus (80) Google Scholar) by limiting the extent of receptor activation. It will be of interest to expand the analysis of the extent to which LRIG1 is lost in human tumors and to determine whether LRIG1 loss enhances the rate of tumor formation and progression in cultured cell and mouse models of human cancer. It is noteworthy that the physical mechanisms by which Kek1 and LRIG1 act may differ significantly. The physical interaction of Kek1 with mammalian ErbB receptors interferes with ligand binding and may also interfere with receptor dimerization 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). On the other hand, LRIG1 appears to suppress ErbB signaling by promoting receptor degradation, leading to lower basal receptor levels and suppressed mitogenic capacity. Interestingly, other LRR domain-containing proteins have also been demonstrated to interact with receptor tyrosine kinases. Decorin, a secreted proteoglycan containing nine LRRs, has been shown to interact with the EGF receptor and ErbB2 to initially enhance their activities (27Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Investig. 1998; 101: 406-412Crossref PubMed Scopus (245) Google Scholar, 28Santra M. Eichstetter I. Iozzo R.V. J. Biol. Chem. 2000; 275: 35153-35161Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Decorin interaction with receptors has been suggested to mediate prolonged receptor down-regulation and the inhibition of cellular growth through enhanced expression of p21waf1 (29De Luca A. Santra M. Baldi A. Giordano A. Iozzo R.V. J. Biol. Chem. 1996; 271: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). FLRT3, a transmembrane protein containing 11 LRRs and a fibronectin type III domain (30Lacy S.E. Bonnemann C.G. Buzney E.A. Kunkel L.M. Genomics. 1999; 62: 417-426Crossref PubMed Scopus (94) Google Scholar), has been demonstrated to interact with fibroblast growth factor receptors to stimulate receptor signaling specifically through the Erk pathway in Xenopus laevis (31Bottcher R.T. Pollet N. Delius H. Niehrs C. Nat. Cell Biol. 2004; 6: 38-44Crossref PubMed Scopus (140) Google Scholar). These observations suggest that some LRR proteins may serve as modulators of receptor tyrosine kinase signaling, to either suppress activity or to selectively regulate subsets of downstream signaling pathways. While this report was under revision, an article presenting similar observations and conclusions was published by Gur et al. (32Gur G. Rubin C. Katz M. Amit I. Citri A. Nilsson J. Amariglio N. Henriksson R. Rechavi G. Hedman H. Wides R. Yarden Y. EMBO J. 2004; 23: 3270-3281Crossref PubMed Scopus (237) Google Scholar). We thank Cary Lai for providing the GST-NRG1 construct, Andrea Morrione for providing HA-tagged ubiquitin, and Ron Wisdom for providing mouse brain." @default.
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• W2049969188 title "The Leucine-rich Repeat Protein LRIG1 Is a Negative Regulator of ErbB Family Receptor Tyrosine Kinases" @default.
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