(Received for publication, October 25, 1995)
From the
Neurotrophins regulate differentiation and survival of
vertebrate neurons through binding to members of the Trk family of
receptor tyrosine kinases and to a common low affinity receptor,
p75. The specificity of neurotrophin action is
determined by their selective interaction with the different members of
the Trk family; TrkA, TrkB, and TrkC serve as cognate receptors for
nerve growth factor, brain-derived neurotrophic factor, and
neurotrophin-3 (NT-3), respectively. Unlike nerve growth factor and
brain-derived neurotrophic factor, NT-3 can to some extent also bind
and activate non-cognate TrkA and B receptors, although the
physiological relevance of these interactions is unclear. Previous
studies established that neurotrophins use an extended surface for
binding to cognate Trk receptors, while binding to p75
is mediated by a localized cluster of positively charged
residues. Here we show that the binding site of NT-3 to its
non-preferred receptors TrkA and TrkB is dominated by two positively
charged residues, Arg-31 and His-33, previously shown to constitute a
main determinant of binding to p75
.
Simultaneous mutation of these two residues into Ala completely
abolished NT-3 binding and signaling through TrkA and greatly
diminished binding and activation of TrkB. However, NT-3 binding and
signaling through its cognate receptor TrkC was unaffected by the
mutation. These results show that binding of NT-3 to
p75
, TrkA, and TrkB is mediated by a common
determinant, which is distinct from that recognized by TrkC and also
different and more localized than the one recognized by TrkA and TrkB
in their cognate ligands. Thus, although homologous regions in all
neurotrophins are used for binding to Trk receptors, a given Trk may
actually contact different residues in different neurotrophins. The
mutant NT-3 described here may be of greater advantage than native NT-3
when a trophic activity needs to be specifically targeted to
TrkC-expressing neurons and provides a monospecific neurotrophin for
future therapeutic development.
Vertebrate neurons are dependent on the continuous supply of a
group of proteins generally called neurotrophic factors for their
development, differentiation, and survival. The neurotrophins are
structurally and functionally related neurotrophic factors that
function in the developing and adult nervous
system(1, 2, 3) . In mammals, the
neurotrophin family presently includes four members, nerve growth
factor (NGF)()(4) , brain-derived neurotrophic
factor (BDNF)(5, 6) , neurotrophin-3
(NT-3)(7, 8, 9, 10, 11, 12) ,
and neurotrophin-4 (NT-4)(13, 14) , also called
NT-5(15) , which share approximately 50% amino acid sequence
identity. An additional neurotrophin molecule with structural and
functional similarities to NGF has recently been isolated from the
platty fish and named NT-6 (16) , despite being only the third
neurotrophin identified in this species. The neurotrophins are
non-covalently linked homodimers of two highly basic 120-residue long
polypeptide chains. Each protomer contains three disulfide bridges
forming a cysteine knot at the bottom of the structure (17) and
three pairs of anti-parallel
-strands connected by
-hairpin
loops(18) . The latter contain most of the variable residues
found among all the neurotrophins(19) .
Neurotrophins
mediate their action by binding to two classes of specific neurotrophin
receptors. The low affinity neurotrophin receptor
(p75) is a transmembrane glycoprotein that
binds all neurotrophins with equal affinity(20, 21) .
p75
is structurally related to the tumor
necrosis factor receptor and to CD40, and it contains a short
cytoplasmic domain of unknown biochemical function. In contrast to
p75
, members of the Trk family of tyrosine
kinase receptors exhibit ligand-dependent activation of endogenous
tyrosine kinase activity and can thus clearly function as signaling
receptors(22, 23) . Specificity of neurotrophin action
is achieved by their selective interaction with cognate members of the
Trk family of receptors. Thus, p140
(TrkA) is
the cognate receptor for NGF, p145
(TrkB)
serves as receptor for BDNF and NT-4, while p145
(TrkC) is the cognate receptor for NT-3(22) . Unlike
the other neurotrophins, NT-3 can also interact with non-cognate Trk
receptors, although the physiological importance of these interactions
is still unclear(24, 25) .
