(Received for publication, October 17, 1994; and in revised form, December 7, 1994)
From the
Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant
similarities and differences of their overall structures and functions.
Biophysical analyses confirmed that all NDF isoforms are monomeric, but
have an extended ellipsoidal shape in solution. All full-length NDFs
are similar in secondary and tertiary structures and they contain no
-helix but are abundant in
-strand structures. A small NDF
fragment containing only the epidermal growth factor domain is also
rich in
-strand structures, but exhibits tertiary structure
different from the long NDF forms. Monoclonal antibodies that
selectively recognize epidermal growth factor domains of human
NDF-
or NDF-
can specifically bind the respective NDF-
and -
isoforms independent of NDF origins. Western blot analysis
and quantitative binding assays further identify that an NDF
preparation produced naturally from Rat1-EJ cells contains both
and
isoforms in a 3 to 2 ratio. In receptor-binding competition
experiments, human and rat NDF-
isoforms have higher affinity than
NDF-
isoforms. NDF-
isoforms can dramatically enhance the
stimulation of DNA synthesis for transfected NIH3T3 cells that
overexpress HER-3 and HER-4 receptors, while NDF-
isoforms can
only stimulate proliferation of HER-4-transfected cells with lower
activity. Taken together, NDF-
and -
isoforms share similar
gross protein conformations but are biologically distinct.
Neu differentiation factors (NDF) ()or
heregulins, which stimulate autophosphorylation of mammary carcinoma
cell lines expressing EGF receptor-like receptors, are soluble,
secreted proteins that are processed from membrane-associated
precursors, pro-NDFs(1, 2, 3, 4) .
Pro-NDFs exist in multiple isoforms which exhibit significant sequence
differences in the third disulfide loop of the EGF domain, the
juxtamembrane region, and the C-terminal cytoplasmic tail(5) .
These isoforms together with two other distinct molecules, glial cell
growth factor (6) and acetylcholine receptor inducing activity (7) are now known to belong to a related gene family, termed as
neuregulin(5) . The molecular diversity of neuregulin has
raised interesting questions on aspects of structural and functional
multiplicity of various isoforms. It is still unknown whether each
molecular species within the family can display distinct biological
actions in its membrane-bound or secreted form. Little is known about
the contribution of the N-terminal Ig unit to the biological function
of NDFs. There has also been no detailed analysis on structural
characteristics of glycosylated NDF. Recent studies have shown that NDF
or heregulins induce tyrosine phosphorylation of HER-4 (8) and
that NDF isoforms bind to both HER-3 and HER-4 receptors, (9) suggesting that instead of HER-2, HER-3 and HER-4 may
function as direct physiological receptors for neuregulin.
In the
preceding paper(10) , we expressed various human and rat
pro-NDF isoforms in CHO cells and isolated different secreted isoforms
to apparent purity. Purified recombinant NDF- and -
isoforms
can stimulate phosphorylation of mammary carcinoma cell lines and exist
as highly glycosylated forms of similar molecular size. The soluble
factors are apparently derived from specific proteolytic processing of
pro-NDFs at both N and C termini, which leads to the secretion of only
two types of isoforms, i.e.
and
(10) . In
this report, we investigate further the molecular, immunological, and
biological properties of these isolated isoforms and compare some of
the properties to the Escherichia coli-derived,
nonglycosylated molecules. All full-length NDF molecules, either
glycosylated or nonglycosylated, and
or
isoforms, displayed
similar structural folding and behaved as though they have a very
extended, elongated shape. However,
and
isoforms are
immunologically and biologically distinct.
Sedimentation velocity experiments were performed at 20 °C and 60,000 rpm and the sedimentation coefficient determined as described previously(16) . Conversion to standard conditions, calculation of Stoke's radii hydration, and axial ratios followed procedures described by Laue et al.(14) , using the V and molecular weight determined by sedimentation equilibrium, and assuming that the hydration of the carbohydrate is 0.43 g/g, as calculated for the polypeptide.
NDF was prepared for infrared spectroscopy by
dialyzing against pure water, lyophilizing, and dissolving the
lyophilized powder in a 20 mM sodium phosphate, 100 mM NaCl buffer prepared in DO (Sigma, 99.9% isotopic
purity), with pD = 7.0. Protein concentrations were
approximately 1.5-2.0% (w/v) for the E. coli-derived and
CHO cell-derived proteins, respectively. Solutions were placed in
infrared cells with CaF
windows and 50-µg Teflon
spacers. All spectra were collected using a Mattson Research Series
FTIR spectrometer using a liquid nitrogen-cooled MCT detector. Four
thousand scans (15 min collection time) were co-added for each
spectrum; resolution was set at 4 cm
. Analysis of
infrared spectra was done as described
previously(17, 18, 19) .
