Neurotrophic factors are important for survival
and maintenance of neurons during developmental and adult stages of the
vertebrate nervous system. The neurotrophins mediate their signal into
the cell by specific interaction with tyrosine kinase receptors of the
Trk family. The extracellular immunoglobulin-like domain of the Trk
receptors adjacent to the membrane has previously been shown to be the
dominant element for specific neurotrophin binding. Using computer
graphics models of the human TrkA and TrkC immunoglobulin-like domains
as a guide, the residues involved in binding to their respective
neurotrophins were mapped by mutational analysis. TrkC primarily
utilizes loop EF, between
-strands E and F, for binding. In
contrast, TrkA utilizes the EF loop as well as additional residues, the
latter being prime candidates for determining the specificity of TrkA
versus TrkC. When selected TrkC and TrkA mutants with reduced binding were expressed on NIH3T3 cells, neurotrophin-induced autophosphorylation was strongly reduced or absent.
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INTRODUCTION |
The neurotrophins form a highly homologous family of growth
factors responsible for differentiation, survival, and function of
neurons sensitive to their presence (reviewed in Ref. 1). The members
of this family include nerve growth factor
(NGF),1 brain-derived
neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5
(reviewed in Refs. 2 and 3), and neurotrophin-6 (4). Neurotrophins bind
to receptor tyrosine kinases encoded by the trk gene family,
which includes trkA (5), trkB (6), and
trkC (7). Each Trk receptor discriminates between the
different neurotrophins: TrkA interacts with NGF (8), TrkC interacts with NT-3 (7), while TrkB binds BDNF and neurotrophin-4/5 (9, 10).
Binding induces autophosphorylation of the Trk receptors, which
triggers the subsequent steps in the signal transduction cascade
(reviewed in Ref. 11). All neurotrophins also bind to a second
receptor, p75, the role of which is still under investigation (reviewed
in Refs. 11 and 12).
The domain organization of the extracellular portion of the Trk
receptors has been proposed based on sequence information (13).
According to this proposal, the extracellular portion of the Trk
receptors is composed of a cysteine-cluster, a leucine-rich motif, and
a second cysteine-cluster, followed by two immunoglobulin-like domains.
Analysis of a series of Trk receptor domain deletions and chimeras
between the different members of the Trk family has shown that the
second immunoglobulin-like domain of TrkC and TrkB confers specificity
and affinity to their respective neurotrophins (14) and that both the
first and second immunoglobulin-like domains are important for TrkA
binding and specificity (14, 15). The leucine-rich motif has been
implicated to bind neurotrophins (16, 17), although no recruitment of
BDNF binding was observed when this motif was transferred into a TrkC
background (14). In contrast, exchanging the second immunoglobulin-like
domain from TrkB into TrkC resulted in high affinity BDNF binding (14). In addition, a fragment of TrkA comprising the two immunoglobulin-like domains has been shown to bind NGF and inhibit neurite outgrowth (18,
19).
In this study, the neurotrophin binding sites on TrkA and TrkC have
been determined using molecular modeling and mutagenesis of the second
immunoglobulin-like domain. Most of the NT-3 binding site on the TrkC
receptor is contributed by residues in a loop connecting
-strands E
and F at the membrane proximal portion of the binding domain. In
contrast, while this same loop is important for TrkA binding of NGF,
numerous residues outside of this loop also make major contributions to
binding, suggesting a mechanism of specificity discrimination.
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EXPERIMENTAL PROCEDURES |
Molecular Modeling of the TrkA and TrkC Binding Domains--
The
models of human TrkA domain 5 (TrkAd5; second immunoglobulin-like
domain) and TrkC domain 5 (TrkCd5) were based on the crystal structure
of the first domain of human vascular adhesion molecule-1 (VCAM) (20).
The sequences of human TrkAd5 and TrkCd5 were compared with the
sequences of immunoglobulin-like domains for which crystallographic or
NMR coordinates are available, e.g. CD4, CD2, telokin,
titin, etc. None of the available structures exceeded 10% identity
with either TrkAd5 or TrkCd5 (TrkAd5 and TrkCd5 have 36% identity).
Since no single immunoglobulin-like domain stood out as a template, the
VCAM structure was chosen as the template, since it contains individual
immunoglobulin-like domains, as opposed to antibody crystal structures
in which individual domains form an interface to another immunoglobulin
domain. Although the percentage of identity of TrkAd5 and TrkCd5 to any
specific immunoglobulin-like domain is low, the wealth of crystal and
NMR structures of immunoglobulin-like domains increases the reliability of models of these domains. Difficulties concerning specific loops or
amino acids are addressed below.
When the sequence of TrkCd5 was aligned with the sequence of human VCAM
(Fig. 1), it became apparent that the buried disulfide bond, which is a
hallmark of immunoglobulin domains, was absent but that the hallmark
tryptophan residue was conserved (residue 304). Cys289 is
in
-strand B, as in VCAM, but the two residues at positions +2 and
2 from Cys289 are glutamic acid (Fig. 1). Since these two
glutamic acids are unlikely to be buried, Cys289 must be on
the exterior of the
-strand. Likewise, Cys331 has a
glutamic acid at position
2 from it and is also likely to be exposed;
in addition, Cys331 is in
-strand E, not in
-strand F
as in VCAM (Fig. 1). Upon building the model, Cys289 and
Cys331 were found to be near one another on the two
adjacent
-strands B and E, again suggesting that the disulfide in
TrkCd5 is exterior, not interior. In domain 5 of both TrkA and TrkC,
the sequence of loop EF can accommodate the conformation found in VCAM
domain 1 (20) or can accommodate an
-helix as found in the CH2 and CH3 domains of IgG (21).
The VCAM domain 1 was transformed into TrkCd5 in three steps. First,
all residues in the
-strands were changed to the TrkCd5 sequence using the INSIGHT-II program (Biosym Technologies, San Diego, CA). If possible, conformations of TrkCd5 side chains were kept similar to those of VCAM; otherwise, they were based on rotamer libraries (22), packing, and hydrogen bond considerations. Second, loop
structures connecting the
-strands were gleaned from a search of
loops of similar size from the Protein Data Bank (23) using the loop
search algorithm in the INSIGHT-II program. Finally, the relatively
large segment connecting
-strands C and E (24 residues long;
His306-Gly330) was modeled in two
conformations: one similar to the conformation found in telokin (24)
and titin (25) and the other similar to the conformation found in IgG
Fc domains CH2 and CH3 (21) (shown in Fig. 2). It is not possible to
discern which conformation will be adopted. The TrkAd5 model was then
generated from the TrkCd5 model using similar methods.
The TrkAd5 and TrkCd5 models were subjected to energy minimization
using the DISCOVER program (Biosym Technologies, San Diego, CA). The
all-atom AMBER forcefield (26) as provided in the DISCOVER program was
used for all calculations, employing a 14-Å cut-off for nonbonded
interactions, a linear dielectric (
= 4.0 × r), and 1-4
atom-atom interactions (i.e. atoms connected by two
intervening covalently bonded atoms) scaled by a 0.5 factor. Prior to
minimization, hydrogen atoms were added to the structure using
INSIGHT-II, and positions of hydrogens on Ser, Thr, and Tyr side chains
were checked visually for proper alignment in hydrogen bonds, if
present. Energy minimization was performed in three stages. In stage
one, 500 cycles of steepest descents minimization was employed with 47 nitrogen donor:oxygen acceptor atom pairs constrained to be within hydrogen bonding distance (2.90 Å; force constant of 50 kcal/Å). The
latter were included to retain the
-strand nature of the immunoglobulin-like domain. In stage two, conjugate gradient
minimization was employed for 2000 iterations, and the hydrogen bond
constraints were retained. In stage three, another 2500 cycles of
conjugate gradient minimization were employed with the hydrogen bond
constraints released.