Studies of
structure-function relationships in the neurotrophins have identified
amino acid residues important for their interaction with cell surface
receptors(26) . The binding site of neurotrophins to
p75 is dominated by positively charged
residues located in two spatially close variable loop
regions(27, 28) . Although the precise nature and
spatial arrangement of the side chains involved vary among the
different proteins, all neurotrophins utilize a similar cluster of
positive charges for binding to
p75
(28) . Disruption of
p75
binding does not interfere with binding
and activation of cognate Trk receptors, although it does appear to
affect responsiveness to some neurotrophins in cells co-expressing
p75
and Trks(28) . (
)In
contrast to p75
, the interaction of
neurotrophins with cognate Trk receptors involves multiple contacts
distributed along the side of the neurotrophin dimer, parallel to the
2-fold symmetry axis (19, 29) . Taken together, data
from different studies on neurotrophin-Trk interactions support a model
in which conserved residues in
-strands provide the contacts with
the highest binding energy, while variable residues in turns and loop
regions along this surface determine biological specificity, either by
contributing directly with contacts of lower energy to cognate
receptors or indirectly, by preventing interaction with inappropriate
receptors. Loss-of-function studies have shown that the binding epitope
of NT-3 to its cognate receptor TrkC appears dominated by the conserved
residue Arg-103, together with Glu-10, Tyr-51, Glu-54, and
Arg-56(29) . In addition, gain-of-function experiments have
established the role of variable residues 39-48 in determining
the specificity of binding of NT-3 to TrkC(30) .
Although
NT-3 can also interact with TrkA and TrkB, this neurotrophin lacks some
of the major determinants of binding found in the preferred ligands of
these receptors, NGF and BDNF(19, 30) , suggesting
that the interaction of NT-3 with its non-preferred receptors may be
mediated by residues conserved in all three neurotrophins. We show here
that, surprisingly, the binding site of NT-3 to TrkA and TrkB is
dominated by two positively charged variable residues, Arg-31 and
His-33, previously shown to constitute a main determinant of binding to
p75. Mutation of these residues does not
affect NT-3 binding or activation of TrkC. Thus, binding of NT-3 to
p75
, TrkA, and TrkB is mediated by a common
epitope distinct from that recognized by its cognate receptor TrkC.
A mutant NT-3 with Arg-31 and His-33 replaced by Ala
(R31A+H33A NT-3) was produced and purified from
baculovirus-infected insect cells. This mutation has previously been
shown to abolish NT-3 binding to the low affinity receptor
p75 but not to affect the ability of the molecule to
induce TrkC autophosphorylation and to promote neuron
survival(28) . We examined the ability of the R31A+H33A
NT-3 mutant to interact with TrkA and TrkB receptors in steady-state
competitive binding assays using stably transfected mouse 3T3
fibroblasts. Iodinated NT-3 was used as tracer, and the abilities of
mutant and wild type NT-3 to displace binding of labeled NT-3 from
fibroblasts expressing TrkA, TrkB, and TrkC, respectively, were assayed
and compared. The mutated NT-3 was totally unable to displace
I-NT-3 from TrkA receptors (Fig. 1A).
Wild type NT-3, in contrast, displaced binding of radiolabeled ligand
with an IC
at 60 ng/ml. Using
I-NGF, wild
type NT-3 appeared about 10-fold less potent than NGF in binding to
TrkA receptors (Fig. 1B), in agreement with previous
observations. Mutant NT-3, however, was again unable to bind TrkA even
at 3 µg/ml, the highest concentration tested (Fig. 1B). The R31A+H33A mutation also impaired
binding to TrkB receptors (Fig. 1C). The IC
of the mutant was approximately 20-fold higher than that of wild
type NT-3, which in this assay is comparable with that of BDNF. Despite
its effects on TrkA and TrkB binding, the mutation had no effect on
binding to TrkC (Fig. 1D). In this assay, the IC
values of mutant and wild type NT-3 were comparable, in agreement
with previous results showing unimpaired ability to induce TrkC
autophosphorylation in fibroblasts(28) .
