(Received for publication, January 10, 1995; and in revised form, August 31, 1995)
From the
The extracellular domains of the TrkA nerve growth factor (NGF)
receptor and its homologs harbor a modular mosaic of potential ligand
binding motifs, namely two immunoglobulin (Ig)-like modules and an
LRM cassette consisting of a tandem array of three
leucine-rich motifs (LRMs) flanked by cysteine-rich clusters
(Schneider, R., and Schweiger, M. (1991) Oncogene 6,
1807-1811). Identification of a structural motif capable of
specifically recognizing the various neurotrophins was achieved by
assessing their affinities to isolated recombinant modules of TrkA and
TrkB. In both receptors the LRM
cassette alone could
mediate the respective neurotrophin selectivities and affinities.
Further tracking down of this NGF-binding site in TrkA strikingly
revealed that a single LRM of 24 amino acids could bind NGF selectively
with nanomolar affinity. Since this is the first example of a single
LRM with a highly specific, well defined function, it might serve as a
valuable tool to elucidate the general structural requirements of
substrate recognition and high affinity binding in the large
superfamily of LRM-containing proteins.
Nerve growth factor (NGF) ()was the first growth
factor to be discovered over 40 years ago(2) . It belongs to
the growing family of neurotrophins that promote the survival and
differentiation of distinct neuronal
populations(3, 4, 5, 6) .
Neurotrophins bind to two discrete receptor types, which can be
distinguished pharmacologically. p75
binds all known
neurotrophins with similar nanomolar
affinities(7, 8) , whereas cells expressing TrkA, a
tyrosine kinase receptor originally identified as a human
oncogene(9) , bind solely NGF and exhibit significantly slower
dissociation kinetics(10, 11) .
There are two
receptors homologous to TrkA, namely TrkB and TrkC, which are
correlated with the binding of other neurotrophins. TrkB is a receptor
for three distinct ligands, brain-derived neurotrophic factor
(BDNF)(12) , neurotrophin-3 (NT-3)(5) , and
neurotrophin-4 (NT-4)(13) . TrkC exclusively binds
NT-3(14) . Although only cells expressing Trk-type receptors
show functional responses upon neurotrophin binding (15, 16) , it is still a matter of debate whether the
binding phenomena observed in different cellular systems (17, 18, 19, 20) are solely due to
the action of these receptors (21, 22) or rather to
larger protein complexes involving the concerted action of additional
components such as p75. Other imponderabilities of cell
culture-based systems arise from internalization events, temperature
dependence, lysosomal degradation, and cell type-specific differences.
This prompted us to conduct a systematic biochemical characterization
of isolated receptor modules to achieve a clear assignment of
affinities and kinetic properties to defined molecular structures.
The extracellular ligand-binding domains of receptor tyrosine kinases harbor various protein-protein interaction motifs that potentially function in ligand binding and/or receptor dimerization(1, 23, 24) . In many cases mutations in the extracellular domains lead to a ligand-independent constitutive activation of the receptor(25, 26, 27) . In the case of Trk-type receptors the modular structure of the extracellular domain was elucidated by the application of sequence comparison algorithms (1) capable of detecting even highly degenerate motifs(28) . Several distinct protein modules were identified that could serve as docking sites for either neurotrophins or other components involved in a functional receptor complex(1) . Among these are immunoglobulin-like domains and leucine-rich motif (LRM)-containing regions.
LRMs are short amino acid sequences (22-30 residues) that are repeated in tandem in an individual protein and contain hydrophobic residues at conserved positions(29) . In the case of Trk-type receptors, there are three tandem LRM repeats of, on the average, 24 residues in the N-terminal region of the molecule that are flanked by two cysteine-rich regions, so-called cysteine clusters(1) . LRM repeats are potent mediators of strong and specific homo- and heterophilic protein-protein interactions. They have been found in proteins as diverse as human platelet glycoprotein IX(30) , Drosophila Toll(31) , and Drosophila Chaoptin(32) , where they mediate cell-cell interactions and communication, and yeast adenylate cyclase, where they form the interaction site with the Ras protein(33) . This clear involvement of LRM repeats in protein-protein interactions made them highly interesting candidates for the NGF binding site within the TrkA receptor.