Mutagenesis and Recombinant DNA Manipulations--
An
immunoadhesin (27) of the extracellular portion of human TrkC, in which
the extracellular portion is fused to the Fc of a human antibody, was
previously constructed, sequenced, and subcloned into a vector that
allows for production of double- and single-stranded DNA in
Escherichia coli as well as expression of mature receptors
in a mammalian system under control of the cytomegalovirus promoter
(28). A similar immunoadhesin was constructed using human TrkA.
Oligonucleotide site-directed mutagenesis (29) on these vectors
generated the specific mutants. After transformation into the E. coli strain XL1-Blue (Stratagene, San Diego, CA), colonies were
screened for the presence of the desired mutation by sequencing
single-stranded DNA using the Sequenase version 2.0 kit (U.S.
Biochemical Corp.). For each selected clone, the entire DNA sequence
coding for the receptor immunoadhesin was verified. Double-stranded DNA
used for transfection of human kidney 293 cells was isolated from XL
1-Blue with the QIAGEN DNA purification kit (Qiagen Inc., Chatsworth
CA).
Design, Construction, Expression, and Purification of Receptor
Mutants--
All receptor mutants were constructed in the context of
the full-length extracellular portion and expressed as immunoadhesins. The extracellular portions of TrkA and TrkC expressed in this fashion
have been shown to display the expected specificities (28) and to
interact with high affinity with their respective neurotrophin ligands
(30). Furthermore, a panel of NT-3 mutants displayed a similar profile
of affinities to TrkC whether the latter was expressed as immunoadhesin
or on NIH3T3 cells (30), indicating that the binding domains of TrkC
have a similar conformation in both systems. Plasmid DNA coding for the
TrkA or TrkC immunoadhesin mutants were introduced into the human fetal
kidney cell line 293 by calcium phosphate precipitation (31). The
proteins were expressed and purified as described previously (14) and
were more than 95% pure as judged from SDS-polyacrylamide gel
electrophoresis (data not shown).
Iodination of NT-3--
NT-3 was labeled with 125I
using lactoperoxidase (Calbiochem). Briefly, 8 µg of purified NT-3 in
20 µl of 10 mM sodium acetate, 140 mM NaCl,
pH 5.5, was mixed with 37.5 µl of 0.4 M sodium acetate, pH 5.6, 0.015 IU of lactoperoxidase, and 2 mCi of 125I. The
reaction was started by adding 15 µl of diluted hydroxyperoxide (1:174,000 of 30% stock) (Mallinckrodt, Phillpsburg, NJ) and was kept
at room temperature for 5 min. An additional 15 µl of diluted hydroxyperoxide was added, and the reaction was kept at room
temperature for another 5 min. The reaction was stopped by the addition
of 15 µl of 20 mM
N-acetyl-L-tyrosine (Sigma) in water. The
labeled NT-3 was isolated from free iodine by use of a PD-10 desalting column (Pierce). The trichloric acetic acid precipitability of the
fractions containing the labeled NT-3 was usually higher than 90%, and
the specific activity was between 2000 and 3000 Ci/mmol.
Binding TrkC Mutants to NT-3--
Competition binding assays
were performed using a 96-well plate format and purified receptor and
ligand preparations. The binding constants (Kd) of
receptor mutants are related to inhibition constants (IC50)
of competition experiments using labeled and unlabeled neurotrophins by
the Cheng-Prusoff equation (Kd = IC50/(1 + L*/KdL*)), where L* and
KdL* are the concentration of the labeled ligand and
its affinity to the receptor, respectively (32). Prior to the
competition binding experiment, the concentration of each receptor
immunoadhesin was adjusted to bind 10-15% of the total labeled ligand
in the absence of competitor to satisfy true competition requirements
(e.g. K Rt < 0.1) and to prevent tracer ligand
depletion due to increased receptor concentrations (33). Native TrkC
bound 10% of the total labeled NT-3 at a concentration of 7.1 ± 1.7 ng/ml (five independent expressions). Some of the mutants
(e.g. N335A/T338A/N342A) did not bind any labeled NT-3 above
background when assayed at 2000 ng/ml concentration; therefore, no
competition experiment could be performed. Hence, the affinities
of these mutants were estimated to be reduced at least 200-fold
and were labeled as NB (no binding) in Table I.
After coating each well with 100 µl of 5 µg/ml goat
F(ab')2 anti-human Fc IgG (Organon Technika, Westchester,
PA) in 0.1 M Tris, pH 9.5, for 15 h at 4-8 °C, the
wells were aspirated, washed three times with PBS, incubated for 2 h with 100 µl of a solution of the receptor immunoadhesin in binding
buffer, and washed with PBS. Binding buffer consists of Leibovitz's
L-15 medium supplemented with 5 mg/ml bovine serum albumin (Intergen,
Purchase, PA), 0.1 mg/ml horse heart cytochrome c (Sigma),
and 20 mM HEPES, pH 7.2. In competition experiments, 50 µl of binding buffer was immediately added to the wells to prevent
drying. Purified, unlabeled NT-3 was serially diluted in binding buffer
to a concentration range of 4096-0.125 pM. 25 µl of
serial dilution was added per well followed by 25 µl of labeled NT-3.
The final concentration of labeled NT-3 in each well was approximately
30 pM. After 3 h of incubation at room temperature,
the wells were washed with PBS, 0.5% Tween 20 (Sigma), and the bound
radioactivity was counted. Competition data were fit to a
four-parameter equation using the Kaleidagraph software (Abelbeck
Software). All competition binding experiments were repeated at least
three times.
Binding of TrkA and TrkC Mutants to Monoclonal
Antibodies--
The monoclonal antibodies against the extracellular
domains of TrkA and TrkC were developed using an analogous protocol to a described procedure (34). Microtiter plates (Nunc, Naperville, IL)
were coated with 100 µl/well of 1 µg/ml goat F(ab')2 anti-human IgG
Fc-specific antibody (Cappel-Organon Teknika, Durham, NC) in 0.05 M carbonate buffer, pH 9.6, overnight at 4 °C. The
plates were washed with PBS, 0.05% Tween 20, and the excess binding
sites were blocked with 200 µl/well of PBS, 0.05% Tween 20, 0.5%
bovine serum albumin (Calbiochem) (buffer A) for 1-2 h at ambient
temperature. The plates were washed again, and 100 µl of 1 µg/ml
Trk or Trk mutant immunoadhesins diluted in buffer A were added to the
appropriate wells. After incubation for 1-2 h at ambient temperature,
plates were washed and 100 µl of human TrkA- or TrkC-specific mouse
mAbs (1 µg/ml) were added, incubated for 1 h at ambient
temperature, and washed. Horseradish peroxidase-conjugated goat
anti-mouse Fc IgG antibody (Cappel-Organon Teknika), 1:5000 in buffer
A, was added to the plate, incubated for 1 h at room temperature, and washed. The color was developed for 10-20 min with 100 µl of
o-phenylenediamine (Sigma) substrate solution (5 mg of
o-phenylenediamine in 12.5 ml of PBS containing 4 mM H2O2), and the reaction was stopped with 100 µl of 2.5 N
H2SO4. Absorbances were recorded at 490 nm with
a 405-nm reference filter (UVMax kinetic microplate reader, Molecular
Devices, Palo Alto, CA).
