©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cDNA Cloning and Characterization of a Cek7 Receptor Protein-tyrosine Kinase Ligand That Is Identical to the Ligand (ELF-1) for the Mek-4 and Sek Receptor Protein-tyrosine Kinases (*)

(Received for publication, December 15, 1994; and in revised form, January 5, 1995 )

Haining Shao Liandi Lou Akhilesh Pandey Michael F. Verderame (1) Doyle A. Siever (1) Vishva M. Dixit(§)

From the Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109 Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a murine cDNA encoding a ligand for the Cek7 receptor protein-tyrosine kinase (RPTK), a member of the Eph/Eck RPTK subfamily. Sequence analysis predicts an open reading frame of 209 amino acids with a predicted molecular mass of 24 kDa. The Cek7 ligand shows a 48% sequence identity at the protein level to B61, a ligand for the related Eck RPTK, 30% to the Cek5 ligand, 59% to the recently cloned Ehk1-L, and identity to ELF-1, a recently described ligand for the Mek4 and Sek RPTKs. The expressed Cek7 ligand is functionally active as it induces autophosphorylation of the Cek7 RPTK.


INTRODUCTION

Ligand-mediated activation of receptor protein tyrosine kinases (RPTK) (^1)represents a vital conduit for the flow of information from the extracellular environment across the cell membrane into the cell to modulate the processes of cell growth, survival, migration, and differentiation.

RPTKs have been divided into families based on structural homology present in their extracellular ligand-binding domains. The family with the largest number of known members is the Eph family with at least 12 distinct members, not counting receptor homologues in other species (1) . The extracellular domain of this subfamily has 19 or 20 highly conserved cysteine residues that are clustered and two fibronectin type III repeats(1) . Expression patterns for some of the Eph family members suggest an important role in vertebrate development. For one group, prominent expression is observed primarily in the developing brain (e.g. Elk(2) , Mek4(3) , Sek1(4) , and Cek7(5) ), whereas for a second group (Eck (6) and Cek5(7) ), expression is seen in a broader range of tissues including brain, lung, intestine, and kidney. Despite the large number of members in the Eph family, they were all initially identified as orphan receptors without known ligands. Only recently, the ligand for Eck was found to be the previously described cytokine-inducible gene product designated B61(6, 7, 8) , and the ligands for Cek5(9) , Elk(10) , Mek4(11) , Sek1(11) , and Ehk-1 (12) were cloned using an expression cloning strategy. Eph family members, like all other tyrosine kinase receptors, have an intrinsic kinase activity that is activated following ligand binding. Since activation of the RPTK by its cognate ligand is the most important criterion for the authenticity of receptor-ligand interaction, it is somewhat disappointing that ligand-induced receptor autophosphorylation (a measure of receptor activation) has only been demonstrated for Eck(8) , Cek5(9) , and Ehk-1 ligands(12) . In all other cases, the assignment of a ligand to a particular receptor has been based solely on binding studies.

Cek7, originally cloned from a 10-day chicken embryonic cDNA library, belongs to the Eph/Eck subfamily and is most highly expressed in the central nervous system and eyes of 10-day chicken embryos(5) .

To identify the Cek7 RPTK ligand, an expression cloning approach was used that employed a chimeric protein consisting of the extracellular domain of Cek7 fused to the Fc portion of human IgG(1)(9, 13) . Upon sequence analysis the cloned Cek7 ligand was found to be a novel protein that, importantly, had 48% sequence identity (at the protein level) to the Eck ligand B61(7) , 30% to the Cek5 ligand(9) , and 59% to the Ehk1 ligand(12) . Additionally, like B61, (^2)the Cek7 ligand activated the Cek7 RPTK as revealed by its ability to induce autophosphorylation of the receptor.


MATERIALS AND METHODS

Cell Lines

The human embryonic kidney cell line 293T was grown in Eagle's minimal essential medium containing 10% bovine calf serum (HyClone, Logan, UT), penicillin, and streptomycin. CHO-Tag(14) , a CHO cell line stably transfected with the polyoma large T antigen (kindly provided by Dr. P. Smith, University of Michigan, Ann Arbor), was propagated in alpha-minimal essential medium (supplemented with deoxyribonucleotides and ribonucleotides, Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum (HyClone), 0.4 mg/ml G418 (active drug, Life Technologies, Inc.), penicillin, and streptomycin. NIH3T3 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (HyClone), penicillin, and streptomycin.

