Neuronal Leucine-rich Repeat Protein-3 Amplifies MAPK Activation by Epidermal Growth Factor through a Carboxyl-terminal Region Containing Endocytosis Motifs

Neuronal leucine-rich repeat protein-3 (NLRR-3) belongs to the LRR superfamily. Expression of rat NLRR-3 gene isolated from c-Ha-ras transgenic rat tumor is regulated mainly through the Ras-MAPK signaling pathway. NLRR-3 was found to enhance phosphorylation of MAPK when COS-7 cells were transfected with NLRR-3 and stimulated with a low concentration (0.01 ng/ml) of epidermal growth factor (EGF), but the amplification of MAPK phosphorylation by NLRR-3 was no longer observed when the carboxyl-terminal 30 amino acid stretch containing clathrin-mediated endocytosis motifs was deleted. A green fluorescent protein-tagged NLRR-3 localized at the plasma membrane was efficiently internalized in COS-7 cells, but internalization of a carboxyl-terminal-deleted version (NLRRΔC) was less efficient. The presence of clathrin-adaptor protein complexes containing NLRR-3 in brain lysate was confirmed by immunoprecipitation and glutathione S-transferase pull-down experiments, and affinity column chromatography revealed that the carboxyl-terminal region of NLRR-3 interacts with β-adaptin. We propose that NLRR-3 potentiates Ras-MAPK signaling by facilitating internalization of EGF in clathrin-coated vesicles. Neuronal leucine-rich repeat protein-3 (NLRR-3) belongs to the LRR superfamily. Expression of rat NLRR-3 gene isolated from c-Ha-ras transgenic rat tumor is regulated mainly through the Ras-MAPK signaling pathway. NLRR-3 was found to enhance phosphorylation of MAPK when COS-7 cells were transfected with NLRR-3 and stimulated with a low concentration (0.01 ng/ml) of epidermal growth factor (EGF), but the amplification of MAPK phosphorylation by NLRR-3 was no longer observed when the carboxyl-terminal 30 amino acid stretch containing clathrin-mediated endocytosis motifs was deleted. A green fluorescent protein-tagged NLRR-3 localized at the plasma membrane was efficiently internalized in COS-7 cells, but internalization of a carboxyl-terminal-deleted version (NLRRΔC) was less efficient. The presence of clathrin-adaptor protein complexes containing NLRR-3 in brain lysate was confirmed by immunoprecipitation and glutathione S-transferase pull-down experiments, and affinity column chromatography revealed that the carboxyl-terminal region of NLRR-3 interacts with β-adaptin. We propose that NLRR-3 potentiates Ras-MAPK signaling by facilitating internalization of EGF in clathrin-coated vesicles. Leucine-rich repeat (LRR) 1The abbreviations used are: LRR, leucine-rich repeat; NLRR-3, neuronal leucine-rich repeat protein-3; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; aa, amino acid; GFP, green fluorescent protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol domains were first identified in an α-2-glycoprotein in human serum (1Takahashi N. Takahashi Y. Putnam F.W. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1906-1910Crossref PubMed Scopus (235) Google Scholar). They contain highly hydrophobic amino acids and a repeated structure consisting of about 24 residues (2Rothberg J.M. Jacobs J.R. Goodman C.S. Artavanis-Tsakonas S. Genes Dev. 1990; 4: 2169-2187Crossref PubMed Scopus (434) Google Scholar), and the LRR motif provides an ideal conformation for binding to other proteins. Neuronal LRR protein (NLRR) genes were first isolated from a mouse brain cDNA library (3Taguchi A. Wanaka A. Mori T. Matsumoto K. Imai Y. Tagaki T. Tohyama M. Mol. Brain Res. 1996; 35: 31-40Crossref PubMed Scopus (56) Google Scholar, 4Taniguchi H. Tohyama M. Takagi T. Mol. Brain Res. 1996; 36: 45-52Crossref PubMed Scopus (35) Google Scholar), and three distinct isoforms, NLRR-1, -2, and -3, have been identified in fish, frog, mouse, rat, and humans (3Taguchi A. Wanaka A. Mori T. Matsumoto K. Imai Y. Tagaki T. Tohyama M. Mol. Brain Res. 1996; 35: 31-40Crossref PubMed Scopus (56) Google Scholar, 4Taniguchi H. Tohyama M. Takagi T. Mol. Brain Res. 1996; 36: 45-52Crossref PubMed Scopus (35) Google Scholar, 5Hayata T. Uochi T. Asashima M. Gene. 1998; 221: 159-166Crossref PubMed Scopus (18) Google Scholar, 6Bormann P. Roth L.W. Andel D. Ackermann M. Reinhard E. Mol. Cell. Neurosci. 