(Received for publication, May 22, 1995; and in revised form, July 28, 1995)
From the
Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer.
The neurotrophins represent a family of structurally conserved,
basic proteins including nerve growth factor (NGF), ()brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT3), and neurotrophin-4/5 (NT4/5). These proteins are critical for
the development and maintenance of a wide range of neuronal populations
in both the peripheral and central nervous system (Purves, 1988;
Thoenen, 1991). Neurotrophins are believed to exist in solution as
non-covalent dimers (Angeletti et al., 1971; McDonald et
al., 1991; Narhi et al., 1993) at physiologically
relevant concentrations (Bothwell and Shooter, 1977). The protomers of
the dimer are held together through non-polar interactions of a flat
interface formed structurally by three anti-parallel pairs of
-strands, which are highly conserved in terms of primary sequence,
between all known neurotrophins (McDonald et al., 1991). The
neurotrophins exert their biological effects through selective
interaction with and proposed dimerization (Jing et al., 1992)
of a family of high affinity tyrosine kinase receptors (Trks) such that
NGF binds primarily to TrkA (Kaplan et al., 1991a, 1991b;
Klein et al., 1991a), BDNF (Klein et al., 1991b;
Soppet et al., 1991; Squinto et al., 1991), and NT4/5
(Berkemeier et al., 1991; Escandon et al., 1993) to
TrkB and NT3 to TrkC (Lamballe et al., 1991; Tsoulfas et
al., 1993). It has been argued that a single neurotrophin dimer
interacts with two Trk receptors to form a functional receptor
homodimer (Jing et al., 1992; Meakin and Shooter, 1992; Ibanez et al., 1993) and that the high affinity interaction between
the receptor subunits and the neurotrophin is the driving force for
receptor dimerization.
We have previously shown that chimeric neurotrophin dimers can be formed from human and mouse NGF and that these act identically to either human or mouse NGF homodimers in bioassays (Moore and Shooter, 1975; Burton et al., 1992; Luo and Neet, 1992; Schmelzer et al., 1992). We and others have also shown that three different COOH-terminal forms of recombinant human NGF are capable of forming chimeric dimers that exhibit biological activity (Moore and Shooter, 1975; Burton et al., 1992; Luo et al., 1992).
Analysis of residues with high
surface accessibility, at the interface of the mouse NGF homodimer,
indicates a strict conservation of this structure between the different
members of the neurotrophin family. ()The high degree of
homology between interface residues led us to hypothesize that it might
be possible to form heterodimeric neurotrophins. Furthermore, since
these molecules would contain the variable domains from two different
neurotrophins with similar tertiary structure, we were interested to
see how active these molecules would be in terms of receptor binding.
Evidence from proteolyzed neurotrophins and mutagenesis has shown that the amino terminus is the most important binding determinant for interaction of NGF with the TrkA receptor (Moore and Shooter, 1975; Burton et al., 1992; Ibanez et al., 1992; Kahle et al., 1992; Luo and Neet, 1992; Shih et al., 1994). Since neurotrophin heterodimers would have two distinct amino termini, it was also necessary to determine whether both amino termini were implicit in a functional interaction with the receptor. To this end, asymmetric neurotrophin heterodimers were generated to act as specific probes to investigate some of the structural elements important for neurotrophin-receptor interactions.
NGF:NT4/5 was also
compared to NT4/5 for its ability to stimulate phosphorylation of Trk
receptors in primary cultures of fetal rat cortex (Wistar, embryonic
age E15-E16; Charles River, Wilmington, MA). Primary cultures
were prepared as previously described (Knusel et al., 1990).
Cultures grown for 6 days were treated with neurotrophin for 4 min and
immediately lysed in 0.5 ml of lysis buffer (137 mM NaCl, 20
mM Tris, pH 8.0, 1% Triton X-100, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 0.5 mM sodium orthovanadate) at
4 °C. The samples were centrifuged for 10 min at 10,000 g, and the supernatant was stored frozen at -70 °C.
Trk receptors were then immunoprecipitated with a pan-Trk receptor
anti-serum (a gift from Dr. David Kaplan). Samples were Western blotted
and probed with an anti-phosphotyrosine antibody as described in detail
elsewhere (Kaplan et al., 1991a, 1991b). Choline
acetyltransferase activity was determined as previously described
(Knusel et al., 1994).