Immunodetection of NDFs in Western blot analysis was done with mAb at a concentration of 5 µg/ml followed by a 1:1000 dilution of peroxidase-labeled goat anti-mouse IgG. The blot was then developed and visualized according to ECL instruction (Amersham, United Kingdom). The real-time biospecific interaction analysis (BIA) was performed using a BlAcore instrument (Pharmacia Biosensor AB, Uppsala, Sweden) as described(20) . Biospecific affinity assays using BIAcore (Pharmacia) were employed to quantitate NDF that binds to specific antibodies as described(21) .
HER-3 or HER-4 transfected cells were plated in 6-well
plates with a cell density of 10 cells/plate. After 24 h,
cells were starved in serum-free medium for 6 h and then changed to
medium containing the indicated concentrations of human or rat NDF
isoforms. After 12 h incubation, the medium was pulse-labeled by
[
H]thymidine (2 µCi/ml) for 2 h. The medium
was then removed and the residual radioactivity washed by
phosphate-buffered saline. Cells on the plate were harvested by cell
scraper and counted for radioactivity.
Recombinant CHO-derived human NDF-1 and rat NDF-
2 were
further analyzed by sedimentation equilibrium. The molecular weight
determined by this technique is 39,100 ± 1000 (38.5 ±
0.5% carbohydrate) for rat NDF-
2 and 37,200 ± 900 (35.6
± 0.5% carbohydrate) for the human NDF-
1, with most of the
uncertainty arising from that in the partial specific volume of the
carbohydrate. Both data obtained from light scattering and
sedimentation equilibrium experiments appear to indicate that the NDF
molecule is monomeric in solution.
With the true molecular weight of
NDF known from light scattering and sedimentation equilibrium, we
decided to confirm its molecular shape using sedimentation velocity.
For CHO cell-derived human NDF-1, the measured s
of 2.65 S implies a Stokes radius
of 3.81 nm, which is 74% larger than expected for a spherical molecule
of this molecular weight, and which would be consistent with a hydrated
prolate ellipsoid with an axial ratio of
8. The 1.90 S
sedimentation coefficient of recombinant human NDF-
2 produced in E. coli implies that it is also elongated, with an axial ratio
similar to that of CHO cell-derived NDF.
Figure 1:
Near UV and far UV
CD analysis (A and B, respectively) of CHO
cell-derived NDF isoforms. Spectra a-d, rat NDF-2,
human NDF-
1, rat NDF-
2, and human NDF-
1,
respectively.
The far UV CD analysis of
both CHO cell-derived human and rat and
isoforms is
illustrated in Fig. 1B. Both NDF-
and -
isoforms displayed a negative CD spectrum at 195 nm. All NDF species
also appear to have a small positive peak in the 220-230-nm
region. This structural characteristic appears in other proteins as
well and is believed to arise from ring stacking of aromatic amino
acids, probably Tyr, in
-sheet containing
proteins(22, 23) . The rest of the far UV CD spectra
for all NDF isoforms are consistent with a protein containing
-sheets and unordered structures.
CD spectra of E.
coli-derived NDF isoforms and NDF-EGF domains were also measured
and shown in Fig. 2, A and B. In the near UV
CD region, these nonglycosylated isoforms also display structural
features similar to glycosylated NDFs produced in CHO cells (Fig. 1A). The NDF- species (Fig. 2A, spectra c and d) also have
stronger and better defined spectra at 270-280 nm, which again
may be due to the two extra tyrosines at the C termini of both human
NDF-
1 and -
2 isoforms. There is an obvious difference between
the spectra of the NDF species derived from E. coli and the
spectra of the NDF's derived from CHO cells in the region near
245-240 nm. Whether human or rat NDF,
or
isoform, all
CHO-derived NDF proteins have spectra which are characterized by an
increase in ellipticity in this region, while the E.
coli-derived molecules all have spectra which continue to be
negative.
Figure 2:
Near UV and far UV CD analysis (A and B, respectively) of E. coli-derived NDF
isoforms and NDF EGF domains. Spectra a-d, human NDF-2,
rat NDF-
2, human NDF-
1, and rat NDF-
4, respectively. Spectrum e, human NDF-
1 EGF
domain.
