(Received for publication, March 27, 1995; and in revised form, June 26, 1995)
From the
The interaction of neu differentiation factor (NDF) with the
extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been
studied using native gels, light scattering, and sedimentation
equilibrium. The full-length NDF2 was shown to bind sHer3 with a
dissociation constant of 26 ± 9 nM, while it showed a
1000-fold weaker binding to sHer2. Taken together, these results
demonstrate that NDF is a high affinity ligand for Her3, but not for
Her2. No increase in affinity of the NDF
2 for sHer3 was observed
upon addition of sHer2 to the NDF
2-sHer3 mixture. Binding of
NDF
2 to sHer3 did not induce receptor dimerization or
oligomerization, the stoichiometry being one sHer3 per one NDF
molecule. This finding suggests that transmembrane and/or intracellular
domains of receptor family members or perhaps additional unidentified
components may be involved in NDF induced dimerization and
autophosphorylation, or alternatively, that dimerization is not the
mechanism for Her3 autophosphorylation and signal transduction.
There have been numerous studies showing that high expression of
erbB2/Her2 tyrosine kinase receptor correlates with poor prognosis in
patients with breast cancer (Slamon et al., 1989; Wright et al., 1989; Gullick et al., 1991; Hartmann et
al., 1994). Her2 is a member of the EGF ()receptor
subfamily, which also includes the EGF receptor, and Her3 and Her4
receptors (Kraus et al., 1989; Plowman et al., 1993).
EGF and transforming growth factor-
are known as ligands for the
EGF receptor but they do not stimulate autophosphorylation of Her2,
Her3, and Her4 receptors.
Receptor dimerization has been implicated as a mechanism of receptor phosphorylation and signal transduction for a variety of ligand-receptor pairs. Using a recombinant extracellular domain of EGF receptor, dimerization and oligomerization of the receptor, induced by EGF, have been observed with a dissociation constant of 100-200 nM and a stoichiometry of one receptor per one EGF molecule (Hurwitz et al., 1991; Brown et al., 1994).
Recently, neu differentiation factor (NDF) or heregulin and other structurally related ligands have been shown to increase tyrosine phosphorylation of the Her2 receptor and, therefore, was initially assumed as a Her2 ligand (Peles et al., 1992; Wen et al., 1992; Holmes et al., 1992; Marchionni et al., 1993; Falls et al., 1993). However, there is evidence that NDF neither binds directly to Her2 nor stimulates its kinase activity (Tzahar et al., 1994), but rather binds to and stimulates tyrosine phosphorylation of Her3 and Her4 (Plowman et al., 1993; Kita et al., 1994). There is also evidence that NDF's stimulatory activity on Her3 is augmented when Her2 was also coexpressed (Sliwkowski et al., 1994). Here we have studied the interaction of NDF with the extracellular domains of Her2 (sHer2) or Her3 (sHer3) using native gels, light scattering, and sedimentation equilibrium in order to further define the binding strengths and stoichiometries and to explore whether binding specificity is solely a property of the receptor extracellular domains.
Simultaneous data analysis on data from multiple samples and speeds was carried out using nonlinear least squares techniques similar to those in NONLIN (Johnson et al., 1981), using the program KDALTON developed in-house. Confidence intervals for the parameters were evaluated using the ``preferred method'' outlined by Johnson and Faunt(1992).
The sedimentation data on the mixtures were fitted to heterogeneous
association models of the forms A + BAB or A +
2B
AB + B
AB
, where A is NDF or a
sHer3
NDF complex, and B is sHer2 or sHer3, accounting
for the different molar extinction coefficients of each species at the
measurement wavelength, using techniques described previously (Philo et al., 1994). During the fitting, the buoyant molecular
weights of each monomeric species were held fixed at the values
determined when run alone. The data for up to 18 samples were globally
fitted to determine the association constants. The fitted parameters
were constrained to values which correctly account for the total
concentrations each protein placed into the cell.
Buffer densities
were measured with a Mettler-Paar DMA-02 density meter. Polypeptide
partial specific volumes of 0.7258, 0.7220, and 0.7272 ml/g, and molar
extinction coefficients at 280 nm of 6.04 10
, 6.76
10
, and 1.56
10
M
cm
for sHer2,
sHer3, and NDF
2, respectively, were calculated from their amino
acid composition (Laue et al., 1992; Gill and von Hippel,
1989).