Binding of TrkA Mutants to NGF--
An NGF/TrkA assay similar to
the one described for NT-3/TrkC was evaluated but was found to lack the
sensitivity required to delineate differences in binding, possibly due
to the covalently bound iodine on the labeled NGF interfering with
binding. Instead, the mAb binding assay described above was modified to
evaluate the binding of TrkA mutants to NGF. The anti-NGF and anti-TrkA mAbs used in the assay were developed using an analogous protocol to
one previously described (34). Plates were coated with 100 µl/well
anti-NGF mAb 911 (1 µg/ml) overnight at 4 °C. The plates were
washed with PBS, 0.05% Tween 20, and the excess binding sites were
blocked with 200 µl/well buffer A for 1-2 h at ambient temperature. NGF (100 µl of a 1 µg/ml solution in buffer A) was then added and
incubated for 1 h. Serial dilutions of TrkA were added in duplicate (100 µl of solutions starting at 1 or 16 µg/ml; 8 dilutions total), and the bound receptor was detected with biotinylated anti-TrkA specific mAb 1487 (100 µl of a 1 µg/ml solution in buffer A) (see Table II). Horseradish peroxidase-conjugated streptavidin (Sigma), 1:1000 in buffer A, was added, and the absorbances were recorded at 490 nm with a 405-nm reference filter (UVMax kinetic microplate reader, Molecular Devices).
A similar enzyme-linked immunosorbent assay-based assay was also
evaluated for NT-3/TrkC to determine if this assay format was more
sensitive than the 125I-labeled NT-3 format. A selected
panel of TrkC mutants showed the same pattern of binding as in the
labeled NT-3 format. The enzyme-linked immunosorbent assay format was
not more sensitive than the labeled NT-3 format, and the rest of the
TrkC mutants were not repeated using the enzyme-linked immunosorbent
assay format.
Construction of Stable Transfected NIH3T3 Cells--
The
cDNA coding for the extracellular domain of TrkC and the mutants
T338A and N335A/T338A/N342A were amplified using specific PCR primers,
which incorporated flanking restriction sites as described previously
(14). Similarly, a fragment coding for the transmembrane and the
intracellular domain of TrkC was amplified using PCR. These fragments
were ligated into the mammalian expression vector pMEXneo (5). NIH3T3
cells were transfected with these constructs using lipofection (DOTAP
reagent; Boehringer Mannheim). At least 10 single colonies for each of
the constructs were selected for G418 resistance, expanded, and assayed
for receptor expression by binding to wheat germ agglutinin or
immunoprecipitation with the pan-Trk antibody 443 as described
previously (35). For all constructs, at least 90% of the selected
colonies expressed the desired receptor.
The cDNA coding for the extracellular domain of TrkA and the
mutants E301A, T292A, and T319A/H320A/N323A were amplified using specific PCR primers, which incorporated flanking restriction sites as
described previously (14). Similarly, a fragment coding for the
transmembrane and the intracellular domain of TrkC was amplified using
PCR. These fragments were ligated into the mammalian expression vector
pMEXneo (5). NIH3T3 cells were transfected with these constructs using
lipofection (DOTAP reagent; Boehringer Mannheim). At least 10 single
colonies for each of the constructs were selected for G418 resistance,
expanded, and assayed for receptor expression by binding to wheat germ
agglutinin or immunoprecipitation with the pan-Trk antibody 45 (a gift
of Dr. Barbara Hempstead) as described previously (35).
Stimulation of TrkC Receptor Autophosphorylation on NIH3T3 Cell
Lines by NT-3--
Approximately 1 × 107 cells were
treated at 37 °C for 5 min with the appropriate neurotrophin. With
NT-3, they were treated at 100 ng/ml or as indicated in Fig. 3; with
NGF, they were treated at the different concentrations shown in Figs. 6
and 7. Nonidet P-40 plate lysis and immunoprecipitation with pan-Trk
antiserum 443 (NT-3/TrkC) or pan-Trk antibody 45 (NGF/TrkA) was
performed as described previously (35). The phosphotyrosine content was analyzed by Western blot using monoclonal antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) as described previously (35, 36).
For each of the constructs, two different cell lines were selected and
assayed for neurotrophin-induced autophosphorylation in triplicate.
Inhibition of NT-3-induced Neurite Extensions of PC12/TrkC Cells
by Receptor Immunoadhesins--
Approximately 103 PC12
cells expressing rat TrkC (35) were plated onto 35-mm collagen-coated
tissue culture dishes and grown overnight. The TrkC immunoadhesin or
TrkC mutant immunoadhesins were mixed with NT-3 at the indicated
concentrations in 2 ml of medium, incubated for 30 min at room
temperature, and then added to the cells. The proportion of
neurite-bearing cells was determined by counting the number of cells
containing processes at least twice the length of the cell body after 3 days. The photographs in Fig. 4 were taken after 3 days. Inhibition
experiments were performed at least three times using soluble receptor
from two expressions.
 |
RESULTS |
The Three-dimensional Model of the TrkC Binding Domain Identifies
Surface-exposed Residues--
To guide mutagenesis and aid in
interpreting the results, computer graphics models of the second
immunoglobulin-like domains of TrkA and TrkC were generated based on
the crystal structure of human vascular cell adhesion molecule-1 domain
1 (20). The model predicts seven
-strands (Fig.
1) in a c-type arrangement, with four
strands (A, B, D, and E) forming sheet I and three strands (C, F, and
G) forming sheet II of the
-sandwich (37). Loops will be denoted by
the two
-strands that the loop connects, e.g. loop AB
connects
-strands A and B.

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Fig. 1.
Alignment of human TrkA (trkAd5)
and TrkC (trkCd5) domain 5 sequence with human vascular
adhesion molecule-1 domain 1 (VCAMd1). Buried residues
in VCAM domain 1 are denoted by a filled circle above the
residue. -Strands in the VCAM crystal structure (20) and
the TrkC model are underlined and labeled. The
lowercase letters at the C terminus of TrkC and TrkA were
not included in the immunoadhesin constructs. TrkC and TrkA residue
numbers are under the amino acid sequence.
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One of the original hallmarks of an immunoglobulin-like fold is a
conserved disulfide bond between strands B and F connecting the two
-sheets in the
-sandwich structure (38). However, many examples
of immunoglobulin-like domains show that the disulfide bond can be
moved within strands B, C, E, and F or may be absent (37). As described
under "Experimental Procedures," the disulfide bond in domain 5 of
the Trks is probably on the exterior of the domain.
Surface-exposed residues were identified by inspection of the models.
For the majority of residues, the contribution to binding was probed by
mutation to alanine (39). Some residues, including native alanine
residues, were changed to amino acids other than alanine,
e.g. the analogous amino acid of a different Trk receptor. Selected residues that showed a pronounced effect when mutated to
alanine were subsequently mutated to other amino acids to determine how
a particular side chain contributes to affinity.
The Binding Site on TrkC for Its Ligand NT-3--
The most
important binding determinants in TrkC were discovered by analysis of
the single alanine mutants E287A, R295D, N335A, T338A, and N342A
(mutants are designated by the one-letter code for the native amino
acid, the residue number, and the substituted amino acid; multiple
mutants are separated by slashes; numbering is that of mature human
TrkA or TrkC). Residue Glu287 is located in loop AB (Fig.