Figure 1:
Binding of wild type and
R31A+H33A mutant NT-3 to TrkA, TrkB, and TrkC receptors expressed
on fibroblast cell lines. A, C, and D,
serial dilutions of purified wild type NT-3 () and mutant NT-3
R31A+H33A (
) were assayed for their ability to displace
I-NT-3 from TrkA (A), TrkB (C), or TrkC (D) expressing fibroblasts. B, serial dilutions of
purified wild type NGF (
), wild type NT-3 (
), and mutant
NT-3 R31A+H33A (
) were assayed for their ability to displace
I-NGF from TrkA-expressing fibroblasts. Each point represents the mean ± S.D. of triplicate
determinations.
To correlate binding with receptor activation, we compared the abilities of mutant and wild type NT-3 to induce tyrosine autophosphorylation of TrkA and TrkB receptors expressed on transfected fibroblasts. We previously showed that both mutant and wild type NT-3 were equally potent in stimulating TrkC autophosphorylation(28) . In agreement with its totally impaired binding, no TrkA autophosphorylation was induced by the mutant NT-3 even when tested at 300 ng/ml (Fig. 2A). Wild type NT-3, in contrast, readily induced TrkA activation at 30 ng/ml. In addition, the ability of the mutant to stimulate TrkB autophosphorylation was reduced by 10-30-fold compared with wild type NT-3 (Fig. 2B).
Figure 2: Autophosphorylation of TrkA and TrkB receptors stimulated by wild type and R31A+H33A mutant NT-3. Serial dilutions of purified wild type (wt) NGF (A), wild type NT-3 (A and B), and mutant NT-3 (R31A+H33A) (A and B) were assayed for their ability to stimulate tyrosine autophosphorylation of TrkA (A) and TrkB (B) expressed in fibroblast cells. Autoradiograms of phosphotyrosine blots are shown, indicating factor concentrations in ng/ml. Similar results were obtained in duplicate experiments.
MG87-3T3 fibroblasts expressing Trk receptors survive and proliferate in serum-free medium if this is supplemented with cognate neurotrophins (31, 32) . We compared the ability of mutant and wild type NT-3 to promote survival and growth of fibroblasts expressing TrkA, TrkB, and TrkC receptors, respectively. The R31A+H33A mutant failed to promote proliferation of MG87-3T3 fibroblasts expressing TrkA or TrkB at all concentrations tested (Fig. 3, A and B). Although less potent than NGF and BDNF, wild type NT-3 did stimulate growth of TrkA- and TrkB-expressing fibroblasts when tested at 1 µg/ml (Fig. 3, A and B). In agreement with previous results(28) , the mutant was unimpaired to promote survival and growth of TrkC-expressing fibroblasts (Fig. 3C).
Figure 3:
Biological activities of wild type and
R31A+H33A mutant NT-3 in fibroblasts expressing TrkA, TrkB, and
TrkC receptors. Serial dilutions of purified wild type (wt)
() and mutant (
) NT-3 were assayed for their ability to
stimulate survival and growth of MG87 fibroblasts expressing TrkA (A), TrkB (B), and TrkC (C) as assessed by
acid phosphatase activity. Wild type NGF (
) (A) and BDNF
(
) (B) were used as controls in the TrkA and TrkB assays,
respectively. The results are expressed as the average optical density (O.D.) of triplicate wells ±
S.D.