Figure 1:
Native gel analysis of
NDF binding to sHer2 and sHer3. A, NDF2 binding to sHer2
and sHer3. Lane 1, 1.0 µg of sHer3; lane 2, 1.0
µg of sHer2; lane 3, 2.5 µg of NDF
2; lane
4, 1.0 µg of sHer3 + 2.5 µg of NDF
2; lane
5, 1.0 µg of sHer2 + 2.5 µg of NDF
2; lane
6, 0.5 µg of sHer3 + 0.5 µg of sHer2 + 2.5
µg of NDF
2; lane 7, 0.625 µg of NDF
2; lane 8, 1.0 µg of sHer3 + 0.625 µg of NDF
2; lane 9, 1.0 µg of sHer2 + 0.625 µg of NDF
2; lane 10, 0.5 µg of sHer3 + 0.5 µg of sHer2 +
0.625 of NDF
2. B, NDF
2 binding to sHer2 and sHer3.
Same as lanes in A using NDF
2 instead of
NDF
2.
Fig. 1A shows the results with
NDF2. In the presence of 6.8- (lane 4) or 1.7-fold (lane 8) molar excess of NDF
2, sHer3 displayed a new band
slightly above the free molecules, indicating formation of a complex.
Under identical conditions, sHer2 gave no complex formation (lanes
5 and 9). In the presence of equal amounts of sHer2 and
sHer3, addition of excess NDF
2 gave the same band seen with sHer3
alone, i.e. no stable ternary complex of
sHer3
NDF
2
sHer2 was observed (lanes 6 and 10). Despite the presence of excess NDF
2 a large portion
of sHer3 remained as a free molecule. Fig. 2B shows the
results with NDF
2 obtained under exactly the same experimental
conditions. Here again, complex formation was observed only when
NDF
2 was mixed with sHer3 and no ternary complex was observed when
sHer2 was also present. Comparing Fig. 1, A and B, it is apparent that a larger portion of sHer3 was
incorporated into complexes with NDF
2 than with NDF
2,
suggesting that the NDF
2 has a higher affinity for sHer3 under the
experimental conditions used.
Figure 2:
Sedimentation equilibrium of sHer3 +
NDF mixtures at 8000 rpm. The natural log of the absorbance at 280 nm
is plotted versus (r - r
)/2, where r is the
radius and r
is the radius at the center
of the sample. In such a plot a single species will give a straight
line whose slope is proportional to molecular weight. The diamonds are for a sample containing 6 µM sHer3 and 6
µM NDF
2; the squares are for 6 µM sHer3 + 3 µM NDF. The dashed line illustrates the slope calculated for a complex containing 2
sHer3/NDF. The solid and dotted lines show the
calculated slope for a 1:1 sHer3
NDF complex. Only data for the
outer half of the cell are shown. Conditions: 25 °C; 10 mM sodium phosphate + 50 mM NaCl, pH
7.9.
Control studies of NDF2 alone were consistent
with a monomer at the sequence M
of 24,694. The
data for sHer3 were also well fitted by a single ideal species, with no
evidence for self-association to dimers or higher oligomers. From the
buoyant molecular weight of 24035 [23976 and 24103] (values
within square brackets indicate 95% confidence intervals), the sequence M
of 68178, and estimates for the carbohydrate
partial specific volume (Philo et al., 1994), it is possible
to calculate that sHer3 contains 17.0% [15.0%, 18.5%]
carbohydrate, for a total mass of 82.2 [80.2, 83.7] kDa.
Fig. 2shows some data for samples containing 6 µM sHer3 and either 3 or 6 µM NDF2. On such a plot,
a single species will give a straight line with a slope proportional to
molecular weight, and for mixtures of species the slope near the outer
portion of the cell will primarily reflect the largest species which
are present (only data for the outer half are shown). The dashed
line on the plot indicates the slope expected for complexes with
two sHer3 bound to one NDF; the solid and dotted lines indicate the slope for one sHer3 per NDF. Clearly both samples
contain primarily the 1:1 complex. Furthermore, there appears to be
little, if any, 2:1 complex in the sample made at that stoichiometry
despite the high protein concentration (which should favor such
complexes even if the binding of a second sHer3 is relatively weak).
For more detailed analysis, sedimentation equilibrium data from 13 experiments were simultaneously analyzed to different binding models. These data covered a range of sHer3 loading concentrations from 400 nM to 6 µM, both 2:1 and 1:1 sHer2:NDF stoichiometries, and 2 rotor speeds. Consistent with the simple analysis presented in Fig. 2, a model in which sHer3 and NDF bind to form 1:1 complexes gives a good fit to all the data, with nearly randomly distributed residuals, as shown in Fig. 3, and returns a dissociation constant of 26 [17, 35] nM.
Figure 3: Sedimentation equilibrium data and fitted curves for sHer3/NDF mixtures. Thirteen data sets for samples loaded at sHer3 concentrations from 400 nM to 6 µM, at both 1:1 and 2:1 sHer3:NDF stoichiometry, and at 8,000 or 12,000 rpm, were simultaneously fitted to a binding model in which a single sHer3 binds per NDF. The fit returns a dissociation constant of 26 nM. For the sake of clarity, the data sets at 12,000 rpm have been shifted upward by 0.6 absorbance unit. Constraints are imposed during fitting to force the computed species distributions to closely match the total number of moles of sHer3 and NDF loaded into the cell. The lower panel shows the residuals from this fit, with different data sets displaced vertically for clarity. The overall root mean square error was 0.0056 absorbance units (AU).