2A), and the affinities of
E287A, E287K, and E287H to NT-3 were reduced 7-, 27-, and 3-fold,
respectively, when compared with TrkC (Table
I). That the most pronounced effect on
the affinity resulted from charge reversal of the side chain suggests
that Glu287 may be interacting with a basic amino acid on
NT-3. Residue Arg295 is in loop BC (Fig. 2A),
and the analogous residues in TrkA and TrkB are aspartic acid and
lysine, respectively. R295A had no effect on binding, but R295D reduced
binding by an order of magnitude (Table I) and suggests that
Arg295 in TrkC might interact with an acidic residue in
NT-3.

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Fig. 2.
The binding site for NGF on the second
immunoglobulin-like domain of TrkA and for NT-3 on the second
immunoglobulin-like domain of TrkC. A, model of second
immunoglobulin-like domain of TrkC. The -strands and connecting
loops are shown in tan. Residues with a >20-fold effect on
binding to NT-3 (Asn335, Thr338, and
Asn342) are shown in green; residues with a
<20-fold effect on binding are shown in yellow. The
disulfide bridge between Cys289 and Cys331 is
shown in purple. B, model of second
immunoglobulin-like domain of TrkA. The -strands and connecting
loops are shown in tan. Residues with a >50-fold effect on
binding to NGF are shown in green; residues with a
10-40-fold effect on binding are shown in yellow. The
disulfide bridge between Cys267 and Cys312
(yellow) is at the middle, right of
the domain. Glu262 and His265 are shown in
purple. Side chain oxygen atoms are show in red; side chain nitrogen atoms are shown in blue.
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Residues Asn335, Thr338, and Asn342
are located in loop EF (Fig. 2A), and the affinities to NT-3
of N335A, T338A, and N342A were reduced by 38-, 30-, and 24-fold,
respectively (Table I). Thr338 and Asn342 are
conserved among the three Trk receptors, while position 335 is aspartic
acid in TrkB and asparagine in TrkA. Two other residues in loop EF at
positions 336 and 340 vary among the three Trk receptors; however,
alteration of these two residues did not affect binding (K336A and
K336A/Y340A, Table I). At position Asn335, the affinities
of N335Q, N335D, and N335S were reduced by 21-, 0.8-, and 1.2-fold,
respectively, compared with native TrkC (Table I). Replacing
Asn335 with a longer side chain with the same functional
group in mutant N335Q resulted in a pronounced decrease of affinity,
which indicates that the space in the binding pocket at this position
may be very limited and that no additional atoms can be accommodated.
Introduction of a charged but isosteric amino acid in N335D as well as
exchanging asparagine for serine did not decrease affinity to NT-3.
These results indicate that Asn335 probably acts as a
hydrogen bond acceptor in the complex.
The affinity of T338S for NT-3 was equivalent to native TrkC, while
T338A resulted in a reduction of 30-fold. When Thr338 was
changed to valine, which has a similar size but no hydrogen bonding
potential, the affinity decreased by 12-fold (Table I). This suggests
that in the complex with NT-3, the major contribution of
Thr338 to the affinity is by forming a hydrogen bond
involving its hydroxyl oxygen, although some steric component is
evident when T338A and T338V are compared. A larger side chain cannot
be accommodated at position 338, since T338N had its affinity reduced
23-fold when compared with native TrkC.
Residue Asn342 was probed by mutations to aspartic acid and
serine with reduction of affinities of 20- and 21-fold, respectively, while mutation to glutamine resulted in no detectable binding (Table
I). As at position 335, replacing Asn342 with a larger side
chain reduced binding. In contrast to position 335, however, making
Asn342 smaller (N342A, N342S) also reduced binding, as did
removal of the side chain NH2 group (N342D). These data
suggest that the Asn342 side chain acts as a hydrogen bond
donor and that the steric requirements for hydrogen bond(s) and/or
packing are quite rigid.
Since individual alanine mutations at positions 335, 338, and 342 did
not abolish binding completely, we constructed all possible combinations of double mutants (N335A/T338A, N335A/N342A, T338A/N342A) as well as the triple alanine mutation (N335A/T338A/N342A). None of
these mutants bound NT-3 at detectable levels (Table I).
In addition to the four major contributors, two other residues showed
minor effects; C331A in
-strand E and H363A in
-strand G had
their affinities reduced 2.9- and 2.0-fold, respectively. All other
residues that were tested displayed affinities that were unchanged
compared with native TrkC (Table I). Notably, alteration of the
cysteine residues to alanine either had a minor effect (C331A) or no
effect (C289A). This supports the hypothesis that the disulfide bond of
TrkC is not involved in maintaining the structure of the domain, unlike
the conserved disulfide bond of canonical immunoglobulin-like domains
(38). The second immunoglobulin-like domain of TrkC contains two
possible N-linked glycosylation sites at positions
Asn344 and Asn357 (Fig. 1). Changing these
asparagines to alanine had no effect on binding (Table I).
In addition to the two immunoglobulin-like domains, the leucine-rich
motif has been previously implicated in binding neurotrophins (16, 17).
However, no recruitment of BDNF binding was observed when the TrkB
leucine-rich motif was transferred into a TrkC background (14). In this
study, a chimeric protein in which the leucine-rich motif of TrkA was
substituted into TrkC (mutant A2C) showed no binding to NGF
but bound NT-3 as well as native TrkC (Table
I).
The Binding Site on TrkA for Its Ligand NGF--
As for TrkC, loop
EF in TrkA contains residues that play a major role in binding NGF.
T319A, H320A, and N323A (Fig. 2B) all exhibited greater than
100-fold reduction in binding. More modest reductions were evident for
other residues in loop EF, V321A (24-fold) and Q317A (6-fold) (Table
I). In contrast to TrkC, several residues in loops other than loop EF
were found to be important. Three residues in loop AB
(Val261, Met263, and His264), one
in loop BC (D273A), one in loop CD (T292A), and three in loop DE
(E306A, T307A, H310A) all exhibited reduced binding compared with
native TrkA (Table I).
Also in contrast to TrkC, several residues in the TrkA
-strands were
shown to significantly reduce binding when altered: P269E (
-strand
B), C312A (
-strand E), T327A/L329A (
-strand F), and S340A/M342A
(
-strand G) (Table I). Less dramatic reductions in binding were
exhibited by other
-strand residues: H258A (
-strand A), C267A and
S271A (
-strand B), and N332A (
-strand F) (Table I). Five
positions within the segment connecting the second immunoglobulin-like domain to the transmembrane sequence showed reduced binding of NGF when
altered to alanine: E351A/N353A, E355A/D356A, and I358A (Table I).
It should be noted that the NGF/TrkA assay used may have overestimated
the reduction in binding of a few mutants because the anti-TrkA mAb
used as the detecting agent in the assay, A1487 (Table
II), also showed slightly reduced binding
to several mutants compared with native TrkA (Table
III). The mAb A1487 was the only mAb that
could be used in the assay, since the other four mAbs either mapped to
domain 5 (A1486 and A1502) or were blocking mAbs (A1488 and A1503)
(Table II). For mutants C267A, P269E, C312A, and T327A/L329A, mAb A1487
showed 11-27% reduction in binding compared with TrkA; therefore, the
IC50mutant/IC50TrkA values in Tables I and III
are probably inflated by these same percentages. Regardless, P269E
remains a major determinant in TrkA binding to NGF, while the other
three, especially C267A and C312A, are of lesser importance.