Figure 1:
A shows the separation of neurotrophins
by HPIEC at A. Panel1, NT4/5; panel2, NGF:NT4/5; panel3, NGF; panel4, pre-HPIEC pool of heterodimer preparation. B, SDS-polyacrylamide gel electrophoresis was performed using
5 µg of each protein on 15% polyacrylamide slab gels with a 4%
stacking gel (both contained 3.8 M urea). Gels were silver
stained (Morrissey, 1981). Lane1, NT4/5; lane2, NGF:NT4/5; lane3, NGF; lane4, low molecular weight standards (Pharmacia). C shows the analytical reverse phase separation of neurotrophins
using a Vydac C
reversed phase HPLC. Panel1, NGF; panel2, NT4/5; panel3, NGF:NT4/5. D shows the thermal-dependent
rates of decay of NGF:NT4/5 and NGF:
9NGF at 37, 25, 4, and
-70 °C.
HPIEC peak ratios during initial production of
heterodimers match the equilibrium peak ratios observed during
rearrangement of heterodimers at 37 °C in the stability studies
carried out. These peak ratios and the rate at which they rearrange
most likely reflect the relative affinities of the different
neurotrophin protomers for each other. It is difficult to compare
rearrangement rates in homodimers due to difficulties in tagging the
individual protomer subunits of the dimer accurately. Stability studies
were carried out on human NGF(120):NGF(118) and show very similar rates
of breakdown to NGF:9NGF (data not shown). NGF(118) homodimer has
the two most proximal COOH-terminal residues removed (Arg-119, Ala-120)
but displays full bioactivity and binding when compared to full-length
NGF (Burton et al., 1992; Schmelzer et al., 1992).
This suggests that while individual neurotrophin protomers have a very
high affinity for each other (Bothwell and Shooter, 1977), they undergo
rearrangement even in their intact homodimeric state. This is important
since it suggests that heterodimers could be formed in any situation
where two or more neurotrophin homodimers exist together. Furthermore,
Jungbluth et al.(1994) showed that co-expression of
neurotrophins (NT-3 and BDNF) in a single cell line can result in the
formation of a mixture of homo- and heterodimers in the culture medium.
Figure 2:
Examples of competition binding isotherms
of NGF:NT4/5 and NGF:9NGF to A875, PC12, and TrkA- or TrkB
expressing NIH 3T3 cells. A, B, C, and E represent A875 (p75 only), PC12 (p75 and TrkA), and TrkA (only)
expressing cell binding of
I-NGF and competition with
unlabeled NGF, NGF:NT4/5, NT4/5, NGF:
9NGF, and homodimeric
9
NGF. D represents TrkB-expressing cell binding of
I-labeled NT4/5 and competition with unlabeled NGF,
NGF:NT4/5, and NT4/5.
In PC12 cells, a rat pheochromocytoma cell
line, which expresses p75 and a smaller number of TrkA receptors,
NGF:9NGF and NGF:NT4/5, were able to displace all specifically
bound
I-NGF, as efficiently as NGF, while homodimeric
NT4/5 displaced a maximum of 80% (probably corresponding to the p75
component) (for example, see Fig. 2B). These results
suggested that both NGF:
9NGF and NGF:NT4/5 were able to displace
not only the p75 component of PC12 cell binding but also the TrkA
specific component. Radioligand-receptor binding was then studied on
NIH 3T3 cells selectively expressing rat TrkA (Kaplan et al.,
1991a, 1991b) or rat TrkB (Soppet et al., 1991) using matched
dilution curves of the homo- and heterodimeric neurotrophins. Both
NGF:NT4/5 (for example, see Fig. 2C) and NGF:
9NGF
(for example, see Fig. 2E) were able to displace
I-NGF from TrkA-expressing NIH 3T3 cells showing an
average 2-fold increase in IC
compared to homodimeric NGF (Table 1), whereas NT4/5 was only able to displace
I-NGF from the TrkA receptor if taken to micromolar
concentrations (for example, see Fig. 2C). NGF:NT4/5
displayed a 20-fold increase in IC
compared to NT4/5 for
displacement of
I-labeled NT4/5 from the rat TrkB
receptor, while no displacement was detectable with NGF (for example,
see Fig. 2D). These results indicate that binding
interactions of NGF:
9NGF and NGF:NT4/5 with the rat TrkA receptor
are very similar to homodimeric NGF. Although NGF:NT4/5 binds rat TrkB
20-fold less efficiently than NT4/5, the binding is clearly significant
compared to homodimeric NGF with the TrkB receptor. Importantly,
preliminary results analyzing the displacement binding properties of
NGF:NT4/5 from cells expressing the human TrkA or human TrkB receptors
indicate only 2-3-fold increases in IC
when compared
to cognate homodimeric neurotrophins. These observations suggest that
the differences seen with NGF:NT4/5 on the rat TrkB-expressing cell
line could be due to species variations in the receptor (data not
shown). Small variations seen in IC
values may be due to
slight variations in conformation of the receptor binding domain of the
heterodimer or perhaps differential involvement of amino termini in
binding to their cognate Trk receptor.