Preparation of Recombinant Ig Chimeras

The extracellular domain of chicken Cek7 was cloned by PCR using full-length Cek7 cDNA as a template and custom oligonucleotide primers(5) . The sense primer, encoding sequences immediately downstream of the signal peptide cleavage site and including a custom NheI site (underlined), had the sequence CACTCGCTAGCCAGCCCGGGCAGCGAG, and the antisense primer, encoding sequences immediately upstream of the transmembrane domain and containing a custom BamHI site (underlined), had the sequence CACTGGATCCTGGCTCTGGTCACTGGATGC. The PCR product, encoding amino acids 31-547 of Cek7(5) , was digested with NheI and BamHI and ligated to similarly digested CD5-IgG(1) vector, which contained in-frame the CD5 signal peptide and sequences encoding the human IgG(1) Fc domain (13) . The Cek7 ligand-Ig chimera was constructed using full-length Cek7 ligand cDNA (^3)as template and custom primers. The sense primer, encoding sequences immediately downstream of the signal peptide cleavage site and including a custom NheI site (underlined), had the sequence CACTCGCTAGCGCGCAACGAGGACCCGG, and the antisense primer, encoding sequences immediately upstream of the carboxyl-terminal hydrophobic domain and containing a custom BamHI site (underlined), had the sequence CACTGGATCCCTGGTGAAGATGGGCTCTGGAG. The PCR product, encoding amino acids 20-182 of Cek7 ligand, was cloned into the same CD5-IgG(1) vector described as above. Since these plasmids did not have a selectable marker for expression in mammalian cells, the Cek7-Fc, Cek7 ligand-Fc, and control-Fc inserts (9) were subsequently subcloned into pCEP4 (Invitrogen), which is an episomal vector that contains the hygromycin resistance gene. These three constructs were used to make Cek7, Cek7 ligand, and control Ig chimeric proteins, respectively.

The Ig chimera expression plasmids were stably transfected into 293T cells by the calcium phosphate method as described previously (15) and selected in Opti-MEM I (Life Technologies, Inc.) containing 100 µg/ml hygromycin (Pharmacia, Uppsala, Sweden) and 2% low IgG fetal bovine serum (HyClone). The supernatant was harvested every 4 days and pooled. 500 ml of this pooled supernatant was applied to a 1-ml protein A-Sepharose column (MAPS II kit, Bio-Rad), and the bound chimera was eluted according to the manufacturer's instructions. The eluate was dialyzed against three changes of PBS, and the presence and integrity of the fusion protein in the eluate were confirmed by immunoblotting with an anti-human IgG(1) Fc antibody (Bio-Rad). The concentration of Ig chimera was estimated by comparison to an albumin standard following SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.

Western Blot Analysis

Conditioned media and cell lysates from transfected cells were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose using an electroblotting apparatus (LKB Multiphor, Pharmacia). For detection of Cek7 and control Ig chimeras, the membranes were blocked at 4 °C overnight in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% nonfat dry milk and then incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Bio-Rad) for 40 min at room temperature. For demonstrating phosphorylation of Cek7-myc RPTK, the membranes were blocked with 1% bovine serum albumin in TBST for 1 h and incubated with 4G10 anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY) at a concentration of 1 µg/ml for 1 h at room temperature, followed by incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad). Membranes were then washed extensively and developed by a chemiluminescent reaction (ECL, Amersham Corp.) according to the manufacturer's instructions.

Adhesion Assay

48-well non-tissue culture-treated cluster plates (Costar, Cambridge, MA) were coated with affinity-purified anti-human IgG(1) Fc antibody (Cappel, Durham, NC) at a concentration of 30 µg/ml in PBS overnight at 4 °C, washed once in PBS, and then blocked with 1% bovine serum albumin in PBS for 1 h at 4 °C. The plates were then incubated with 1 µg/ml of either Cek7 or control Ig chimera in PBS for 3 h at 4 °C. To detach CHO-Tag cells for the adhesion assay, dishes were incubated with a low trypsin/EDTA buffer (Life Technologies, Inc.) for 20 s at room temperature and resuspended in PBS. The cell suspension was added to the coated wells for 20 min at room temperature, the wells were washed 4 times with PBS, and the adherent cells were observed under an inverted microscope. In all assays, the number of cells adhering to the Cek7 Ig chimera-coated plates was compared with the number adhering to the control Ig chimera-coated plates.

cDNA Library Construction

An oligo(dT)-primed cDNA library in the mammalian expression vector pcDNA1 (Invitrogen) was prepared using poly(A) RNA isolated from day 15-17 mouse embryonic brain. The library contained 7.4 times 10^5 independent recombinants with an average insert size of approximately 1.6 kilobase pairs.