Several of the TrkA mutants deserve closer analysis. Compared with
TrkC, where altering the cysteine residues to alanine resulted in
little or no effect on binding, in TrkA altering the cysteines to
alanine did effect reduction in binding. However, the reduction in
binding (C267A, <14-fold; C312A, <28-fold) was less dramatic than for
several other TrkA mutants, e.g. P269E at >100-fold, H310A
at 79-fold and T319A at >100-fold (Table I). The second immunoglobulin-like domain of TrkA contains four possible
N-linked glycosylation sites at positions
Asn285, Asn290, Asn305, and
Asn325 (Fig. 1). Changing these asparagines to alanine had
little or no effect on binding to NGF (Table I), suggesting that if
carbohydrate is present it does not play a role in binding. At three of
these sites, altering the threonine side chain 2 positions downstream from the asparagine (part of the consensus Asn-X-(Thr/Ser)
glycosylation sequence) did effect a relatively large reduction in
binding, 20-24-fold at each site (Table I). This implies that these
three threonine residues interact directly with NGF; if lack of
carbohydrate were the cause for these mutants not binding well, then
the same should have been exhibited by changing the coupled asparagines to alanine. Finally, as with TrkC, residues in TrkA loop EF, which individually abrogated binding when changed to alanine were combined in
double and triple mutants, and these showed no detectable binding (Table I).
One of the crystal structures of murine NGF showed the presence of a
zinc-binding site in the central
-strand bundle involving His84 and Asp105 as zinc ligands (40). The
model of TrkA (Fig. 2B) shows that in
-strand A and loop
AB, four residues (His258, Glu262,
His264, and His265) could provide one or more
possible zinc-binding ligands to complement the zinc-binding site in
NGF. Alternatively, His310 in loop EF (important for the
NGF/TrkA interaction but not the NT-3/TrkC interaction), could also be
a possible zinc-binding ligand. When the TrkA binding assay was
performed in the presence of 2 mM and 10 mM
EDTA, no abrogation of binding was apparent (Table I). Hence, though
zinc was bound to NGF in the crystal structure, it apparently is not
involved in the NGF/TrkA interaction.
Binding of a Panel of Monoclonal Antibodies Confirms the Structural
Integrity of TrkA and TrkC Mutants--
A loss in activity of a
protein upon mutation of a side chain can be attributed either to the
importance of this side chain to the function of the protein or to an
involvement in maintaining the structural integrity of the protein. A
method to verify the structural integrity of mutants with reduced
activity is to determine their reactivities against a panel of
monoclonal antibodies (41).
A panel of five mAbs reactive against the extracellular domain of human
TrkA was generated. To determine the domain(s) on the extracellular
part of TrkA that interacts with each of these mAbs, their affinities
to previously described TrkA mutants (14) were determined. All of the
mAbs were specific for TrkA and did not bind to TrkB or TrkC (Table
II). The domain mapping of the mAbs is provided in Table II. The
blocking mAb A1502 only bound well to constructs in which domain 5 was
present (Table II); the epitope of mAb A1502 was further resolved to
include the TrkA segment connecting domain 5 to the transmembrane
sequence, since the mutants E355A/D356A, I358A, D360A, and T361A showed
reduced binding to this mAb (Table III).
Of the two mAbs that map to domains 1-4, i.e. A1503 and
A1487, one blocks NGF binding, while the other does not. Of the two mAbs that map to domain 5, i.e. A1486 and A1502, one blocks,
and the other does not. Two of the three blocking mAbs include domains 2 and 4 in their epitope (A1488 and A1503), but the third blocking mAb
maps only to domain 5 (A1502). Hence, the pattern of blocking versus nonblocking mAbs does not unambiguously resolve which
domains need to be occluded to block NGF binding by TrkA.
The panel of anti-human TrkA mAbs was used to test the structural
integrity of the TrkA mutants that exhibited reduction in NGF binding.
Most of the TrkA mutants bound to the five mAbs as well as native TrkA;
those that showed reduced binding (i.e. <95% compared with
native TrkA) to at least one of the mAbs are listed in Table III. Four
mutants showed reduced binding only to mAb A1502, and these are
probably part of the epitope of this mAb. Two mutants, T307A and N332A,
showed reduced binding only to mAb A1486 and may comprise part of the
epitope of this mAb. Three mutants (P269E, C312A, and T327A/L329A)
showed reduced binding to mAbs A1488, A1487, and A1486, although
binding was still 51-83% compared with native TrkA. These residues
may interact directly with an mAb (e.g. A1486, which maps to
domain 5), may play a role in interdomain interaction, or may perturb
the local structure of domain 5. That these three mutants bound well to
the other two mAbs, however, suggests that any structural deformation
is not global.
A panel of four mAbs reactive against the extracellular domain of human
TrkC was also generated. To determine the domain(s) on the
extracellular part of TrkC that interacts with each of these
antibodies, their affinities to previously described TrkC mutants (14)
were determined. All of the mAbs were specific for TrkC and did not
bind to TrkB, although mAb C1435 did show some residual binding to TrkA
(Table IV). The domain mapping of the
mAbs is provided in Table IV.
The panel of anti-human TrkC mAbs was used to test the structural
integrity of the TrkC mutants E287A, N335A, T338A, N342A, and
N335A/T338A/N342A, all of which showed reduced binding to NT-3. These
mutants bound all four mAbs with affinities similar to TrkC,
i.e. between 84 and 100% (Table
V). Hence, the observation that all four
mutants bind the mAbs C1435, C1437, and C1438 demonstrates that the
first three domains of the TrkC mutants are structurally intact and
that these mutations do not lead to global unfolding of the protein.
Most importantly, these mutants also bound to mAb C1436, which was
mapped to the two immunoglobulin-like domains, confirming that the
mutations did not lead to unfolding of the binding domain.
In addition to binding by the panel of mAbs, it should be noted that
the TrkA and TrkC mutants were expressed as immunoadhesins (27),
i.e. with an immunoglobulin Fc fused to the C terminus of
the Trk extracellular portion. Since these proteins were purified by
interaction of the Fc with Staphylococcus aureus protein A and then analyzed for proper molecular size by SDS-polyacrylamide gel
electrophoresis (data not shown), the entire Trk extracellular portion
was necessarily expressed and folded such that it was not degraded
during expression.
Neurotrophin-induced Autophosphorylation of TrkC Receptor
Mutants--
To test the ligand interactions of the receptor mutants
in a cellular environment, stable NIH3T3 cell lines expressing TrkC and
the mutants T338A and N335A/T338A/N342A were constructed. The
expression of the receptors was demonstrated by immunoprecipitation with the pan-Trk antibody 443 (5) (Fig.
3A). As reported for the
rodent Trk receptors (6, 35, 42), the apparent molecular weights of the
full-length human TrkC and TrkC mutants indicate the presence of a
significant amount of glycosylation (calculated molecular mass of
full-length, unglycosylated Trk receptors is about 95 kDa). For each of
the constructs, two cell lines with different levels of expression were
chosen for NT-3-induced autophosphorylation: C-18 and C-3 for TrkC;
48-2 and 48-4 for T338A; and 92-3 and 92-5 for N335A/T338A/N342A. The
addition of NT-3 at 100 ng/ml resulted in rapid autophosphorylation of
TrkC (C-18/+ and C-3/+; Fig. 3B), while no signal was
detectable in the absence of NT-3 (C-18/
and C-3/
; Fig.