On the other hand, immunoglobulin-like domains are firmly established as potent ligand binding domains. The keratinocyte growth factor receptor(34) , the macrophage colony-stimulating factor receptor(35) , and the intercellular adhesion molecule 1 (ICAM-1) (36) are prominent examples of receptors that utilize Ig-like domains for ligand binding. Recently two groups have shown independently that the immunoglobulin-like domains of TrkA, TrkB, and TrkC play quantitatively important roles in the binding of the neurotrophin ligands and in the activation of the receptors(37, 38) .
Essentially the same experiment was
conducted with the other receptor domains of TrkA and TrkB (using I-BDNF). For the binding reactions 1.25 pmol or 2.5 pmol
of receptor protein were used.
Calculations of the K and the
kinetic values were performed according to Rodriguez-Tebar and Barde (42) using the GraFit program (Erithacus).
The goal of this study was to investigate the distinct roles these individual structural modules of Trk-type receptors play in the binding of neurotrophin and how strong of a contribution these receptors make in generating high affinity binding. For an initial experiment, the entire extracellular domains of TrkA and TrkB were expressed in recombinant soluble form and purified as described under ``Experimental Procedures.'' Immobilized on a column, these recombinant domains were found to exhibit the same ligand binding specificities observed in the cellular system(5, 12, 18) , i.e. NGF bound to TrkA, and BDNF and NT-3 bound to TrkB. This is direct evidence that Trk proteins can specifically bind neurotrophins in the absence of their intracellular kinase domain and without membrane attachment or contribution of other receptor components.
Quantification of the
affinities of neurotrophins to the recombinant extracellular domains
using binding assays with I-NGF and
I-BDNF
(see ``Experimental Procedures'') led to K
values of
10
M for the
respective ligand-receptor pairs (NGF-TrkA and BDNF-TrkB) (Table 1). No affinity of
I-NGF to TrkB and of
I-BDNF to TrkA was detected (Table 1), which
verifies the finding of high binding specificity in Trk receptors
described above. These results directly show that the NGF binding sites
with nanomolar K
values observed in NIH 3T3, COS,
and NR18 cells ectopically expressing TrkA (16, 18) can be attributed entirely to the affinity of
the TrkA receptor. The same is true for the BDNF-TrkB interaction. The
nanomolar affinities observed by us for this ligand-receptor pair are
in good agreement with the data obtained by Dechant et al.(43) for A293 cells ectopically expressing chick TrkB and
by Soppet et al.(12) for NIH 3T3 cells ectopically
expressing rat TrkB, the receptor used in this work.
Scatchard plot
analyses of the interactions of I-NGF with the TrkA
extracellular domain (Fig. 1) and of
I-BDNF with
the respective region of TrkB (data not shown) revealed no binding
sites with affinities in the range of 10
M as observed on NIH 3T3-derived cell lines overexpressing TrkA (44) or even in the picomolar range as exhibited by chick
embryonic sensory neurons (17) and other neuronal cells. No
affinities of
I-NGF to TrkB and of
I-BDNF
to TrkA could be detected (Table 1). In vivo more than
50% of the extracellular domains of Trk receptors are made up of
carbohydrates. Our studies with non-glycosylated recombinant proteins
suggest that glycosylation does not enhance the affinity and
specificity of the Trk-neurotrophin interactions but they do not
exclude the possibility that glycosylation may reduce ligand-binding
affinity, possibly even in a regulatory mechanism.
Figure 1:
Scatchard plot analysis of the
equilibrium binding of I-NGF to the recombinant soluble
TrkA extracellular domain. The experiments were carried out with
immobilized recombinant receptor proteins as described under
``Experimental Procedures.'' Final concentrations of
I-NGF ranged from 7.8125
10
to 4
10
M. Each binding
reaction was carried out in duplicate, and for each concentration of
I-NGF a MBP-
Gal control was made to detect
nonspecific binding to MBP. Differences between duplicate values were
generally very small.