To further investigate the
promiscuity of the amino termini between neurotrophins with regard to
their potential involvement with binding domains on the other protomer
of the dimer, a small quantity of heterodimeric 9NGF:NT4/5 was
generated, and some preliminary binding studies were carried out.
9NGF:NT4/5 displaces
I-labeled NT4/5 bound to human
TrkB similar to homodimeric NT4/5 and
I-NGF from human
TrkA similar to
9NGF (300-fold less than homodimeric NGF). The
loss of an amino terminus from NGF does not appear to alter the
interaction of NT4/5 with TrkB, and the presence of an amino terminus
of NT4/5 in the dimeric structure of
9NGF:NT4/5 does not
facilitate
9NGF binding to the TrkA receptor, consistent with the
data generated with NGF:NT4/5.
Figure 3:
Characterization of biological activity of
heterodimeric neurotrophins. A and B are Western
blots that show the dose dependence of Trk receptor phosphorylation to
four concentrations of neurotrophins. A shows the response to
1) NGF, 2) NGF:NT4/5, and 3) NT4/5 of TrkA- and TrkB-expressing 3T3
cells and ``primed'' PC-12 cells, respectively. B shows the response to 1) NGF, 2) NGF:9NGF, and 3)
9NGF
of TrkA-expressing 3T3 cells. C shows the dose-dependent
phosphorylation of Trk receptors in rat cortical cultures after
stimulation with either NGF:NT4/5 or NT4/5. D shows the
time-dependent response of primed PC12 cells to NGF exposure using a
wash-out paradigm and counting at 24 h after initial stimulation. E
shows the dose-dependent response in neurite outgrowth of primed PC12
cells to NGF, NGF:NT4/5, NGF:
9NGF, or
9 NGF but not to
NT4/5.
Proteolytic cleavage of the amino termini from other neurotrophin
family members leads to equivalent increases in IC of
neurotrophin displacement from the cognate Trk receptor, as seen with
NGF and
9NGF interactions with the TrkA receptor, suggesting a
general requirement for this structural feature (data not shown). At
present, it is unclear whether both amino termini of the neurotrophins
act solely as a site of Trk interaction or whether they might also
contribute to the stability of neurotrophin dimeric structure.
Amino-terminal-deleted neurotrophins remain competent in terms of p75
binding, which probably indicates little effect on overall structure.
In this report, we demonstrate the capability of neurotrophin family members to form heterodimers that appear to be relatively stable. These molecules are asymmetric neurotrophin dimers, in contrast to symmetric dimers of neurotrophin produced by mutagenesis, and as such can be used as unique structural probes for ligand-receptor binding interactions and activation. Surprisingly, their demonstrated specificity for receptor interactions and biological activity appears to be largely predictable from the homodimer equivalents of their subunit parts. Furthermore, analysis of the amino terminus of NGF suggests that only one intact amino terminus is necessary for NGF to elicit significant actions through the TrkA receptor. These findings suggest that the binding surface(s) sufficient for NGF receptor-ligand interactions can be largely carried on each protomer of the dimeric molecule.
A structural model of NGF-Trk receptor interactions has
been proposed based upon extensive mutagenesis of variable sequence
regions identified within the neurotrophin family (Ibanez et
al., 1991, 1992, 1993; Suter et al., 1992; Shih et
al., 1994). In our present work and in previous studies (Moore and
Shooter, 1975; Burton et al., 1992; Kahle et al.,
1992; Luo and Neet, 1992; Shih et al., 1994), we have observed
that the amino-terminal region of NGF is critical for high affinity
binding and biological activity. In agreement with this, Ibanez et
al.(1993) suggest that the amino terminus may be the most
significant contributor to the binding energy of NGF with TrkA. Other
sites of receptor interaction, i.e. between variable regions
I, II, IV, and V, although helping to stabilize the ligand-receptor
complex, appear to be secondary. In contrast to the proposed nature of
the amino terminus-receptor interaction, the other variable regions,
particularly region II, may be involved in receptor activation more
than receptor binding (Ibanez et al., 1993). The proposed
models of NGF-Trk interaction suggest that multiple specific contacts
within the variable regions of NGF, described above, form a continuous
binding surface on both sides of the molecule (Ibanez et al.,
1993). The bivalent nature of this proposed binding surface is
consistent with the 2-fold symmetry of NGF suggested to be responsible
for Trk dimerization and signal transduction (Fig. 4B, model1) (Jing et al., 1992). Structural
elements from both protomers have been implied to contribute to the
binding surface. For example, part of the Trk binding/activation
surface of NGF has been suggested to be a patch of residues that
contains variable regions I, IV, and V from one protomer and region II
and possibly the amino terminus from the other protomer. NGF:9NGF
has similar biological activity to homodimeric NGF, suggesting that
this proposed domain on NGF involving the amino termini and variable
regions does not have to be bilateral for receptor binding, signal
transduction, and thus receptor dimerization to occur. It is important