1999; 13: 167-179Crossref PubMed Scopus (42) Google Scholar, 7Fukamachi K. Matsuoka Y. Kitanaka C. Kuchino Y. Tsuda H. Biochem. Biophys. Res. Commun. 2001; 287: 257-263Crossref PubMed Scopus (19) Google Scholar). These isoforms constitute a novel LRR protein family containing 11 or 12 LRRs, one immunoglobulin-like domain, and one fibronectin type III-like domain (5Hayata T. Uochi T. Asashima M. Gene. 1998; 221: 159-166Crossref PubMed Scopus (18) Google Scholar, 6Bormann P. Roth L.W. Andel D. Ackermann M. Reinhard E. Mol. Cell. Neurosci. 1999; 13: 167-179Crossref PubMed Scopus (42) Google Scholar, 7Fukamachi K. Matsuoka Y. Kitanaka C. Kuchino Y. Tsuda H. Biochem. Biophys. Res. Commun. 2001; 287: 257-263Crossref PubMed Scopus (19) Google Scholar). Although, based on their structural features, NLRRs have been proposed to function as adhesion molecules or soluble ligand binding receptors, little is known about their biological activities. The 170-kDa epidermal growth factor receptor (EGFR) exerts its biological effects in response to binding of specific polypeptide ligands, including epidermal growth factor (EGF) and transforming growth factor-α. Binding of the ligands leads to activation of the EGFR catalytic tyrosine kinase domain, autophosphorylation of specific residues in its carboxyl terminus, and recruitment and phosphorylation of heterologous signaling proteins such as Shc (8McCormick F. Nature. 1993; 363: 15-16Crossref PubMed Scopus (445) Google Scholar, 9van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1245) Google Scholar). Shc binds to activated EGFR and becomes phosphorylated on Tyr317, creating a binding site for Grb2-Sos (10Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar). Once Ras has been activated by Sos, GTP-bound Ras interacts with and facilitates activation of target enzymes, including the serine/threonine kinase Raf (11Marshall C.J. Nature. 1996; 383: 127-128Crossref PubMed Scopus (100) Google Scholar). Activated Raf phosphorylates and activates the downstream kinase, the MAPK/ERK kinase, which in turn phosphorylates and activates MAPK/ERK (12Marshall C. Curr. Opin. Cell Biol. 1999; 11: 732-736Crossref PubMed Scopus (142) Google Scholar). A consequence of EGFR activation is the clustering of ligand-receptor complexes in clathrin-coated pits, which increases the rate of receptor internalization (13Chang C.P. Lazar C.S. Walsh B.J. Komuro M. Collawn J.F. Kuhn L.A. Tainer J.A. Trowbridge I.S. Farquhar M.G. Rosenfeld M.G. J. Biol. Chem. 1993; 268: 19312-19320Abstract Full Text PDF PubMed Google Scholar). Endocytosis of the receptor has been recognized as an attenuation mechanism that affects long-term EGFR function (14Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Crossref PubMed Scopus (343) Google Scholar,15Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (828) Google Scholar). However, it has been demonstrated that EGF remains associated predominantly with EGFR in sorting endosomes and that internalized EGF-EGFR complexes retain equal tyrosine phosphorylation stoichiometry as well as competency in binding, phosphorylating, and activating signaling proteins (16Di Guglielmo G.M. Baass P.C., Ou, W.J. Posner B.I. Bergeron J.J. EMBO J. 1994; 13: 4269-4277Crossref PubMed Scopus (299) Google Scholar, 17Haugh J.M. Huang A.C. Wiley H.S. Wells A. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 34350-34360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Haugh J.M. Schooler K. Wells A. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 8958-8965Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 19Sorkina T. Bild A. Tebar F. Sorkin A. J. Cell Sci. 1999; 112: 317-327Crossref PubMed Google Scholar). This suggests that meaningful signal transduction might be extended after endocytosis of EGF, and several studies have revealed that EGFR internalization does indeed amplify MAPK phosphorylation (17Haugh J.M. Huang A.C. Wiley H.S. Wells A. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 34350-34360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Haugh J.M. Schooler K. Wells A. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 8958-8965Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 20Sato Y. Takahashi M. Shibukawa Y. Jain S.K. Hamaoka R. Miyagawa J. Yaginuma Y. Honke K. Ishikawa M. Taniguchi N. J. Biol. Chem. 2001; 276: 11956-11962Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 21Schoeberl B. Eichler-Jonsson C. Gilles E.D. Muller G. Nat. Biotechnol. 2002; 20: 370-375Crossref PubMed Scopus (773) Google Scholar). In this study we found that when COS-7 cells transfected with NLRR-3 were stimulated with a low concentration of EGF, NLRR-3 enhanced phosphorylation of MAPK with no effect on EGFR or Shc phosphorylation; the carboxyl-terminal region of NLRR-3, which contains two endocytosis motifs, seemed responsible for this effect. COS-7 and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were transfected overnight using FuGENE6 (Roche Molecular Biochemicals) according to the manufacturer's directions, and after starving in serum-free medium (Opti-MEM I, Invitrogen) for 24 h, they were exposed to EGF (Invitrogen). To construct NLRR-3 protein tagged at the carboxyl terminus with green fluorescent protein (GFP), the TAG stop codon of rat NLRR-3 cDNA (7Fukamachi K. Matsuoka Y. Kitanaka C. Kuchino Y. Tsuda H. Biochem. Biophys. Res. Commun. 2001; 287: 257-263Crossref PubMed Scopus (19) Google Scholar) was removed by PCR with the forward primer 5′-TTAAGCTTTAAGATGAAGGACGCACCAC-3′, containing the HindIII site (underlined), and the reverse primer 5′-TACCCGGGACATACTTGTCGGCACAC-3′, containing theSmaI site (underlined). A truncated NLRR-3 construct lacking aa 676–707 (NLRRΔC) was prepared by PCR using the above forward primer and the reverse primer 5′-TTGGATCCAGCTCACTGAATGCCAAGGT-3′, containing theBamHI site (underlined). The PCR product was excised withHindIII/SmaI orHindIII/BamHI and inserted into pEGFP-N1 (Clontech), and the sequence was then confirmed. To construct the carboxyl terminus of rat NLRR-3 (amino acids 638–707) tagged with glutathione S-transferase (GST) at the NH2 terminus (GST-W), rat NLRR-3 cDNA (7Fukamachi K. Matsuoka Y. Kitanaka C. Kuchino Y. Tsuda H. Biochem. Biophys. Res. Commun. 2001; 287: 257-263Crossref PubMed Scopus (19) Google Scholar) was excised with Sau3AI (blunting)/NotI and inserted into pGEX-6P-1 (Amersham Biosciences). A NLRR-3 construct lacking amino acids 676–707 (GST-ΔC) was prepared by PCR-based mutagenesis using the primer 5′-TTGTCGACTAAAGCTCACTGAATGCCAAGG-3′ containing theSalI site (underlined). To construct NLRR-3 protein containing the extracellular region of rat NLRR-3 (aa 401–577) tagged with GST at the NH2 terminus (GST-NLRR3ex), rat NLRR-3 cDNA was excised with EcoRI/Bst1107I and inserted into pGEX-6P-1. GST fusion proteins were expressed in Escherichia coli strain BL21. Protein expression was induced by 1.0 mmisopropyl-β-d-thiogalactopyranoside