The first step in
the identification of the ligand binding sites of TrkA and TrkB was the
expression of the two major structural components of their
extracellular domains, the LRM cassette and the Ig2 domain.
As mentioned above, both types of structures have been shown to be
capable of exerting strong and specific protein-protein
interactions(29, 34, 35, 36, 45, 46) and
thus were good candidates for the ligand binding site. The recombinant
domains were immobilized on column matrices, and their ability to bind
NGF, BDNF, and NT-3 was tested (Fig. 2).
Figure 2:
Panel A, the E. coli-expressed
TrkA and TrkB Ig2-domains exhibit no neurotrophin binding. The
experiments were carried out using affinity columns with immobilized
recombinant receptor modules as described under ``Experimental
Procedures.'' The respective neurotrophin was loaded onto the
column, and the flow-through was analyzed by SDS-polyacrylamide gel
electrophoresis and silver staining. A reduction in the strength of the
neurotrophin band as compared with the flow-through of the
corresponding control column (with immobilized Gal-MBP) represents
specific binding of the neurotrophin to the respective Trk domain.
Binding of all neurotrophins to the Ig2-domains of their non-natural
receptors (i.e. NGF to TrkB and BDNF/NT-3 to TrkA) was also
tested, but no affinities were detected. A,
MBP-TrkA-Ig2-domain; B, MBP-TrkB-Ig2-domain; -, controls
(MBP-
Gal). B, selective neurotrophin binding by the
LRM
cassettes of TrkA/TrkB. The experiments were performed
as described in the legend to panel A. A,
MBP-TrkA-LRM
cassette; B, MBP-TrkB-LRM
cassette; -, controls
(MBP-
Gal).
The binding
experiments with the recombinant Ig2-domains revealed no detectable
affinity of NGF, BDNF, or NT-3 to this segment of any of the two Trk
receptors investigated (Fig. 2A). Additional binding
assays using I-NGF and
I-BDNF (Table 1) confirmed these results. Therefore, our experiments did
not support a role of the Ig2-domain in the binding of NGF to TrkA and
the binding of BDNF and NT-3 to TrkB, but rather suggested the
LRM
cassette as the major neurotrophin binding site. Since
the immunoglobulin-like domains have recently been shown to
significantly contribute to ligand binding in Trk receptors (37, 38) we assume the lack of glycosylation or some
other system-inherent problem prevented the binding of NGF to the
immunoglobulin-like domains in our assays. We therefore concentrated on
investigating the role of the N-terminal LRM
cassette in
Trk receptor function.
This structural entity of TrkA and TrkB could
still discriminate between NGF, BDNF, and NT-3 in that the
LRM cassette of TrkA specifically bound NGF (but not BDNF
and NT-3) and the LRM
cassette of TrkB specifically bound
BDNF and NT-3 (but not NGF) (Fig. 2B). These results
demonstrate that the LRM
cassettes of TrkA and TrkB contain
neurotrophin binding sites displaying the same ligand binding
specificities as the entire receptors. Quantitative binding assays
revealed that the LRM
cassettes of TrkA and TrkB bind
I-NGF and
I-BDNF, respectively, with the
same nanomolar affinities as the complete recombinant extracellular
domains (Table 1).
Since the expression of trkB-derived proteins is significantly hampered by their high
toxicity to E. coli, we chose TrkA to systematically trace
down the exact location of a neurotrophin binding site. Creating
appropriate expression vector constructs, the two cysteine clusters
flanking the LRM cassette were removed first
(L
) followed by the elimination of the first
(L
C
) and the third
(C
L
) LRM repeat in two separate
approaches. All these soluble recombinant receptor proteins showed the
same nanomolar affinities and specificities for
I-NGF (Table 1), suggesting that the 24 amino acids of the middle LRM
are sufficient to constitute a ligand binding site.
