HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 46, 43549-43552, November 15, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/43549    most recent C200502200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fukamachi, K. Articles by Tsuda, H. Search for Related Content PubMed PubMed Citation Articles by Fukamachi, K. Articles by Tsuda, H. Social Bookmarking                      What's this?

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

Katsumi Fukamachi^§, Yoichiro Matsuoka^¶, Hiroshi Ohno^**, Tetsuya Hamaguchi^§, and Hiroyuki Tsuda^¶

From the  Experimental Pathology, Chemotherapy Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, the  Division of Molecular Membrane Biology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan, and the ^** RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan

Received for publication, September 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (NLRRC) 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 -adaptin. We propose that NLRR-3 potentiates Ras-MAPK signaling by facilitating internalization of EGF in clathrin-coated vesicles.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Leucine-rich repeat (LRR)¹ domains were first identified in an -2-glycoprotein in human serum (1). They contain highly hydrophobic amino acids and a repeated structure consisting of about 24 residues (2), 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 (3, 4), and three distinct isoforms, NLRR-1, -2, and -3, have been identified in fish, frog, mouse, rat, and humans (3-7). These isoforms constitute a novel LRR protein family containing 11 or 12 LRRs, one immunoglobulin-like domain, and one fibronectin type III-like domain (5-7). 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-. 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 (8, 9). Shc binds to activated EGFR and becomes phosphorylated on Tyr³¹⁷, creating a binding site for Grb2-Sos (10). Once Ras has been activated by Sos, GTP-bound Ras interacts with and facilitates activation of target enzymes, including the serine/threonine kinase Raf (11). Activated Raf phosphorylates and activates the downstream kinase, the MAPK/ERK kinase, which in turn phosphorylates and activates MAPK/ERK (12).

A consequence of EGFR activation is the clustering of ligand-receptor complexes in clathrin-coated pits, which increases the rate of receptor internalization (13). Endocytosis of the receptor has been recognized as an attenuation mechanism that affects long-term EGFR function (14, 15). 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 (16-19). 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 (17, 18, 20, 21). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Transfection-- 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).

Plasmid Constructs-- To construct NLRR-3 protein tagged at the carboxyl terminus with green fluorescent protein (GFP), the TAG stop codon of rat NLRR-3 cDNA (7) was removed by PCR with the forward primer 5'-TTAAGCTTTAAGATGAAGGACGCACCAC-3', containing the HindIII site (underlined), and the reverse primer 5'-TACCCGGGACATACTTGTCGGCACAC-3', containing the SmaI site (underlined). A truncated NLRR-3 construct lacking aa 676-707 (NLRRC) was prepared by PCR using the above forward primer and the reverse primer 5'-TTGGATCCAGCTCACTGAATGCCAAGGT-3', containing the BamHI site (underlined). The PCR product was excised with HindIII/SmaI or HindIII/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 (7) was excised with Sau3AI (blunting)/NotI and inserted into pGEX-6P-1 (Amersham Biosciences). A NLRR-3 construct lacking amino acids 676-707 (GST-C) was prepared by PCR-based mutagenesis using the primer 5'-TTGTCGACTAAAGCTCACTGAATGCCAAGG-3' containing the SalI 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.

Expression of GST Fusion Proteins-- GST fusion proteins were expressed in Escherichia coli strain BL21. Protein expression was induced by 1.0 mM isopropyl--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 mM PMSF, 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, 2 M 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 mM PBS. The bound proteins were then eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0.

Generation of Anti-NLRR-3 Polyclonal Antibodies-- 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.

Immunofluorescence-- 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).

Immunoprecipitation-- 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 mM EGTA, 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--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 mM NaCl 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-- Western blot analysis was performed as described elsewhere (22). Anti--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.