Transfection of CHO-Tag Cells and cDNA Library Screening

Transfection of CHO-Tag cells was carried out using a commercially available transfection reagent (DOTAP, Boehringer Mannheim) as described previously with modifications(14) . A transfection efficiency of 5-10% was obtained as determined by transfection with a beta-galactosidase reporter construct. Twenty-three 100-mm plates were transfected with 2 µg each of CsCl-banded plasmid DNA prepared from the mouse embryonic brain library. Approximately 44 h following transfection, cell suspensions from each 100-mm dish (3 times 10^6 cells/dish) were panned over 60-mm bacterial culture dishes (Fisher Scientific Co., Canada) precoated with Cek7 Ig chimera (1 µg/ml) that had been immobilized by prior binding to affinity-purified anti-human IgG(1) Fc antibody (Cappel). Nonadherent cells were removed by washing 4 times with PBS. Plasmids were extracted from adherent cells by the Hirt procedure (16) and introduced by electrotransformation into the Escherichia coli strain MC1061/P3(17) . Amplified plasmids were used to transfect 10 plates of CHO-Tag cells for two additional rounds of screening by the same procedure. After the third round, the resulting bacterial colonies were divided into pools of 400-2500 colonies, and plasmids representing these pools were transfected into CHO-Tag cells and tested in the adhesion assay. Panning-directed sib-selection (18) was carried out to isolate a single plasmid capable of conferring upon transfected CHO-Tag cells the ability to adhere to Cek7 Ig chimera-coated plates.

DNA Sequence Analysis

Plasmids were purified by CsCl ultracentrifugation and sequenced on both strands using the Sequenase kit (U. S. Biochemical Corp.) and custom synthetic oligonucleotide primers. The sequence was assembled and analyzed using MacVector release 4.1.2 (IBI, New Haven, CT) and aligned by the Clustal method using MegAlign version 1.02 (DNASTAR Inc. Madison, WI). Homology searching and computation were performed at the National Center for Biotechnology Information using the BLAST network service.

Metabolic Radiolabeling and Immunoprecipitation

NIH3T3 cells were transiently transfected by the calcium phosphate method as described previously(15) . Transfected cells were metabolically labeled with [S]cysteine and methionine (TranS-label, ICN) at 100 µCi/ml for 4.5 h. Cells were subsequently lysed in a buffer containing 1% Nonidet P-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA and a protease inhibitor mixture (5 µg/ml leupeptin, 5 µg/ml aprotinin, 50 µg/ml soybean trypsin inhibitor, and 5 µg/ml pepstatin) for 30 min at 4 °C. Following centrifugation for 20 min at 4 °C to pellet insoluble material, the supernatants (1 ml) were transferred to fresh tubes, and 3 µg of Cek7 Ig chimera was added, and the mixture was incubated for 4 h at 4 °C. Immune complexes were precipitated by adding 50 µl of a 50% slurry of protein A-agarose (Life Technologies, Inc.), and then the complexes were incubated at 4 °C overnight. After three washes with lysis buffer, the beads were pelleted, resuspended in sample buffer containing beta-mercaptoethanol, boiled, and resolved on a 10% SDS-polyacrylamide gel. The fixed and dried gel was developed after exposing to film at -70 °C overnight.

