Ligand-mediated activation of receptor protein tyrosine kinases
(RPTK) (
)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
(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, (
)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
-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
vector, which contained in-frame the CD5 signal
peptide and sequences encoding the human IgG
Fc domain (13) . The Cek7 ligand-Ig chimera was constructed using
full-length Cek7 ligand cDNA (
)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
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
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
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
10
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
-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
10
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
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
(Tran
S-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
-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
-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
10
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
HPO
, 1.5 mM KH
PO
, 1 mM sodium orthovanadate
plus the protease inhibitor mixture as described above. Lysates were
centrifuged at 12,000
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
-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 (Trans
S-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.
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.