We have also looked for evidence for the binding of a second sHer3
to the same NDF molecule by analyzing data using a binding model in
which NDF has two sites that bind sHer3 independently with different
dissociation constants. This analysis clearly showed that no
significant amount of 2:1 complex (<3%) exists in these
samples, but could not rule out a very weak second binding site with a
dissociation constant of 100-300 µM. To further
explore the possibility of a second very weak interaction, we carried
out further experiments at higher protein concentrations, reaching
sHer3 concentrations >35 µM (>2 mg/ml). The
inclusion of a second binding interaction in these high concentration
data does not significantly improve the fit over the 1:1 stoichiometry
model, and the analysis indicates that any such second binding site, if
it exists at all, has a dissociation constant >1 mM.
Simultaneous analysis of 18 data sets gave a
reasonably good fit to the 1:1 stoichiometry model with a dissociation
constant of 19.6 µM [17.3, 22.0], i.e. about 1000-fold weaker binding than seen for sHer3. This fit does,
however, show some systematic deviation from the experimental data,
especially at the highest concentrations where the apparent M is somewhat below that predicted by the fit.
Consistent with this pattern of deviations, the inclusion of a second
interaction (to allow 2:1 complexes) only makes the fit worse, and fits
to the two-site independent model will not converge because the fitting
routine attempts to make the second dissociation constant become zero.
The deviations from the model are consistent with some solution
non-ideality (which would not be surprising at these protein
concentrations), but might also be due to some heterogeneity related to
glycosylation. We therefore conclude that the 1:1 binding model is
correct.
To interpret the data on the ternary
mixtures, there are three general cases we need to consider. We have
already demonstrated that NDF will bind strongly to sHer3 and weakly to
sHer2, but does not induce dimerization of either receptor. Those
studies cannot tell us, however, whether sHer3 and sHer2 are binding to
the same, or different, sites on NDF. Therefore the first possibility
to consider is one where the sites are the same or largely overlapping,
in which case the low affinity of sHer2 will make it unable to compete
with sHer3, so essentially no interaction with sHer2 will occur. A
second possibility is that sHer2 may bind independently to a different
``face'' of NDF, and then we would expect to see some
formation of sHer3NDF
sHer2 ternary complexes, but the
amount of ternary complex would be strongly limited by the weak
NDF-sHer2 interaction. The last, and most interesting, possibility is a
synergistic binding of sHer2 and sHer3 to the same NDF molecule, giving
much more ternary complex than expected based on the weak binding of
sHer2 alone.
We can again get a good overall view of the results by
looking at Fig. 4, a linearized data plot like that in Fig. 2, for a sample loaded with 3 µM of each
protein. The solid line shows the slope expected for a
sHer3NDF complex, while the dashed line shows that for a
ternary sHer3
NDF
sHer2 complex. Clearly there is no large
amount of ternary complex formed, and the data are consistent with
essentially complete formation of sHer3
NDF complexes, as expected
at this high protein concentration. Therefore we can eliminate the case
of significant synergistic binding of sHer2 and sHer3. On the other
hand, if sHer2 is not bound at all, its lower molecular weight would be
expected to reduce the slope below that for 100% sHer3
NDF, which
is not seen. Therefore the data would be consistent with a very weak
formation of ternary complexes.
Figure 4:
Sedimentation equilibrium of 3 µM sHer2 + 3 µM sHer3 + 3 µM NDF
at 8000 rpm. The data are plotted as in Fig. 2. The solid
line shows the slope calculated for a sHer3NDF complex (a
sHer2
NDF complex would have nearly the same slope); the dashed line shows the slope calculated for a
sHer3
NDF
sHer2 complex.
We have not attempted to directly
fit to an appropriate ternary association model. Such a model would
require consideration of 6 species with different molecular weights,
and at least 3 binding constants, and is therefore exceedingly complex
and unlikely to give unique results. However, to further confirm our
interpretation that the sHer2 is binding only weakly, if at all, we
first tried an analysis of the data for the higher concentration
samples as a mixture of two non-interacting species: sHer2, and a
sHer3NDF complex. During this analysis, the concentrations were
constrained to give the correct 1:1 molar ratio averaged over the cell.
This model gave a moderately good description of the data (not shown),
but seemed to somewhat underestimate the average molecular weight,
particularly at the highest concentrations.