Autophosphorylation Assay

Cek7-myc construct was made by PCR using full-length Cek7 cDNA as a template and custom oligonucleotide primers(5) . The sense primer, encoding sequences immediately upstream of the initiator methionine and including a custom NheI site (underlined), had the sequence CACTCGCTAGCGACCCATGCGCTGAGGGG; the antisense primer, encoding a sequence immediately upstream of the termination codon and including a myc epitope sequence EQKLISEEDLN(19) , had the sequence CACTGGATCCTTAGTTCAAGTCTTCTTCAGAAATAAGCTTTTGTTCCAATGGCACCATCCCATTCAC (custom BamHI site underlined). The insert containing full-length myc epitope-tagged Cek7 was cloned into the expression vector pCEP4. NIH3T3 cells were transiently transfected with this construct using LipofectAMINE reagent (Life Technologies, Inc.) 2 days prior to the autophosphorylation assay. 100-mm dishes precoated (overnight, 4 °C) with goat anti-human IgG(1)-Fc (30 µg/ml, Cappel) were incubated with 5 ml of either Cek7 ligand Ig chimera or control Ig chimera at a concentration of 1 µg/ml for 3 h. Approximately 6 times 10^6 Cek7-myc-transfected NIH3T3 cells were detached with PBS/EDTA buffer, and half of them were added to either Cek7 ligand or control Ig chimera-coated dishes, respectively, and incubated at room temperature for 20 min. Both attached and floating cells were collected and lysed on ice in a buffer containing 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 0.38 mM NaCl, 2.7 mM KCl, 8.1 mM Na(2)HPO(4), 1.5 mM KH(2)PO(4), 1 mM sodium orthovanadate plus the protease inhibitor mixture as described above. Lysates were centrifuged at 12,000 times g for 20 min at 4 °C, and the supernatants were incubated with an anti-myc monoclonal antibody Ab-1 (Oncogene Science, Inc. Uniondale, NY) at a concentration of 2 µg/ml for 4 h, followed by incubation with protein A-agarose beads for 2 h at 4 °C. Following washing three times with lysis buffer, the beads were boiled in sample buffer plus beta-mercaptoethanol, resolved on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with the 4G10 anti-phosphotyrosine antibody at a concentration of 1 µg/ml. Equivalent numbers of Cek7-myc-transfected NIH3T3 cells were metabolically labeled with [S]cysteine and methionine (TransS-label, ICN) and subjected to immunoprecipitation analysis using the same anti-myc antibody as described above.


RESULTS AND DISCUSSION

Expression Cloning of the Murine Ligand for Cek7

To clone the ligand for the Cek7 RPTK, an expression cloning strategy was devised, the first step of which entailed the expression of the extracellular ligand-binding domain of Cek7 (5) as an immunoglobulin chimera(9) . The expression construct encoding the Ig chimera was stably transfected into 293T cells, and the Cek7 Ig chimera was detected in the supernatant by immunoblotting with an anti-human Fc antibody (data not shown). The Cek7 Ig chimera was used to capture transfected cells expressing the Cek7 ligand, since such cells should bind to the ligand-binding domain present in the Cek7 Ig fusion protein. Since Cek7 is predominantly expressed in embryonic and adult brain(5) , it seemed appropriate to screen a mouse embryonic brain cDNA library for expression cloning of the ligand. The expression library was constructed in the pcDNA1 vector, which contains the SV40 origin of replication, allowing for amplification to a high copy number in cells such as CHO-Tag (14) that constitutively express the SV40 large T antigen. Additionally, the CHO-Tag cells did not attach to immobilized Cek7 Ig chimera, making them an ideal recipient for expression cloning, since only transfected cells expressing the ligand should attach to the immobilized chimera. Following the first round of panning, plasmid rescue, and amplification, the procedure was repeated two more times. After the third round, the resultant colonies were divided into different sized pools, and plasmid from each pool was transfected into CHO-Tag cells and tested by panning. The smallest positive pool consisting of 170 transformants was subjected to a sib-selection protocol, resulting in the identification of a single plasmid that on transfection conferred upon the recipient cells the ability to bind to Cek7 Ig chimera-coated plates.

Sequence Analysis of Cek7 Ligand

The nucleotide and derived amino acid sequence of the cDNA encoding the Cek7 ligand showed that the indicated initiator methionine was found in the context of a Kozak consensus sequence (20) for initiation of translation with a purine at the -3 position and a G at the +4 position and was followed by an open reading frame of 627 nucleotides.^3

The predicted open reading frame encoded a polypeptide of 209 amino acids with an estimated molecular mass of 24 kDa. An analysis of hydrophilicity (21) (data not shown) showed that the Cek7 ligand contained a hydrophobic region at the N terminus (amino acids 1-20) and a second hydrophobic region at the C terminus (amino acids 187-209), presumably representing a signal peptide (22) and a signal for the addition of a GPI tail, respectively(23, 24) . Cleavage of both the putative N-terminal signal peptide following the alanine residue at position 20 (according to the(-3, -1) rule of von Heinje(22) ) and C-terminal hydrophobic peptide around amino acid residue 187 (according to the , + 2 rule for GPI anchor signal sequences(23, 24) ) would result in a mature protein of approximately 166 amino acids, presumably linked to a GPI anchor. The rest of the molecule is predominantly hydrophilic. A single consensus sequence for N-glycosylation (NX(S/T)) is found at amino acids 38-40.