This could be
demonstrated by the expression of three further fusion proteins, the
isolated second LRM (L) and two proteins disrupting the
second LRM in the very center from opposite sides
(C
L
and
L
C
). The L
region
exhibited full binding affinity (K
for
I-NGF
10
M) and
specificity, being able to discriminate between NGF, BDNF, and NT-3 in
that no affinity of L
for the latter two neurotrophins was
detected (Table 1). Scatchard plot analysis revealed the
existence of a single type of binding site (data not shown). In
contrast, C
L
and
L
C
showed no detectable affinity for
I-NGF and
I-BDNF (Table 1). These data
unambiguously identify the 24-amino acid L
leucine-rich
motif as an NGF binding site within the TrkA receptor.
The sequence of this region of rat TrkA as well as of the corresponding region of rat TrkB is shown in Fig. 3. The two sequences are 54% identical, leaving 11 residues to account for the ligand binding specificity observed in our experiments. The second LRM is the only repeat in Trk-type receptors that matches perfectly with the general LRM consensus sequence making it a classic structural and functional unit.
Figure 3: Leucine-rich motifs and the NGF binding site of TrkA. The first two lines show an alignment of the second LRM from TrkA and TrkB. The third line is the consensus of both sequences. The last line shows the general consensus sequence of a leucine-rich motif.
To investigate in more detail what part of the binding
characteristics of the NGF receptor complex observed in the cellular
system can be attributed to TrkA or, more precisely, to an
unprecedentedly small ligand binding site of TrkA, the binding kinetics
of the recombinant TrkA extracellular domain as well as of L were examined and compared with the data that have been
determined for different in vivo systems.
The kinetics of
association of L with
I-NGF were found to be
complex (Fig. 4A). The relationship between the observed
association rate (k
) and the ligand concentration is
linear at low concentrations of NGF (5
10
to 4
10
M), allowing the
calculation of a k
close to 1
10
M
s
(Fig. 4A). At higher concentrations of ligand
(around the K
) a mechanism of negative
cooperativity seems to take effect again, leading to a linear
relationship between k
and NGF concentration,
allowing the calculation of a second k
value
of about 3
10
M
s
(Fig. 4A). These data correlate
well with the ones obtained with the recombinant extracellular domain.
The on rate that was observed at low concentrations of ligand is akin
to that observed on PC12 cells(48) . Experiments by Mahadeo et al.(49) using mutants of PC12 cells lacking
p75
indicated that in vivo p75
may in some way assist TrkA in recruiting ligand since only in
the presence of this second neurotrophin receptor were k
values in the range of 10
M
s
observed. Our
results indicate that TrkA may be sufficient to generate on rates in
this range even though such a mechanism seems to only be effective at
low concentrations of ligand.
Figure 4:
The binding kinetics of I-NGF to the 24-amino acid second LRM of TrkA are
equivalent to the ones observed for the entire recombinant
extracellular domain. A, kinetics of association. The observed
association rates (k
) were calculated from the
times at which half of the equilibrium binding was reached (k
= ln 2/t) and plotted against
the total concentrations of NGF. The k
values were obtained from the slopes of the regression lines.
Binding reactions were prepared as described under ``Experimental
Procedures.'' The reactions were brought to final concentrations
of
I-NGF ranging from 5
10
M to 2
10
M.
Aliquots were taken at the different time points and processed as
described above. All data points are means of duplicates. The data are
corrected for nonspecific binding. The k
values for the second LRM of TrkA (L
,
) and the
TrkA extracellular domain (
) were (8.54 ± 1.37
10
)/(2.87 ± 0.68
10
) M
s
and (7.03 ±
1.03
10
)/(2.53 ± 0.72
10
) M
s
,
respectively. B, kinetics of dissociation. Binding reactions
were prepared and brought to equilibrium binding as described under
``Experimental Procedures.'' Concentrations of
I-NGF ranged from 5
10
to 4
10
M. Dissociation of
I-NGF was induced by the addition of a 100-fold excess of
unlabeled NGF. Aliquots were taken at the different time points and
processed as described above. All data points are means of duplicates.
Two separate curves can be drawn through the above data leading to two
different t
and k
values
for each data set. The data for the second LRM of TrkA (L
,
) and the TrkA extracellular domain (Ex,
) were
almost identical, with t
and k
values of
16/110 min and (
5
10
)/(1
10
s
), respectively.