GST Affinity Column Chromatography-- 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 insoluble material was removed by centrifugation at 125,000 × g for 45 min. The supernatant was precleared for 2 h with CNBr-activated Sepharose 4B (Amersham Biosciences) coupled with GST and then was incubated overnight with either the GST-W or GST-C resin (5 mg protein/ml gel). Each column was washed with 1 ml of lysis buffer and eluted into 0.3-ml fractions with a step gradient of 150, 250, and 500 mM NaCl (3 ml each). All procedures were performed at 4 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Carboxyl-terminal 30-Amino Acid Stretch of NLRR-3 Enhances MAPK Activation by EGF-- Because the carboxyl terminus of NLRR-3 contains two putative endocytosis motifs (7) and the importance of EGFR internalization to Ras-MAPK activation has already been established (17, 18, 20, 21), we investigated the effect of overexpression of NLRR-3 on MAPK activation through EGFR. COS-7 cells, which express little, if any, endogenous NLRR-3, were transfected with GFP or full-length NLRR-3 tagged with GFP at the carboxyl end; after serum starvation for 24 h, they were challenged with different concentrations of EGF (0.01, 0.1, and 10 ng/ml). Phosphorylation of endogenous MAPK was visualized by immunoblotting with anti-phospho-MAPK antibody. Elevation of MAPK phosphorylation by a low concentration of EGF (0.01 ng/ml) was detected only in cells transfected with NLRR-3, but robust increases in phosphorylation in response to higher concentrations of EGF were observed in both mock transfected and NLRR-3 transfected cells (Fig. 1A and data not shown). No EGFR autophosphorylation (at Tyr¹¹⁷³) or Shc phosphorylation (at Tyr³¹⁷) was detected at 0.01 ng/ml of EGF, but phosphorylation was readily observed at the higher concentrations (Fig. 1A and data not shown). It has been demonstrated that Ras activation with EGF concentrations above 0.02 ng/ml requires phosphorylation of EGFR and/or Shc (12, 23). We now show that, in the presence of NLRR-3, much lower levels of EGF (i.e. 0.01 ng/ml) stimulates MAPK phosphorylation downstream of Ras activation by mechanisms that do not require phosphorylation of EGFR and Shc. Overexpression of NLRR-3 alone had no effect on the levels of expression of MAPK or EGFR or on the phosphorylation levels of MAPK, EGFR, or Shc (Fig. 1). The same results were obtained using cells transfected with untagged NLRR-3 (data not shown).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   NLRR-3 enhancement of MAPK activation is dependent on its carboxyl-terminal region. A, COS-7 cells were transfected with GFP () or NLRR3-GFP (+). After serum deprivation for 24 h, the cells were exposed to 10 or 0.01 ng/ml EGF for 15 min. MAPK activity was determined by Western blotting with anti-phosphpo-MAPK antibody. Specific amplification of MAPK activity in the NLRR3-GFP transfected cells was observed when exposed to 0.01 ng/ml EGF. Anti-phospho-EGFR antibody and phospho-Shc antibody are specific for the corresponding proteins phosphorylated at Tyr1137 (Y1173) and Tyr317 (Y317), respectively. A 10-µg sample of cell lysate was loaded in each lane. B, COS-7 cells were mock transfected or transfected with NLRR3-GFP (NLRR-3) or NLRRC-GFP (NLRRC). Cells were stimulated with 0.01 ng/ml EGF for 15 min, 1 h, or 4 h. Stimulation of MAPK phosphorylation was observed at 15 min, 1 h, and 4 h in the cells transfected with NLRR-3 but not in the mock transfected or NLRRC transfected cells. Note that expression of none of the NLRR-3 constructs had any effect on the level of MAPK and EGFR. The expression level of GFP was at least 10-fold higher in mock (GFP) transfected cells than that of GFP fusion proteins in NLRR-3 or NLRRC transfected cells (data not shown). A 10-µg sample of cell lysate was loaded in each lane.

When COS-7 cells were transfected with full-length NLRR-3 and challenged with 0.01 ng/ml of EGF, high phosphorylation levels of MAPK were sustained for at least 4 h, whereas phosphorylation of MAPK was neither elevated nor sustained in the cells expressing the carboxyl-terminal deleted form of NLRR-3, NLRRC (Fig. 1B). We concluded that the cytoplasmic carboxyl-terminal 30 amino acids (aa) are responsible for NLRR-3 amplification of MAPK phosphorylation.

The Carboxyl-terminal Stretch of NLRR-3 Possesses Clathrin-Adaptor Protein Complex Binding Motifs-- NLRR-3 possesses a stretch of 9 amino acids (YPPLIN/SLWE) containing two clathrin-mediated endocytosis motifs, YXX (where  is bulky hydrophobic amino acid) and a dileucine-type motif (24-27), which is well conserved in the NLRR family (Fig. 2A). Indeed, most of the GFP-tagged NLRR-3 was co-localized with clathrin at the ventral surface of plasma membrane of the COS-7 and NIH3T3 cells (data not shown).