3B). The cell lines expressing T338A at low level (48-2) and
high level (48-4) did show neurotrophin-dependent
activation at 100 ng/ml of NT-3 (48-2/+ and 48-4/+; Fig.
3B), but the intensity of the signal was reduced when
compared with the TrkC cell lines. Note that the expression levels of
C-3 and 48-4 are similar (Fig. 3A), but the response to 100 ng/ml NT-3 was strongly reduced in 48-4 when compared with C-3 (Fig.
3B). Finally, no NT-3-dependent activation of
the N335A/T338A/N342A mutant receptor was observed when the cell lines
92-3 and 92-5 were tested at 100 ng/ml NT-3 (92-3/+ and 92-5/+, Fig.
3B). Dose-response autophosphorylation experiments showed
that the TrkC receptor reaches saturation at 50 ng/ml (Fig.
3C). In agreement with the reduced binding affinity of the
T338A mutant for NT-3, the neurotrophin-induced autophosphorylation was
significantly less efficient and reaches saturation at approximately 500 ng/ml (Fig. 3D). These results establish that in a
cellular environment a TrkC receptor mutant with the point mutation
T338A does interact with NT-3 less efficiently than native TrkC and that the triple mutation N335A/T338A/N342A resulted in a TrkC receptor
mutant that does not respond to NT-3 stimulus by
autophosphorylation.

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Fig. 3.
NIH3T3 cell lines expressing TrkC receptor
mutants and induction of autophosphorylation by NT-3. A, the
expression of receptors is shown after immunoprecipitation with the
pan-Trk 443 antiserum. Two independent cell lines with different levels of expression were selected for each of the constructs and assayed for
neurotrophin-induced autophosphorylation. C-18 and C-3 are expressing
human TrkC; 48-2 and 48-4 are expressing the TrkC mutant T338A; and
92-3 and 92-5 are expressing the TrkC triple mutant N335A/T338A/N342A.
Each of the lanes represents the same amount of total protein and the
same exposure to the film; therefore, the intensity of the signal is a
relative measure of expression levels. B, NT-3-induced
autophosphorylation of receptor mutants. NIH3T3 cell lines expressing
human TrkC at high (C-18) and low (C-3) levels at 0 and 100 ng/ml NT-3
(lanes 1-4), the human TrkC mutant T335A at low (48-2) and
high (48-4) levels at 0 and 100 ng/ml NT-3 (lanes 5-8), and
the triple mutant N332A/T335A/N342A at high (92-3) and low (92-5)
levels at 0 and 100 ng/ml NT-3 (lanes 9-12). The receptors
were immunoprecipitated with the pan-Trk antiserum 443, and bands were
stained with the anti-phosphotyrosine antibody 4G10 after Western
transfer as described (35, 36). C, NT-3-induced dose
response of TrkC (cell line C-3) autophosphorylation. Concentrations of
NT-3 are given in ng/ml. The gel was exposed to film for 10 s.
D, NT-3-induced dose response of autophosphorylation of the
human TrkC mutant T335A (cell line 48-4). Concentrations of NT-3 are
given in ng/ml. The gel was exposed to film for 2 min.
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Soluble TrkC Mutants Do Not Inhibit NT-3-induced Neurite Extension
on PC12/TrkC Cells--
PC12 cells that were engineered to
constitutively express rat TrkC respond to NT-3 by forming neurite
extensions (35). This terminal differentiation event is
dose-dependent (35), and consequently PC12/TrkC cells have
become a useful tool for the analysis of TrkC receptor agonists (30).
In this study, soluble immunoadhesins of TrkC and mutants of TrkC were
used to compete for NT-3 and test for inhibition of NT-3 induction of
neurites on PC-12/TrkC cells in analogy to experiments where the
soluble Trk receptor immunoadhesins were used to inhibit
neurotrophin-induced survival of peripheral neurons (28). In this
assay, the level of inhibition of neurite outgrowth by a particular
receptor mutant is inversely proportional to its relative affinity to
soluble NT-3; reduction in a mutant's ability to compete for and
prevent NT-3 binding to TrkC on the cell results in increased neurite
outgrowth (compared with soluble native TrkC).
When NT-3 was added to the PC12/TrkC cells at concentrations of 1 ng/ml
or 250 pg/ml, 67 ± 6 and 59 ± 3% of the cells
differentiated and extended neurites (Fig.
4, A and B). When
NT-3 was absent, no differentiation of neurons occurred (Fig.
4C). The addition of 40 or 100 ng/ml soluble TrkC to 1 ng/ml
NT-3 completely blocked the neurite extension response of PC12/TrkC
cells (Fig. 4, D and E; Table
VI). The same was observed when the ratio
of TrkC to NT-3 was even higher (400 ng/ml TrkC, 250 pg/ml NT-3; Fig.
4F). A concentration of 40 ng/ml soluble TrkC was the
minimal concentration at which the saturating amount of 1 ng/ml NT-3
was inhibited completely (data not shown). Similarly, a series of TrkC
mutants were tested for their ability to block NT-3 action on PC12/TrkC
cells. The triple mutant N335A/T338A/N342A did not inhibit NT-3 at any
of the three receptor/NT-3 ratios (Fig. 4, G-I; Table VI),
and the PC12/TrkC cells differentiated similarly as when NT-3 was added to the cells in the absence of receptor. This supports the results from
the binding studies where it was shown that this TrkC mutant was unable
to interact with NT-3. Even the single alanine mutants of these
residues (N335A, T338A, and N324A) as well as N335Q, T338V, N342D,
N342Q, and E287K completely lost the ability to inhibit NT-3 at all
three receptor/NT-3 ratios (Table VI) and resulted in differentiated
PC12/TrkC cells equivalent to Fig. 4, G-I. In addition to
mutations of the residues Asn335, Thr338,
Asn342, and Glu287, we tested mutants that may
be close in the three-dimensional structure to the main binding
determinants. While all of these mutants inhibited NT-3 action at high
receptor/NT-3 ratios (similar to native TrkC), the mutants E283A,
R285A, L333A, N341A, and N307A were less potent than TrkC at lower
ratios (Table VI). The observation that N307A and N341A did not inhibit
neurite outgrowth at the lowest ratio tested (1 ng of NT-3/40 ng of
receptor) suggests that this assay is more sensitive than the
plate-based competition assay in which N307A and N341A showed binding
equivalent to native TrkC (Table I).

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Fig. 4.
Inhibition of NT-3-induced neurite outgrowth
of PC12/TrkC cells by soluble TrkC and TrkC mutants. A,
B, and C show responses to NT-3 at 1 ng/ml, 250 pg/ml, and 0 ng/ml, respectively. D, E, and
F are responses to NT-3 and TrkC at 1 ng/ml:40 ng/ml, 1 ng/ml:100 ng/ml, and 250 pg/ml:400 ng/ml, respectively. Note that the
neurite extension response is completely blocked by the presence of
TrkC. G, H, and I are responses to
NT-3 and the TrkC triple mutant N335A/T338A/N342A at 1 ng/ml:40 ng/ml,
1 ng/ml:100 ng/ml, and 250 pg/ml:400 ng/ml, respectively. Note that the
neurite extensions in G, H, and I are
similar to the response in A and B.