Finally, we assayed Trk-specific biological activities of mutant and wild type NT-3 in explanted peripheral ganglia from chick embryos. In this assay, ganglion explants are cultured for 48 h in the absence or presence of neurotrophins, and total RNA is subsequently extracted and analyzed for the presence of TrkA, TrkB, and TrkC mRNAs by RNase protection analysis(30, 33) . When embryonic day 8 chick nodose ganglion and dorsal root ganglion sensory neurons are cultured in the presence of 100 ng/ml NT-3, a prominent TrkC mRNA signal is recovered, indicating specific rescue of TrkC mRNA containing neurons by NT-3 (Fig. 4). No TrkC mRNA is recovered if the ganglia are cultured in medium without NT-3 (Fig. 4). In addition to TrkC mRNA, low but significant amounts of TrkB mRNA are also recovered from these cells after NT-3 treatment (Fig. 4; see also (33) ). The effects of NT-3 on TrkB mRNA containing sensory neurons could be due to TrkB and TrkC receptor mRNA coexpression, TrkB mRNA up-regulation, or genuine action of NT-3 through neuronal TrkB receptors. The possibility that NT-3 may in certain circumstances act through TrkB has previously been suggested(34) . However, when chick dorsal root ganglion and nodose ganglion neurons were treated with 100 ng/ml of the R31A+H33A NT-3 mutant deficient in TrkB binding, both TrkC and TrkB mRNA signals were recovered at relative levels, which were comparable to those seen with wild type NT-3 (Fig. 4). Identical results were obtained when mutant and wild type NT-3 were tested at 20 ng/ml (not shown). Given that the mutant was previously found to be as potent as wild type NT-3 on sensory neurons(28) , these results suggest that recovery of TrkB mRNA after NT-3 treatment is likely due to receptor coexpression and not to direct action of NT-3 on TrkB receptors.
Figure 4: Trk-specific biological activities of wild type and R31A+H33A mutant NT-3 in embryonic chick sensory neurons. RNase protection analysis of TrkB and TrkC mRNA in cultures of explanted embryonic day 8 chick dorsal root (DRG) and nodose (NG) ganglia 48 h after incubation with native NT-3, mutant NT-3 (R31A+H33A), or medium. RNA from explanted ganglia prior to culture (control ganglia) was used as a positive control. Because the treatments affect the amounts of RNA recovered from each sample, each lane represents an equivalent amount of ganglia.
In this study, a mutant NT-3 previously characterized with
respect to its interactions with TrkC and p75 was used
to investigate the role of positively charged amino acid residues in
the interaction of NT-3 with its non-preferred receptors TrkA and TrkB.
Unexpectedly, the same mutation that abolished binding of NT-3 to the
low affinity receptor p75
(28) also impaired
binding to and activation of TrkA and TrkB. The fact that NT-3 binding
and signaling through its cognate receptor TrkC was unaffected by the
mutation indicates that NT-3 interacts with p75
, TrkA,
and TrkB through a common and localized determinant, which is distinct
from that recognized by TrkC. Previous studies of TrkC binding
determinants in NT-3 established the importance of conserved residues,
especially Arg-103(29) , as well as variable residues,
particularly positions 39-48 in variable region II(30) .
Thus, like NGF and BDNF(19) , binding of NT-3 to its cognate
Trk receptor appears to be mediated by an extended surface containing
variable and conserved residues from different regions of the molecule.
Although our results show that Arg-31 and His-33 do not appear to be
essential functional epitopes for TrkC binding, their location in the
TrkC binding surface suggests they could nevertheless form part of
structural binding epitopes, involving multiple Van der Waal and
hydrogen bonding contacts. Unlike functional epitopes, such
determinants, taken individually, may not contribute sufficient binding
energy to be easily identified from mutagenesis experiments.
Despite their importance for NT-3 binding to TrkA and TrkB, positively charged residues at the two positions studied here are not essential for binding of NGF or BDNF to these receptors. Mutation of positively charged residues Lys-32 and Lys-34 in NGF, corresponding to Arg-31 and His-33 in NT-3, affected only marginally the binding of NGF to TrkA (27) . This indicates that TrkA interacts with NGF and NT-3 in different ways, the latter being recognized through a more localized epitope, which only has marginal importance for the binding of the cognate ligand. Interestingly, BDNF lacks positively charged residues at these positions, indicating that TrkB may actually contact different residues in BDNF and NT-3. Despite the lack of positive charges in this region, three positively charged residues (Lys-95, Lys-96, Arg-97) in a spatially close loop in BDNF have been shown to play a role in binding to and activation of TrkB(19, 28) . A recently reported crystal structure of a BDNF/NT-3 heterodimer (35) suggests that the side chains of Arg-31 and His-33 in NT-3 could occupy positions equivalent to those of the positively charged residues in BDNF and therefore may be able to provide similar contacts with the TrkB receptor. In any case, while mutation of Arg-31 and His-33 in NT-3 resulted in a 20-fold decrease in the affinity of binding to TrkB, only a 3-5-fold reduction was observed after simultaneous mutation of Lys-95, Lys-96, and Arg-97 in BDNF(19) , indicating that the TrkB binding site of this neurotrophin includes additional determinants.