View larger version (58K): [in this window] [in a new window]   Fig. 2.   The carboxyl-terminal region of NLRR-3 is important to endocytosis of the protein. A, alignment of carboxyl-terminal regions of the NLRR family. A stretch of 11 amino acid is highly conserved in all NLRR family members identified thus far. This region contains two clathrin-mediated endocytosis motifs, YPPL and LIN/SLWE. Conserved amino acids are highlighted. B, COS-7 cells were transfected with NLRR3-GFP (NLRR-3) or NLRRC-GFP (NLRRC) for 24 h and incubated at 4 or 37 °C with anti-NLRR-3ex antibody for 4 h. After the incubations, the cells were fixed and incubated with Alexafluor 568-conjugated goat anti-rabbit antibody, and the distribution of NLRR3-GFP (left panels) and anti-NLRR-3ex antibody (middle panels) was visualized by confocal microscopy. The right panels show the merged images, with yellow indicating internalized-NLRR-3. Bar, 10 µm.

The carboxyl-terminal 30-aa stretch was found to be essential for effective endocytosis of NLRR-3. Full-length NLRR-3 tagged with GFP was efficiently internalized when COS-7 cells expressing the protein were incubated at 37 °C (Fig. 2B), whereas considerable amounts of the carboxyl-terminal deletion mutant (NLRRC) remained at the plasma membrane after incubation for 4 h at 37 °C (Fig. 2B, arrows in bottom panel). The plasma membranes of the cells expressing the full-length NLRR-3 and NLRRC remained labeled by the anti-NLRR-3 extracellular domain antibody when incubated at 4 °C (Fig. 2B), indicating that both forms of the fusion protein were transported to the plasma membrane and properly oriented at the membrane. Similar results were obtained when the cells had been incubated for 1 h (data not shown).

Carboxyl-terminal 30 Amino Acid-dependent Interaction of NLRR-3 with Clathrin-Adaptor Protein Complex-- Immunoprecipitation, GST pull-down, and affinity column binding assays were carried out to investigate the physical association between NLRR-3 and the clathrin-adaptor protein complex. A complex isolated from rat brain lysate with anti--adaptin antibody contained NLRR-3 and clathrin (Fig. 3A, upper panels), and in a reverse experiment, clathrin and -adaptin were identified in a complex precipitated by anti-NLRR-3 antibody (Fig. 3A, lower panels). This association was confirmed by GST pull-down experiment using a GST fusion protein containing the entire cytoplasmic domain described below (data not shown). Thus NLRR-3 can associate with the clathrin-adaptor protein complex under physiological conditions.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   The carboxyl-terminal of NLRR-3 binds to clathrin-adaptor protein complex. A, rat brain lysate (3.9 mg) was incubated without () or with (+) anti-NLRR-3 (2 µg) or -adaptin (1.4 µg) antibody, and then protein G-Sepharose beads were added. The immunoprecipitates were analyzed by Western blotting with anti-NLRR-3, clathrin, and -adaptin antibody. B, GST-W containing aa 638-707 or GST-C containing aa 638-675 was immobilized on CNBr-activated Sepharose 4B (5 mg protein/ml gel) and incubated with 33 mg of rat brain lysate. The column was eluted with a step gradient of 150, 250, and 500 mM NaCl, and 0.3-ml fractions were collected and analyzed by Western blotting with anti--adaptin antibody.