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Table VI
NT-3-induced neurite outgrowth of PC12/TrkC cells in the presence of
soluble TrkC and TrkC mutants
The proportion of neurite-bearing PC12/TrkC cells was determined by
counting the number of cells containing neurites at least twice the
length of the cell body 3 days after application of NT-3 and soluble
TrkC receptors at the indicated concentrations. The percentage of cells
that extended neurites was as follows: +++, >50%; ++, 25-50%; +,
5-25%; , <5%. When PC12/TrkC cells were incubated in the absence
of soluble receptor with 1 ng/ml and 250 pg/ml NT-3, 66.8 ± 5.8%
and 59.2 ± 3.2%, respectively, of the cells extended neurites.
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NGF-induced Autophosphorylation of TrkA Receptor Mutants--
To
test the ligand interactions of the receptor mutants in a cellular
environment, stable NIH3T3 cell lines expressing TrkA and the mutants
T292A, E301A, and T319A/H320A/N323A were constructed. The expression of
the receptors was demonstrated by immunoprecipitation with the pan-Trk
antibody 45 (Fig. 5). As with the TrkC
receptors, the apparent molecular weights of the full-length human TrkA
and TrkA mutants indicate the presence of a significant amount of glycosylation. For each of the constructs, two cell lines with different levels of expression were chosen for NGF-induced
autophosphorylation: A-3 and A-5 for TrkA; 10-6 and 10-9 for E301A;
40-2 and 40-5 for T292A; and 51-4 and 51-7 for T319A/H320A/N323A. The
addition of NGF at 100 ng/ml (+ in Fig.
6) resulted in autophosphorylation of
TrkA and all TrkA mutants except for the triple mutant (51-4 and 51-7;
Fig. 6). Dose-response autophosphorylation experiments showed that the
TrkA receptor reaches saturation at 25 ng/ml (Fig. 7, top). The E301A mutant,
with NGF binding reduced by 3-fold, also showed saturation at 25 ng/ml
(Fig. 7, middle), while the T292A mutant, with NGF binding
reduced by 20-fold, showed saturation at 50 ng/ml (Fig. 7,
bottom). Hence, as also found with the TrkC mutants, reduced
binding of NGF by TrkA mutants correlated with reduced
autophosphorylation in a cellular environment.

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Fig. 5.
NIH3T3 cell lines expressing TrkA receptor
mutants. The expression of receptors is shown after purification
with wheat germ agglutinin and immunoprecipitation with the pan-Trk 443 antiserum. Two independent cell lines were selected for each of the
constructs. A-3 and A-5 are expressing human TrkA; 10-6 and 10-9 are
expressing the TrkA mutant E301A; 40-2 and 40-5 are expressing the TrkA
mutant T292A; and 51-4 and 51-7 are expressing the TrkA triple mutant T319A/H320A/N323A. Each of the lanes represents the same amount of
total protein and the same exposure to the film; therefore, the
intensity of the signal is a relative measure of expression levels.
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Fig. 6.
NGF-induced autophosphorylation of receptor
mutants. NIH3T3 cell lines expressing human TrkA or TrkA mutants
were exposed for 5 min to 0 ( ) or 100 (+) ng/ml NGF. After cell
lysis, the receptors were immunoprecipitated with the pan-Trk antibody
45, and bands were stained with the anti-phosphotyrosine antibody 4G10
after Western transfer as described (35, 36).
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Fig. 7.
NGF-induced dose response of TrkA
autophosphorylation. NIH3T3 cell lines expressing human TrkA
(top), E301A TrkA mutant (middle), or T292A TrkA
mutant (bottom) were exposed for 5 min to 0, 10, 25, or 50 ng/ml NGF. After cell lysis, the receptors were immunoprecipitated with
the pan-Trk antibody 45, and bands were stained with the
anti-phosphotyrosine antibody 4G10 after Western transfer as described
(35, 36). The gels were exposed to film for 1 min.
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DISCUSSION |
Neurotrophins mediate their signal into the cell through specific
interactions with tyrosine kinase receptors of the Trk family (reviewed
in Refs. 11 and 43). Mutational analyses of human NT-3 (30) and mouse
and human NGF (44, 45) have defined the binding sites on these
neurotrophins for TrkC and TrkA, respectively. In addition, the regions
controlling specificity of the neurotrophins have been localized to the
central
-strand bundle (46) and at least one loop connecting the
-strands (47). Notably, the residues that are involved in binding
are not identical to those controlling specificity (46).
While the binding sites on NGF and NT-3 for their cognate Trk receptors
have been determined by mutational analysis, the details of the binding
site on the Trk receptors have remained unexplored. It has been shown
previously that the second immunoglobulin-like domain of the
extracellular portion of TrkC is the main determinant for interaction
with NT-3 (14) and that both the first and second immunoglobulin-like
domains are important for TrkA binding of NGF (14, 15). The present
study involved a more detailed analysis of the second
immunoglobulin-like domain of TrkA and TrkC using molecular modeling
and surface scanning mutagenesis.
Loop EF Plays a Major Role in Binding Neurotrophins--
TrkC is
the most stringent member of the Trk family in terms of specificity; it
binds NT-3 with high affinity but not the other neurotrophins (7). TrkA
preferably binds NGF, and TrkB preferably binds BDNF and NT-4/5, but at
least in vitro, they can bind NT-3 with lower affinity (36,
48-53). This study has shown that both TrkA and TrkC utilize loop EF
as a dominant component of their binding site (Table I). All three Trk
receptors possess a conserved sequence in loop EF: abPTHcNNGd, where a
is N or D; b is Q, N, or K; c is V, M, or Y; and d is N or D (Fig. 1).
While the threonine and second conserved asparagine residues are
important for both TrkA and TrkC (Table I), the two receptors differ in their utilization of the other residues in loop EF. In TrkA,
Gln317, His320, and Val321 are
involved in binding, whereas in TrkC the analogous Lys336,
His339, and Tyr340 are uninvolved; in TrkA,
Asn316 is unimportant, while the analogous TrkC residue
Asn335 is important (Table I). This difference in
utilization of residues in this loop suggests that loop EF may play a
role in determining the specificity, as well as binding, of the
neurotrophins.
Inspection of the three-dimensional structures of immunoglobulins and
immunoglobulin-like domains determined by x-ray crystallography reveals
that loop EF may have one of two basic conformations:
-helical as in
immunoglobulin CH2 and CH3 domains (21) or with a side chain:main chain
hydrogen bond as in VCAM domain 1 (20), CD4 domain 2 (54, 55), and
telokin (54). The amino acid sequence of loop EF in the Trks could
accommodate either conformation. In all three Trk receptors, loop EF
has a hydrophobic residue (Val321 in TrkA;
Tyr340 in TrkC), which could anchor a putative
-helix to
the domain, similar to the function of the loop EF tryptophan residues
in immunoglobulin CH2 and CH3 domains (21). In the alternative conformation, the first conserved asparagine residue
(Asn322 in TrkA; Asn341 in TrkC) could provide
the side chain:main chain hydrogen bond as found in VCAM domain 1, for
example (20). However, changing this asparagine residue to alanine in
both TrkA and TrkC had little or no effect on binding (Table I); this
would argue against this conformation, since alteration of the
analogous EF loop asparagine in VCAM domain 1 affected both binding to
receptor and anti-VCAM mAbs (56). If loop EF adopts this alternative
conformation, then the loop EF hydrophobic residue would be
solvent-exposed and therefore could be important for determination of
specificity of the Trk receptors by direct interaction with
neurotrophins. The results presented here do not allow a definitive
choice between the two possibilities.