to note that these variable regions and the amino-terminal sequences
are very different between NGF and NT4/5, yet NGF:NT4/5 has been shown
to be nearly equipotent to homodimeric NGF in its interactions with and
through TrkA. This is consistent with the suggestion that the amino
terminus from one protomer is not involved in a binding domain on the
other protomer. The significant variability in the primary sequence of
the amino termini and regions I-V between neurotrophins brings into
question the cooperation of variable domains across protomers in the
formation of an extended binding/activation domain in NGF:NT4/5 and by
analogy, therefore, in the NGF dimer structure. Matched dilution curves
of NGF:NT4/5 displayed significant TrkB receptor binding and
autophosphorylation activity compared to NGF but a 10-20-fold
loss in comparison to NT4/5. This could reflect differences either in
the binding domains of NT4/5 and TrkB compared to NGF and TrkA or
simply a species-related difference between rat and human TrkB
receptors. Competition binding indicates that
I-labeled
NT4/5 displacement is only shifted 2-3-fold with NGF:NT4/5
compared to NT4/5 when analyzed on cells expressing the human TrkB
receptor (data not shown). Another heterodimer, that of
9NGF:NT4/5, was able to displace
I-labeled NT4/5
from the human TrkB receptor similar to homodimeric NT4/5 but unable to
displace NGF from the human TrkA receptor effectively. This is
consistent with the data from NGF:NT4/5 and further indicates that the
amino termini are not promiscuous in their interactions between
protomers to form binding domains. The amino termini do appear to be a
very mobile domain in solution as the lack of electron density
indicates in the murine NGF crystallographic structure (McDonald et
al., 1991; Holland et al., 1994). However, the exact
role(s) of the amino termini in NGF dimer stability and in binding
domains remains unclear.
Figure 4:
Representation of the tertiary structure
of murine NGF and models of possible neurotrophin-receptor
interactions. A, this sketch represents a single protomer of
the NGF dimer (McDonald et al., 1991). The dimer is formed by
non-polar interactions of residues situated on three anti-parallel
pairs of -strands (B-D). These residues are highly
conserved between all known neurotrophins, and any substitutions tend
to be the same in terms either of size or of nature. The four variable
domains are marked I-IV. The amino and carboxyl termini are
marked as well as the regional nomenclature described by Ibanez et
al.(1993). B, the present results suggest the possibility
of alternative models of ligand-induced receptor dimerization of the
neurotrophin family with the high affinity Trk receptor family. Models1 and 2 indicate a cross-bridging
dimerization of the Trk receptors, whereas models3 and 4 indicate an interaction with a single receptor
leading to dimerization with and trans-phosphorylation of the other
receptor.
The current data suggest an alternative mechanism for NGF-Trk receptor signal transduction in which the NGF binding/activation domains are contained primarily upon a single protomer (Fig. 4B, model2). Co-crystallization of the soluble human growth hormone receptor-ligand complex reveals that a single protomeric hormone can dimerize two identical receptors through interactions with two distinct binding domains (Cunningham et al., 1991). Our preliminary results with other heterodimeric neurotrophin combinations indicate that they display dual activities consistent with their subunit parts and this model. The potential involvement of conserved residues upon binding interactions with Trk receptors is under further study. It will be interesting to produce heterodimers containing mutated protomers to analyze whether dimerization of Trk receptors is induced by cross-bridging with the neurotrophin (Fig. 4B, model1 and 2) or perhaps by other receptor interactions which in turn induce receptor dimerization and trans-phosphorylation (Fig. 4B, models3 and 4).
In addition to providing an approach to investigating Trk receptor signal transduction, the relative stability and biological activity of heterodimeric neurotrophins suggests that they could represent novel pharmacological agents in vivo. For example, in models of peripheral neuropathy, certain neuronal populations, such as dorsal rat ganglia sensory neurons (McMahon et al., 1994), express multiple Trk receptors and may be responsive to pluripotent heterodimeric neurotrophins. Interestingly, multiple neurotrophins can be co-expressed in some neurons (Philips et al., 1990) and glioma cell lines (Hamel et al., 1993). Jungbluth et al.(1994), using the co-expression of NT-3 and BDNF in a rabbit kidney cell line (RK13) by vaccinia virus, have shown that heterodimers can be purified from serum-free conditioned medium and show some biological activity. Our stability data, generated in vitro under physiological conditions, indicate that heterodimers rearrange to an equilibrium containing significant amounts of heterodimer (40-50%), which suggests the possibility that neurotrophin heterodimers may exist in vivo. Whether or not neurotrophin heterodimers exist in a neuronal context remains an intriguing question.