The site of physical contact of NLRR-3 with the clathrin-adaptor protein complex was located in the carboxyl-terminal 30 aa containing the endocytosis motifs. Two GST fusion proteins, one containing the entire cytoplasmic domain (GST-W) and the other lacking the carboxyl-terminal 30 aa (GST-C), were coupled to CNBr-activated Sepharose 4B, and elution of the clathrin-adaptor protein complex was monitored by immunoblot with anti--adaptin antibody (Fig. 3B). The protein complex bound to GST-W eluted out at 250 mM NaCl, and there was another class of interaction with higher affinity, with an additional -adaptin peak observed at 500 mM NaCl (Fig. 3B, upper panel). By contrast, most of the protein complex was washed out of the column coupled with GST-C at 150 mM NaCl, and none was detected in the eluates at the higher concentrations of NaCl. Based on the above findings, we concluded that the carboxyl-terminal 30-aa region of NLRR-3 is responsible for the amplification of MAPK phosphorylation through association with endocytotic vesicles.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Wennström and Downward (23) demonstrated that low concentrations of EGF (0.02-0.2 ng/ml) stimulate MAPK through Ras activation in COS-7 cells. They suggested that phosphorylation of Shc and subsequent association between Shc and Grb2, rather than binding of these adaptor molecules to EGFR, is the mechanism responsible for Ras activation induced by this range of EGF concentrations (23). Interestingly, we observed stimulation of MAPK phosphorylation in the parent COS-7 cells by the 0.02 ng/ml concentration of EGF (data not shown) but not by the 0.01 ng/ml concentration (Fig. 1). At the latter concentration, EGF clearly increased MAPK phosphorylation in the presence of NLRR-3 with no detectable amplification of EGFR (Tyr¹¹⁷³, an Shc binding site) and Shc (Tyr³¹⁷, the Grb2 binding site) phosphorylation (Fig. 1A). Therefore, it is less likely that NLRR-3 directly affects the signaling components at the plasma membrane that lead to MAPK stimulation.

The results of our study demonstrated that NLRR-3 amplified MAPK activation by an extremely low concentration of EGF with no effects on the levels of expression of EGFR or MAPK or on the phosphorylation of EGFR and Shc. The carboxyl-terminal 30-aa stretch of NLRR-3 was found to be responsible for this effect, and the same region bound to the clathrin-adaptor protein complex. Recent evidence suggests that receptor tyrosine kinase complexes such as EGFR (16-19), Trk A (28), platelet-derived growth factor receptor (29), and c-Ret (30) continue to signal in endosomal compartments after their internalization. Schoeberl et al. (21) used a computational modeling to demonstrate that EGFR internalization has a dual role: signal attenuation at high EGF concentrations (above the K_d = 1-2 nM of EGFR) and signal amplification after internalization at low EGF concentrations (below the K_d of EGFR). The concentration of EGF (0.01 ng/ml = 1.7 pM) at which NLRR-3 increased MAPK phosphorylation was far below the K_d of EGFR. The internalized receptors contributed substantially to activation of MAPK at this concentration as demonstrated previously (21, 31). No changes in phosphorylation of EGFR and Shc after exposure to a low concentration of EGF were observed as a result of expression of NLRR-3 (Fig. 1A), nor was any direct interaction observed between NLRR-3 and EGFR (data not shown). It is likely that NLRR-3 stimulates MAPK phosphorylation at extremely low concentrations of EGF by associating with clathrin-coated vesicles containing EGFR complexes.

	ACKNOWLEDGEMENT

We are grateful to Dr. Gary S. Goldberg for critical reading of the paper and for discussions.

	FOOTNOTES

^* This study was supported in part by a grant-in-aid for scientific research on priority area from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant-in-aid for the second-term Comprehensive 10-Year Strategy for Cancer Control, a grant-in-aid for cancer research from the Ministry of Health, Labor and Welfare of Japan and CREST, Japan Science and Technology Corporation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipients of research resident fellowships from the Foundation for Promotion of Cancer Research in Japan.

^¶ To whom correspondence should be addressed: Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Tel.: 81-3-3542-2511; Fax: 81-3-3542-3586; E-mail: yomatsuo@gan2.res.ncc.go.jp or htsuda@gan2.res.ncc.go.jp.

Published, JBC Papers in Press, September 23, 2002, DOI 10.1074/jbc.C200502200

	ABBREVIATIONS

The 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				T. Bando, K. Sekine, S. Kobayashi, A. M. Watabe, A. Rump, M. Tanaka, Y. Suda, S. Kato, Y. Morikawa, T. Manabe, et al. Neuronal Leucine-Rich Repeat Protein 4 Functions in Hippocampus-Dependent Long-Lasting Memory Mol. Cell. Biol., May 15, 2005; 25(10): 4166 - 4175. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/43549    most recent C200502200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fukamachi, K. Articles by Tsuda, H. Search for Related Content PubMed PubMed Citation Articles by Fukamachi, K. Articles by Tsuda, H. Social Bookmarking                      What's this?