We then tried a model
where sHer2 binds to the sHer3NDF
2 complex with the same low
affinity seen for sHer2 binding to NDF alone. This model clearly
predicts too much ternary complex, and gives a variance 3 times higher
than the non-interacting model. Last, we allowed the affinity of sHer2
for the sHer3
NDF complex to vary during the fitting. This latter
model gives quite a good fit of the data, as shown in Fig. 5,
and returns a dissociation constant of 99 µM [77,
128], i.e. 5-fold weaker binding than when sHer3 is not
already bound to NDF. Thus this analysis confirms that there is no
enhanced binding of sHer2 in the presence of sHer3. Instead, the
binding of sHer2 actually appears to be weaker in the presence of
sHer3, perhaps as a result of some overlap of their binding sites on
NDF, or steric interference between the 2 receptors, or some negative
cooperativity through the common ligand.
Figure 5:
Sedimentation equilibrium data and fitted
curves for equimolar sHer2/sHer3/NDF mixtures. Data are for samples
containing either 3 or 1.5 µM each of sHer2, sHer3, and
NDF, at 8,000 and 12,000 rpm. The curves represent a simultaneous fit
to a model in which sHer2 is associating with a pre-formed
sHer3NDF complex (essentially no dissociation of sHer3 from NDF
is expected at these concentrations; see Fig. 3). The fitted
dissociation constant between sHer2 and the sHer3
NDF complex is
99 µM.
Native gel, light scattering, and sedimentation equilibrium
experiments have all demonstrated binding of NDF2 to sHer3, while
native gels and sedimentation equilibrium detected little binding of
NDF
2 (or NDF
2) to sHer2. Both native gels and size exclusion
chromatography/light scattering showed only partial formation of
sHer3
NDF
2 complexes, while sedimentation equilibrium showed
stoichiometric 1:1 binding at moderately high (26 nM)
affinity. We do not regard these results as contradictory; rather, they
probably indicate that NDFb2 is rather easily dissociated by competing
interactions with the polyacrylamide and dextran matrices.
The 26
nM dissociation constant found here for the interaction of
sHer3 with NDF2 is significantly different from the 1-2
nM values reported for the EGF domain of NDF
1 binding to
Her3 expressed on insect or COS-7 cells (Carraway et al.,
1994; Sliwkowski et al., 1994). It is likely that the
difference in temperature during the binding experiments (0 versus 25 °C) accounts for a large part of the lower affinity in the
present experiments, but there also may be significant differences
between different NDF isoforms, between full-length NDFs and their
EGF-like domains, and/or between the extracellular domain and the
full-length receptor in a solubilized membrane.
The present studies showing extremely weak sHer2-NDF interactions reinforce the proposal that Her2 is not a receptor for NDFs (Peles et al., 1993), and for the first time demonstrate and quantitate an interaction between Her2 and a ligand by physical techniques.
Sliwkowski et
al.(1994) have reported that co-expression of Her2 and Her3 in
COS-7 cells creates a binding site for NDF with a dissociation constant
130 pM, about 10-fold lower than when Her3 is expressed alone.
They also observed chemical cross-linking of Her2 to NDF when Her3 was
also present. These data provide strong support for a model that a
Her2/Her3 heterodimer represents a high affinity binding site for NDF.
In contrast, our studies using purified receptor extracellular domains
show no evidence for enhanced binding by the receptor heterodimer.
Indeed, we actually find weaker binding of sHer2 to NDF2
when sHer3 is already bound. This striking difference in binding
behavior may indicate that enhanced binding by a Her2/Her3 heterodimer
requires the transmembrane and/or intracellular domains. Alternatively,
it could indicate that the enhanced binding requires a third component
which is constitutively expressed in COS-7 cells. It is interesting
that the cross-linked complexes seen by Sliwkowski et
al.(1994) have an apparent molecular mass in excess of 500 kDa,
much higher than expected for a Her2
NDF
Her3 complex. They
also observe complexes of this size when only Her3 is present, whereas
we find no formation of Her3
NDF
Her3 or higher complexes
even at very high protein concentrations. In this regard, it should be
noted that Kita et al.(1994) have shown aggregation of Her3
receptor by cross-linking when Her3-expressing cells were stimulated
with NDF. All this data suggests that a third component (presumably a
protein) might be promoting oligomerization of Her family members on
cell surfaces.
In many cases, soluble receptors have been shown to dimerize upon binding to their cognate ligand, although the number of ligand molecules incorporated into the complex differs for different receptors. It is, therefore, quite significant that NDF does not dimerize the soluble receptor at least under the conditions used here, suggesting the possibility that dimerization is not the mechanism of Her3 autophosphorylation and signal transduction. However, if in fact, receptor dimerization is essential for receptor kinase activation, then these results suggest that components required for dimerization are missing in the experimental conditions used here. Alternatively, one or both of the soluble receptors studied here may be altered in such a way to have lost the capability to dimerize without losing the ability to bind ligand. The methods we have used in this study are not able to distinguish this presumed defect from the participation of other receptor domains or ancillary factors.