for 3 h at 37 °C. The bacterial cultures were centrifuged at 4200 rpm for 20 min, and the pellets were resuspended in buffer A (0.5 mg/ml lysozyme, 10 mm PBS, pH 7.0, 2 mm EGTA, 1 mmPMSF, 20 mm MgCl2, 25 mg/ml DNase I, 1% Triton X-100, 1 mm DTT). After centrifugation at 4200 rpm for 20 min, the pellets containing GST fusion protein were lysed in buffer B (10 mm PBS, pH 7.0, 8 m urea, 2 mm EGTA, 1 mm PMSF, 1 mm DTT) and dialyzed against buffer C (10 mm PBS, pH 7.0, 2m urea, 2 mm EGTA, 1 mm DTT, 0.1% NaN3) at 4 °C overnight. The lysates were clarified by centrifugation at 100,000 × g for 30 min, and after incubation with glutathione-Sepharose 4B (Amersham Biosciences) for 1 h at room temperature, they were washed (three times) with 1% Triton, 1 mm DTT, 0.1% NaN3, 10 mmPBS. The bound proteins were then eluted with 10 mm reduced glutathione in 50 mm Tris-HCl, pH 8.0. A peptide with the sequence KATAIGVPISMS (aa 696–707) was synthesized and conjugated to keyhole limpet hemocyanin (Pierce) for use as the antigen to obtain polyclonal antibody against the cytoplasmic region of rat NLRR-3. And GST-NLRR3ex was used as the antigen to obtain polyclonal antibody against the extracellular region of NLRR-3. Three rabbits were immunized using these antigens and TiterMax Gold (CytRx Corp., Norcross, GA) as an adjuvant. The serum containing the antibody against the extracellular region of NLRR-3 was precleared with glutathione-Sepharose 4B coupled with GST. The specific antibodies were purified by affinity chromatography on HiTrap affinity columns (Amersham Biosciences) coupled with the corresponding antigens. The cells were rinsed twice with PBS and then fixed for 5 min in 3.7% formalin in PBS. Fixed cells were blocked in 10% goat serum in PBS for 10 min at room temperature and then incubated with Alexafluor 568-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) at a 1:500 dilution. Coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were collected by confocal microscopy (Fluoview FV300, Olympus Optical Co., Tokyo, Japan). A rat brain was lysed in 3 ml of lysis buffer (1% Triton X-100, 10% glycerol, 50 mm NaCl, 50 mm Hepes, pH 7.3, 1% sodium deoxycholate, 1 mmEGTA, 1 mm sodium orthovanadate, 1 mm PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin) by 20–30 strokes in a tight-fitting Potter homogenizer. The lysates were centrifuged at 100,000 × g for 20 min and precleaned with 20 μl of protein G-Sepharose 4 Fast Flow (Amersham Biosciences)/300 μl of lysate for 1 h. After centrifugation at 15,000 rpm for 1 min, supernatants were incubated with 20 μl of anti-NLRR3 (against cytoplasmic region) or anti-β-adaptin (Santa Cruz Biotechnology, Santa Cruz, CA) antibody for 8 h followed by overnight incubation with 20 μl of the protein G-Sepharose beads. Immunoprecipitates were washed twice with lysis buffer supplemented with 100 mmNaCl and then five times without NaCl. Immunocomplexes were resuspended in 40 μl of SDS sample buffer for Western blot analysis. All procedures were carried out at 4 °C. Western blot analysis was performed as described elsewhere (22Matsuoka Y., Li, X. Bennett V. J. Cell Biol. 1998; 142: 485-497Crossref PubMed Scopus (173) Google Scholar). Anti-β-adaptin (20 ng/ml), anti-clathrin (100 ng/ml) (Santa Cruz), anti-GST (125 ng/ml) (Amersham Biosciences), anti-EGFR (200 ng/ml), anti-phospho-EGFR (Y1173) (1 μg/ml), anti-MAPK (16 ng/ml) (Upstate Biotechnology, Lake Placid, NY), anti-phospho-MAPK (1 μg/ml), and anti-phospho-Shc (1 μg/ml) (Cell Signaling Technology, Beverly, MA) antibodies were used. Horseradish peroxidase-conjugated anti-rabbit, anti-goat, and anti-mouse IgG antibodies (Southern Biotechnology Associates, Birmingham, AL) and ECL plus Western