Although NT-3 can
interact with TrkA and TrkB and induce receptor activation and survival
in fibroblasts ectopically expressing these two
receptors(36, 37, 38) , NT-3 does not appear
to be an efficient agonist of TrkA and TrkB in primary neurons and
neuronal cell
lines(24, 25, 32, 39) . It has been
proposed that the efficiency of the interaction of NT-3 with
non-cognate receptors may depend on the cellular context (32) and on the presence of accessory receptor molecules such
as p75(24, 25) . Efficient interaction
of NT-3 with TrkA appears also to depend on the specific isoform of
TrkA expressed. In particular, a TrkA splice variant containing a
6-amino acid insert in the proximal part of the extracellular
domain(40) , but not TrkA lacking the insert, has been shown to
mediate NT-3-dependent neurite outgrowth when overexpressed in PC12
cells(25) . A recent study reported TrkB-dependent neurotrophic
activities of NT-3 on developing sensory neurons isolated from mouse
embryos homozygous for a null mutation in the trkC
gene(41) . Our analysis of the survival of subpopulations of
embryonic chick sensory neurons suggests that in normal cells NT-3 acts
predominantly through its cognate receptor TrkC. Comparison of the
effects of wild type and mutant NT-3 did not reveal specific actions of
NT-3 through TrkB; the TrkB-expressing neurons that responded to NT-3
appeared to co-express TrkC. The differences between our study and the
one with neurons from trkC -/- mice may be due to
the high doses of NT-3 (over 20-fold higher or up to 100 nM)
used in the latter and suggest that, in the absence of TrkC, NT-3 may
function through TrkA and TrkB albeit with low efficiency. A more
localized binding site of NT-3 to TrkA and TrkB may be responsible for
the lower affinity and more limited biological actions of NT-3 on these
two receptors.
Neurotrophins are likely to have diverged from a
common ancestor. Present time neurotrophins may still share common
determinants of binding to different Trk family members, while their
variable regions have probably evolved through the acquisition of
specific binding determinants that allow specificity. Why have the
interactions of NT-3 with TrkA and TrkB been preserved during
neurotrophin evolution? Are these molecular relics of interactions
between ancestral neurotrophins and Trks? The fact that the
R31A+H33A mutation did not affect binding to TrkC suggests that
these residues could not have been maintained because of their
importance in TrkC binding as they are dispensable for binding to this
receptor. On the other hand, the complete inability of the
R31A+H33A mutant to interact with p75 suggests
that a need to maintain binding to the low affinity receptor could have
imposed evolutionary constraints to the rate of divergence of these
residues. Thus, NT-3 binding to TrkA and TrkB may be an indirect
consequence of the overlap with the p75
binding site,
suggesting that non-cognate interactions of NT-3 with TrkA and TrkB may
be physiologically irrelevant. Alternatively, NT-3 could have yet
unknown functions, required for normal development and survival,
mediated via TrkA and TrkB. The physiological relevance of the
interactions of NT-3 with p75
, TrkA, and TrkB receptors
could directly be addressed by introducing the R31A+H33A mutant
gene into NT-3 -/- mice(42) .
In this report, we describe a monospecific mutant NT-3 that exhibits normal activities toward TrkC, although it is unable to bind or to activate TrkA and TrkB. Because neurotrophins are being developed as therapeutic agents for nerve injury and neurodegenerative diseases, it is important to consider the potential effects of NT-3 on neurons expressing TrkA and TrkB in addition to those that express TrkC. In this respect, the mutant NT-3 described here may be of greater advantage than native NT-3 when a therapeutic treatment needs to be specifically targeted to TrkC-expressing neurons.