Comparison of the translated Cek7 ligand sequence with protein sequence data bases using the BLAST algorithm identified B61 (7) and Cek5 ligand (9) as having significant homology (p < 0.00015) (Fig. 1). Alignment of the Cek7 ligand with the recently cloned Ehk1 ligand (12) also showed a substantial degree of homology. The protein sequence identity in the conserved region between B61 and the Cek7 ligand was 48%, between Cek7 ligand and Cek5 ligand 30%, and that between Cek7 ligand and the Ehk1 ligand 59%, with all 4 cysteine residues being conserved (Fig. 1).


Figure 1: Comparison of Cek7 Ligand to B61, Cek5 ligand, and Ehk1 ligand. Conserved regions between these ligands are shown. Dashes signify gaps introduced to maximize similarity scores. Amino acid residues identical in two or more sequences are shaded, and conserved cysteine residues are boxed. The putative Cek7 ligand signal peptide is underlined.



The Cek7 Ligand Activates the Cek7 RPTK

A genuine ligand should not only bind to its cognate receptor but also cause its activation, which for RPTKs results in the induction of autophosphorylation. NIH3T3 responder cells transfected with a Cek7 RPTK-myc expression vector were added to either Cek7 ligand Ig chimera-coated dishes or control Ig chimera-coated dishes. After a short incubation, cells were collected and lysed, and cell lysates were subjected to immunoprecipitation with an anti-myc monoclonal antibody. The phosphorylation status of the precipitated myc epitope-tagged Cek7-RPTK was assessed by immunoblotting with an anti-phosphotyrosine antibody. As shown in Fig. 2, the Cek7 RPTK underwent dramatic autophosphorylation when exposed to a Cek7 ligand Ig chimera but not when exposed to control Ig chimera while the absolute amount of Cek7 RPTK remained unchanged. This confirmed that the ligand obtained by expression cloning was indeed the Cek7 ligand, capable of both binding and activating the Cek7 RPTK.


Figure 2: Activation of Cek7 receptor autophosphorylation by Cek7 ligand. Cek7-myc-transfected or vector control-transfected NIH3T3 responder cells were incubated with either Cek7 ligand (+) or control Ig chimera(-) coated dishes. Attached and floating cells were lysed and subjected to immunoprecipitation with anti-myc monoclonal antibody followed by immunoblotting with an anti-phosphotyrosine antibody. Cek7 ligand induced Cek7 RPTK autophosphorylation (arrow, toppanel). Similarly transfected cells were metabolically labeled with [S]cysteine and methionine and immunoprecipitated using anti-myc antibody (bottompanel). An approximately equivalent amount of Cek7 RPTK (arrow) was present in the Cek7-myc transfectants. No Cek7 RPTK was detectable in the vector control-transfected cells.



Recently, a molecule identical to the Cek7 ligand was obtained by expression cloning and shown to bind Mek4 and Sek1 RPTKs, which are also members of the Eph family(11) . Taken at face value, this suggests that three receptors in the Eph family, namely Cek7, Mek4, and Sek1, share a single ligand. Unfortunately, the biochemical evidence for Mek4 and Sek1 was based exclusively on the measurement of binding affinities; no data were presented to demonstrate activation of the receptor (induction of autophosphorylation) by the ligand. Since this is an essential criterion for the establishment of functionality of a ligand for RPTKs, a reappraisal of the work would lead one to conclude that while the Cek7 ligand can bind to Cek7, Mek4, and Sek1 RPTKs(11) , it has only been shown to induce activation of the Cek7 RPTK. Unless this criterion is adhered to strictly, it may lead to the erroneous conclusion of ligand promiscuity and discount other viable models such as that the Cek7 ligand is the functional ligand for the Cek7 RPTK (as it induces activation) but a potential dominant negative inhibitor of the Mek4 and Sek1 RPTKs (if it bound without inducing receptor activation). At this juncture, however, we cannot discount the possibility that the ligand we have cloned is also a ligand for Mek4/Sek1. Inasmuch as these are homologous RPTKs and a family of related ligands, it is clear that substantial further in vitro and in vivo work will be necessary before compelling conclusions are possible regarding the identity of the physiologic partnerships.

Regardless, the availability of recombinant Cek7 ligand in a functional form should allow examination of its role in the developing central nervous system, a proposition made all the more attractive with the discovery that ligands for another family of RPTKs (the Trk family) (25) encode neurotrophic factors of potential therapeutic significance in the treatment of neurodegenerative diseases.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK39255 (to V. M. D.), CA52791 (to M. F. V.), and CA60395 (to D. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine St., Ann Arbor, MI 48109-0602. Tel.: 313-747-0264; Fax: 313-764-4308. vishva_dixit{at}mailqm.pds.med.umich.edu.