blotting detection reagents (Amersham Biosciences) were employed to detect the bound first antibodies. A rat brain was lysed in 15 ml of lysis buffer (50 mm HEPES, pH 7.3, 1% Triton X-100, 10% glycerol, 100 mm NaCl, 1 mm EDTA, 2 mm EGTA, 1 mm PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm Na3VO4, 10 mm NaF), and was removed by centrifugation at × g for The was precleared for 2 h with 4B (Amersham Biosciences) coupled with GST and then was incubated overnight with the or column was washed with 1 ml of lysis buffer and eluted into with a of and mm ml All procedures were performed at 4 °C. the carboxyl terminus of NLRR-3 contains two endocytosis motifs (7Fukamachi K. Matsuoka Y. Kitanaka C. Kuchino Y. Tsuda H. Biochem. Biophys. Res. Commun. 2001; 287: 257-263Crossref PubMed Scopus (19) Google Scholar) and the of EGFR internalization to Ras-MAPK activation has been (17Haugh J.M. Huang A.C. Wiley H.S. Wells A. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 34350-34360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Haugh J.M. Schooler K. Wells A. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 8958-8965Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 20Sato Y. Takahashi M. Shibukawa Y. Jain S.K. Hamaoka R. Miyagawa J. Yaginuma Y. Honke K. Ishikawa M. Taniguchi N. J. Biol. Chem. 2001; 276: 11956-11962Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 21Schoeberl B. Eichler-Jonsson C. Gilles E.D. Muller G. Nat. Biotechnol. 2002; 20: 370-375Crossref PubMed Scopus (773) Google Scholar), we the effect of of NLRR-3 on MAPK activation through COS-7 cells, which NLRR-3, were transfected with or NLRR-3 tagged with at the carboxyl after serum for 24 h, they were with of EGF and 10 of MAPK was by with anti-phospho-MAPK of MAPK phosphorylation by a low concentration of EGF (0.01 ng/ml) was in cells transfected with NLRR-3, but increases in phosphorylation in response to of EGF were observed in transfected and NLRR-3 transfected cells 1 A and EGFR autophosphorylation or Shc phosphorylation was at of EGF, but phosphorylation was observed at the 1 A and has been demonstrated that Ras activation with EGF above phosphorylation of EGFR Shc (12Marshall C. Curr. Opin. Cell Biol. 1999; 11: 732-736Crossref PubMed Scopus (142) Google Scholar, S. J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). We in the presence of NLRR-3, of EGF ng/ml) MAPK phosphorylation downstream of Ras activation by that phosphorylation of EGFR and of NLRR-3 no effect on the of expression of MAPK or EGFR or on the phosphorylation of MAPK, EGFR, or Shc The were using cells transfected with NLRR-3 COS-7 cells were transfected with NLRR-3 and with of EGF, phosphorylation of MAPK were for at 4 h, phosphorylation of MAPK was in the cells the carboxyl-terminal of NLRR-3, We that the cytoplasmic carboxyl-terminal 30 amino acids responsible for NLRR-3 amplification of MAPK NLRR-3 a stretch of amino acids containing two clathrin-mediated endocytosis motifs, is hydrophobic amino and a motif H. D. D. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. H. I. S. T. A. T. Science. PubMed Scopus Google Scholar, T. Annu. Rev. Biochem. PubMed Scopus Google Scholar, I. P. T. EMBO J. 1998; PubMed Scopus Google Scholar), which is well in the family 2 of the NLRR-3 was with at the of plasma membrane of the COS-7 and NIH3T3 cells The carboxyl-terminal stretch was found to be for endocytosis of NLRR-3. NLRR-3 tagged with was efficiently internalized when COS-7 cells the protein were incubated at 37 °C 2 of the carboxyl-terminal (NLRRΔC) at the plasma membrane after incubation for 4 h at 37 °C in The plasma of the cells the NLRR-3 and by the extracellular domain antibody when incubated at 4 °C 2 that of the fusion