Mutations to amino acids other than alanine at positions within loop EF
in TrkC point to rather stringent requirements for binding. TrkC
residue Asn342 was the most sensitive to mutation, since
all replacements at this position resulted in a reduction of TrkC
receptor function (Table I). Mutations at Asn335 and
Thr338 were tolerated if they involved amino acids that
were isosteric or similar in chemical character, but exchanges to
larger side chains reduced binding significantly, indicating that the
spatial requirements at this part of the binding site may be very
restricted.
Loop EF and loop AB, which play a role in the interaction of TrkA and
TrkC with their ligands, have previously been shown to be important for
molecular recognition in immunoglobulins and immunoglobulin-like
domains with their receptors. Mutational analysis of the interaction of
IgE with its high affinity receptor demonstrated that, among others,
loop EF residues in the IgE C
3 domain are major contributors to
affinity (57). The three-dimensional structure of the complex between
S. aureus protein A and IgG Fc (domains CH2 and CH3)
revealed that major contacts are formed by residues in loop EF
(Gln311 and Leu314) and loop AB
(Met252, Ile253, and Ser254) of the
CH2 domain (21); these same two loops are likewise utilized when IgG
binds to the neonatal Fc receptor (58). In VCAM, residue
Asn65 in loop EF is important for binding to the human
receptor but not the murine receptor (56). Although other
immunoglobulin-like domains may utilize loops other than AB and EF,
e.g. mucosal addressin cellular adhesion molecule (59) and
intercellular adhesion molecule (60), the common usage of loops EF and
AB by functionally quite different members of the immunoglobulin
superfamily suggests that this arrangement of functional side chains
provides an example of a structural motif optimal for protein-protein
interaction.
The three-dimensional relationship of the five domains in the Trk
receptors is unknown. The complexity of the epitopes for the anti-TrkA
and anti-TrkC antibodies in conjunction with blocking versus
nonblocking character (Tables II and IV) suggests that two or more of
the domains may interact with one another. For example, it has been
shown previously that TrkC domain 5 alone can bind NT-3 and undergo
autophosphorylation (14). However, the anti-TrkC mAb C1435, which binds
to domains 1-3, blocks NT-3 binding to full-length TrkC (Table IV).
Similarly, some TrkA mutants in domain 5 affect binding to mAb A1488,
which does not map to TrkA domain 5 (Tables II and III), Since both
domain 4 and domain 5 are important for NGF binding in TrkA (14, 15)
the TrkA domain 5 mutants P269E and T327A/L329A (Tables I and III)
could affect binding by disrupting the domain 4-domain 5 interface
(rather than by direct interaction with NGF). The short link between
domains 4 and 5 also suggests that domain 5 loops BC, DE, and/or FG
could form interdomain contacts with domain 4 (similar to the loop
interdomain contacts in VCAM (20)); mutations in these loops might also disrupt the domain-domain interface.
Specificity Determinants of Neurotrophin/Receptor
Interactions--
While both TrkA and TrkC utilize loop EF as a major
component of the binding site, the two receptors differ in their
interaction with the amino acids in loop EF, as noted above, as well as
in their utilization of the rest of the second immunoglobulin-like domain. TrkC is the most stringent among the Trk receptors in its
binding of neurotrophins, yet only a few residues outside of loop EF
seem to be involved in binding: Glu287, Cys331,
and His363 (Table I, Fig. 2A). In contrast, in
TrkA numerous residues outside of loop EF were major
(Pro269, His310,
Ser340/Met342) or minor (e.g.
Val261, His264, Thr292,
Thr307, Thr327/Leu329,
Asn332, Glu355/Asp356) contributors
to binding (Table I, Fig. 2B). Two modes of neurotrophin/Trk interaction, which are not mutually exclusive, are possible. First, it
has been shown previously that both NGF and NT-3 use residues in their
central
-strand bundle for binding (30, 44). However, NGF also
utilizes residues at its N terminus (44, 45, 61), whereas NT-3 does not
(30). This suggests that the central
-strand residues in the
neurotrophins might interact with loop EF in the Trk receptors, while
the NGF N-terminal residues interact with other portions of the TrkA
second immunoglobulin-like domain (interactions not present in the
NT-3/TrkC interaction). One candidate for control of binding and
specificity is
-strand B/loop AB. In TrkC, one residue in the
N-terminal portion of
-strand B (Glu287) is important
for binding NT-3, while in TrkA, several residues in loop AB
(Val261, Met263, and His264) are
important. Second, while portions of the TrkC second
immunoglobulin-like domain outside of loop EF may not play a role in
binding, they may play an important role in
specificity by providing negative interaction with the
noncognate neurotrophin ligands. Hence, NGF may be prevented from
binding to TrkC not by residues in loop EF (most of which are
conserved) but by residues external to loop EF; similarly, NT-3 may be
prevented from binding to TrkA by negative interaction with residues
outside of loop EF. This possibility is supported by the difference in
binding affinity exhibited by the TrkC R295A and R295D mutants. The
TrkC R295A mutant showed no reduction in binding, but a charge
reversal, TrkC R295D (TrkA has an aspartic acid at this position),
effected a 12-fold reduction in binding (Table I). Further support
comes from previous findings that residues in the neurotrophins
involved in binding may be segregated from those involved in
specificity (46) and that changing specific residues in NT-3 altered
binding to TrkA and TrkB but not TrkC (62).
TrkA and TrkC also differ in the nature of residues in the segment
connecting the second immunoglobulin-like domain to the transmembrane
sequence (Fig. 1). Several residues in the TrkA segment affected NGF
binding when mutated (Table I), and these may provide additional
binding/specificity determinants not present in TrkC. A naturally
occurring TrkA receptor isoform carries an insert of six amino acids
between the second immunoglobulin-like domain and the transmembrane
sequence (63), and a similar isoform has also been detected in human
TrkC (28). When tested in fibroblasts, the presence or absence of the
insert did not affect the ligand specificity of the receptor (63).
However, when the TrkA isoform containing the six-amino acid insert was
expressed in PC12nnr5 cells and analyzed for NT-3-induced biological
responses, significantly more neurite outgrowth was observed than in
the cell line expressing a variant lacking the insert (64). This
suggests that the presence of this insert might modulate the
specificity of TrkA in a PC12 cell environment. Although the mechanism
of this effect is unknown, it is conceivable that the insert may
influence the orientation of the nearby binding domain, or,
alternatively, amino acids in the insert may interact directly with
neurotrophins.
While this study has provided a high resolution map of the residues in
the second immunoglobulin-like domain of TrkA and TrkC involved in
neurotrophin binding, it has only provided a glimpse of possible modes
of specificity determination for these receptors. The data presented
here show that TrkC may have fewer positive interactions with its
cognate ligand NT-3 than does TrkA with its cognate ligand NGF and
suggest that negative interactions may dominate the specificity. To
more fully understand the mechanism of specificity, mutants of the Trks
that can recruit binding of the noncognate neurotrophin ligands need to
be evaluated, similar to studies on the specificity of neurotrophins
(30, 46, 47).
We thank Susan Spencer and David Shelton
(Genentech) for making the cDNAs for the human TrkA and TrkC
receptor immunoadhesins available; Gene Burton and Evelyn Martin
(Genentech) for purified recombinant neurotrophins; Eva Szöny and
Enrique Escandón (Genentech) for labeled NT-3; and Mark Vasser,
Parkash Jhurani, Peter Ng, and Kristina Azizian (Genentech) for
oligonucleotide synthesis.