The dissociation kinetics revealed a
similarly complex scenario apparently displaying a curve composed of
two separable components (Fig. 4B). For this reason, as
for the association rates, two different half-lives (16 and
110 min) for the ligand-receptor complex and two different k
(
5
10
s
and
1
10
s
) values could be calculated, again with
L
exhibiting the same behavior as the entire extracellular
domain of TrkA (Fig. 4B). Remarkably, not only this
biphasic behavior but also the measured values are in good agreement
with the ones observed by Meakin et al.(19) in COS
cells expressing rat TrkA (half-lives of 10 and 90 min, respectively).
All of these values clearly represent slow dissociation kinetics,
indicating that the second leucine-rich motif may be involved in
determining the complex kinetics of dissociation that define TrkA as
the ``slow NGF receptor''(50) .
The molecular
mechanism of this complex behavior with respect to the kinetics of
association and dissociation is as yet unclear. The observed negative
cooperativity may somehow be correlated to the formation of di- or
oligomeric receptor complexes. Preliminary results from our laboratory
show that the L fusion protein is capable of forming
oligomers in solution, albeit probably with lower efficiency than the
entire extracellular domain. (
)Taking into account the
concentration of the recombinant proteins in the assays it seems quite
possible that also in this system most of the L
proteins
are present in di-/oligomeric form. These dimers could bind two NGF
dimers with different kinetics, the first being bound with a faster on
rate than the second. Whatever the mechanism, the measured values are
consistent with the equilibrium binding constant determined by us.
Therefore, as summarized in Fig. 5, a single leucine-rich
motif of TrkA not only shows the same affinities for NGF as the
full-length recombinant TrkA receptor but also displays the same
extraordinary kinetic properties. The binding characteristics of the
TrkA receptor are apparently not dependent on glycosylation as far as
the LRM cassette is concerned. The E. coli expressed modules show functionality, and the middle LRM is active
even though it is normally located between two potential glycosylation
sites(1) . The carbohydrate chains attached to these sites may
be a reason why other groups (37, 38) using different in vitro and heterologous in vivo systems were unable
to detect the interaction observed by us. On neurons there may be a
mechanism during receptor activation that exposes the second LRM so it
is accessible for NGF and can fulfill its biological role.
Figure 5: The structure of Trk-type receptors and NGF binding. A, schematic structural organization of Trk-type tyrosine kinase receptors(1) . B, mapping of an NGF binding site in TrkA to a 24-amino acid leucine-rich motif. The abbreviations in this figure are the same as in Table 1.
Our
studies, however, did not demonstrate binding affinities in the range
of 5-10 pM as have been described for some cell lines
overexpressing TrkA receptors(10, 44) . It has been
suggested that this high affinity binding may be a result of the
formation of di-/oligomeric complexes that represent the activated
state of most receptor tyrosine kinases(10) . Even though the
recombinant protein corresponding to the Trk extracellular domains is
capable of forming homodi-/oligomers in vitro, we
cannot, with respect to the fairly high ratio of available MBP docking
sites on the immobilizing matrix to the amount of Trk-MBP protein
present, exclude the possibility that the number of active Trk
di-/oligomers in this experimental system is too low for high affinity
in the picomolar range to be detected. The observation of negative
cooperativity in binding, however, is a good indication that
di-/oligomers are indeed formed in our system.
An interesting
possibility arising from our results is that the promiscuity observed
in Trk-type receptors might be based on the allocation of the binding
sites for the different neurotrophins to distinct LRMs within the
LRM cassette. This would, for example, allow different
neurotrophins to simultaneously bind to one and the same Trk-type
receptor.
Finally, the recently determined three-dimensional structure of an LRM protein (51) offers ideal opportunities for molecular modelling studies on the detailed interaction mechanism not only in the case of Trk-type receptors and neurotrophins but in general for the whole superfamily of LRM containing proteins and their substrates. Hence, the precise mapping of this binding site for NGF in TrkA receptors could facilitate the design of new neurotrophic drugs with enhanced performance and a wider range of supported neurons for the efficient treatment of damaged nerves after injuries or in the course of neurodegenerative diseases.