protein were to the plasma membrane and at the were when the cells been incubated for 1 h GST and affinity column binding were carried out to the NLRR-3 and the clathrin-adaptor protein A isolated from rat brain lysate with anti-β-adaptin antibody NLRR-3 and and in a reverse and were identified in a by antibody This was confirmed by GST pull-down using a GST fusion protein containing the cytoplasmic domain described NLRR-3 with the clathrin-adaptor protein The site of of NLRR-3 with the clathrin-adaptor protein was in the carboxyl-terminal 30 aa containing the endocytosis GST fusion one containing the cytoplasmic domain and the other lacking the carboxyl-terminal 30 aa were coupled to and of the clathrin-adaptor protein was by with anti-β-adaptin antibody The protein bound to eluted out at mm NaCl, and was of with with an observed at mm 3 of the protein was washed out of the column coupled with at mm NaCl, and was in the at the of NaCl. on the above we that the carboxyl-terminal region of NLRR-3 is responsible for the amplification of MAPK phosphorylation through with vesicles. and S. J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar) demonstrated that low of EGF ng/ml) MAPK through Ras activation in COS-7 They that phosphorylation of Shc and Shc and binding of these molecules to EGFR, is the mechanism responsible for Ras activation induced by this of EGF S. J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). we observed of MAPK phosphorylation in the COS-7 cells by the concentration of EGF but by the concentration the EGF MAPK phosphorylation in the presence of NLRR-3 with no amplification of EGFR an Shc binding and Shc the binding phosphorylation 1 it is less that NLRR-3 affects the signaling at the plasma membrane that to MAPK The of study demonstrated that NLRR-3 MAPK activation by an low concentration of EGF with no effects on the of expression of EGFR or MAPK or on the phosphorylation of EGFR and The carboxyl-terminal stretch of NLRR-3 was found to be responsible for this and the region bound to the clathrin-adaptor protein suggests that receptor tyrosine kinase complexes such as EGFR (16Di Guglielmo G.M. Baass P.C., Ou, W.J. Posner B.I. Bergeron J.J. EMBO J. 1994; 13: 4269-4277Crossref PubMed Scopus (299) Google Scholar, 17Haugh J.M. Huang A.C. Wiley H.S. Wells A. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 34350-34360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Haugh J.M. Schooler K. Wells A. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1999; 274: 8958-8965Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 19Sorkina T. Bild A. Tebar F. Sorkin A. J. Cell Sci. 1999; 112: 317-327Crossref PubMed Google Scholar), A 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), growth factor receptor F. S. T. B. S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to signal in after their B. Eichler-Jonsson C. Gilles E.D. Muller G. Nat. Biotechnol. 2002; 20: 370-375Crossref PubMed Scopus (773) Google Scholar) used a to that EGFR internalization has a signal attenuation at EGF the of and signal amplification after internalization at low EGF of The concentration of EGF (0.01 at which NLRR-3 MAPK phosphorylation was the of The internalized to activation of MAPK at this concentration as demonstrated B. Eichler-Jonsson C. Gilles E.D. Muller G. Nat. Biotechnol. 2002; 20: 370-375Crossref PubMed Scopus (773) Google Scholar, J.M. Lauffenburger D.A. J. Biol. 1998; PubMed Scopus Google Scholar). in phosphorylation of EGFR and Shc after to a low concentration of EGF were observed as a of expression of NLRR-3 1 was observed NLRR-3 and EGFR is that NLRR-3 MAPK phosphorylation at low of EGF by with clathrin-coated containing EGFR We to S. for of the and for