(^1)
The abbreviations used are: RPTK, receptor protein-tyrosine kinase; GPI, glycosylphosphatidylinositol; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.

(^2)
Shao, H., Pandey, A., O'Shea, K. S., Seldin, M., and Dixit, V. M.(1995) J. Biol. Chem.270, in press.

(^3)
GenBank accession number U14752[GenBank].


ACKNOWLEDGEMENTS

We gratefully acknowledge Peter Smith for providing the CHO-Tag cells, Karen O'Rourke for helpful technical assistance, and Ian M. Jones for help in the preparation of the manuscript.


REFERENCES

  1. Van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10, 251-337 [CrossRef]
  2. Lhotak, V., Greer, P., Letwin, K., and Pawson, T. (1991) Mol. Cell. Biol. 11, 2496-2502 [Medline] [Order article via Infotrieve]
  3. Sajjadi, F. G., Pasquale, E. B., and Subramani, S. (1991) New Biol. 3, 769-778 [Medline] [Order article via Infotrieve]
  4. Gilardi-Hebenstreit, P., Nueto, M. S., Frain, M., Mattei, M.-G., Chestier, A., Wilkinson, D. G., and Charnay, P. (1992) Oncogene 7, 2499-2506 [Medline] [Order article via Infotrieve]
  5. Siever, D. A., and Verderame, M. F. (1994) Gene (Amst.) 148, 219-226 [CrossRef][Medline] [Order article via Infotrieve]
  6. Lindberg, R. A., and Hunter, T. (1990) Mol. Cell. Biol. 10, 6314-6324
  7. Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838 [Medline] [Order article via Infotrieve]
  8. Bartley, T. D. et al. (1994) Nature 368, 558-560 [CrossRef][Medline] [Order article via Infotrieve]
  9. Shao, H., Lou, L., Pandey, A., Pasquale, E. B., and Dixit, V. M. (1994) J. Biol. Chem. 269, 26606-26609 [Abstract/Free Full Text]
  10. Beckmann, M. P., Cerretti, D. P., Baum, P., Bos, T. V., James, L., Farrah, T., Kozlosky, C., Hollingsworth, T., Shilling, H., Maraskovsky, E., Fletcher, F. A., Lhotak, V., Pawson, T., and Lyman, S. D. (1994) EMBO J. 13, 3757-3762 [Abstract]
  11. Cheng, H. J., and Flanagan, J. G. (1994) Cell 79, 157-168 [Medline] [Order article via Infotrieve]
  12. Davis, S., Gale, N. W., Aldrich, T. H., Maisonpierre, P. C., Lhotak, V., Pawson, T., Goldfarb, M., and Yancopoulos, G. D. (1994) Science 266, 816-819 [Medline] [Order article via Infotrieve]
  13. Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B., and Seed, B. (1990) Cell 61, 1303-1313 [Medline] [Order article via Infotrieve]
  14. Smith, P. L., and Lowe, J. B. (1994) J. Biol. Chem. 269, 15162-15171 [Abstract/Free Full Text]
  15. Opipari, A. W., Hu, H. M., Yabkowitz, R., and Dixit, V. M. (1992) J. Biol. Chem. 267, 12424-12427 [Abstract/Free Full Text]
  16. Hirt, B. (1967) J. Mol. Biol. 26, 365-369 [Medline] [Order article via Infotrieve]
  17. Seed, B., and Aruffo, A. (1987) Proc. Natl. Acad. Sci. U. S. A. 87, 3365-3369
  18. Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231 [Abstract]
  19. Munro, S., and Pelham, H. R. (1986) Cell 46, 291-300 [Medline] [Order article via Infotrieve]
  20. Kozak, M. (1989) J. Cell Biol. 108, 229-241 [Abstract]
  21. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  22. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690 [Abstract]
  23. Gerber, L. D., Kodukula, K., and Udenfriend, S. (1992) J. Biol. Chem. 267, 12168-12173 [Abstract/Free Full Text]
  24. Kodukula, K., Gerber, L. D., Amthauer, R., Brink, L., and Udenfriend, S. (1993) J. Cell Biol. 120, 657-664 [Abstract]
  25. Klein, R., Nanduri, V., Jing, S. A., Lamballe, F., Tapley, P., Bryant, S., Cordon-Cardo, C., Jones, K. R., Reichardt, L. F., and Barbacid, M. (1991) Cell 66, 395-403 [Medline] [Order article via Infotrieve]

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