The far UV CD spectra of E. coli-derived human
NDF- and -
isoforms and
-NDF EGF domain are shown in Fig. 2B. A strong negative CD band near 193-196
nm found in CHO cell-derived NDFs (Fig. 1B) is also
common in E. coli-derived NDF isoforms (Fig. 2B,
spectra a-d). However, there is an obvious spectral
difference around 220-235 nm. The CHO proteins all display broad
but distinct CD spectra in this region, which are missing from the E. coli-derived proteins. Nonetheless, the far UV CD spectra
of NDF isoforms as described above appear to indicate that these
molecules are not typical
helical proteins. The spectra of rat
NDF-
2 in 1 M NaCl, 10 mM phosphate buffer (pH
7.1) are identical to those obtained in phosphate-buffered saline,
indicating that salt does not effect the conformation in solution.
The main feature of the spectra of human NDF-1 EGF domain
consists of a trough from 320 to 250 nm (Fig. 2A, spectrum
e), possibly arising from major contributions of the three
disulfide bridges, with some fine structure from Tyr and Phe
superimposed on it. This structural characteristic is thus very
different from the spectra of the longer isoforms of glycosylated or
nonglycosylated NDFs as studied above. Human NDF-
1 EGF domain also
displays significantly different far UV CD spectrum than the
full-length NDF's. There is a positive and broad 220-235-nm
CD band and a negative CD band around 197 nm (Fig. 2B,
spectrum e). Human NDF-
EGF domain also displays CD spectra
similar to those of the type-
EGF domain (data not shown).
The
conformational stability of several NDF species was studied using
thermal stability as a probe. This was determined by following changes
in the far UV CD region upon heating as indicators for loss of
secondary structure. As listed in Table 2, NDF- EGF domain,
like EGF itself, does not show a single cooperative transition upon
melting and therefore its T
(the transition
midpoint) could not be determined. The T
of
various full-length NDF species were determined from changes in the
ellipticity at 208 nm. E. coli-derived NDF isoforms have a T
between 44 and 50 °C. There appears to be a
slight increase in T
(=52 and 53 °C)
for CHO-derived NDF isoforms. The thermal denaturation of NDFs is
irreversible, so a thorough thermodynamic analysis was not possible.
Most of the
-sheet was recovered after cooling, but the 230-nm
feature was never fully regained. However, all of the full-length NDF
forms melted with a single cooperative transition which ranged over 10
°C.
The second derivative infrared spectra in the amide I region
(1700-1620 cm) of E. coli-derived NDF
and CHO cell-derived NDF isoforms are shown in Fig. 3. These
spectra are related to the polypeptide backbone conformation (24, 25) and are essentially identical for both
and
isoforms, as well as for both nonglycosylated and
glycosylated isoforms (spectra a-d). Human NDF-
2 EGF
domain also displays a spectrum similar to the full-length NDF
molecules (spectrum e). These spectra contain a strong band at
1631-1634 cm
and a clear 1673-1678
cm
band. Curve fitting of the deconvoluted infrared
spectra was used to provide quantitative estimates of secondary
structures, and the resulting band assignments for various isoforms are
listed in Table 3. Band assignments are based on previous
literature reports(24, 25, 26) . Thus, by
infrared studies, it is estimated that the conformations of the
recombinant NDFs are essentially identical and consist of no
-helix, 42-52%
-sheet, a moderate amount of reverse
turns, and the remaining disordered structures.
Figure 3:
FTIR
analysis of NDF isoforms. Spectra a-e, E.
coli-derived human NDF-2, CHO cell-derived rat NDF-
2, E. coli-derived human NDF-
1, CHO cell-derived human
NDF-
1, and E. coli-derived human NDF-
2 EGF
domain.
Figure 4:
Western blot analysis of NDF isoforms by
specific monoclonal antibodies against E. coli-derived human
NDF-2 (A) and human NDF-
1 EGF domain (B).
Unless mentioned, each sample containing 50 ng of NDF was loaded onto
the gels. A, lanes 1-4: E. coli-derived human
NDF-
2, human NDF-
2 EGF domain, human NDF-
1, and human
NDF-
1 EGF domain, respectively. Lanes 5-6 and 8-9: CHO cell-derived human NDF-
1, rat NDF-
2,
human NDF-
1, and rat NDF-
4, respectively. Lane 7:
Rat1-EJ cell-derived NDF. B, Lane 1: low molecular weight
standards; lanes 2-3: E. coli derived human
NDF-
1 and NDF-
2, respectively; lanes 4-7: CHO
cell-derived human NDF-
1, human NDF-
2, human NDF-
1, and
rat NDF-
4, respectively; and lanes 8-10: Rat1-EJ
cell-derived NDF at 50, 100, and 200 ng loading,
respectively.
Western blot analysis of Rat 1-EJ
cell-derived NDF with the monoclonal antibodies was also studied. Both
1H7A3 and 10-125A antibodies can recognize this natural rat NDF
preparation; however, stronger binding was found with mAb 1H7A3 than
with 10-125A at the same sample loading (Fig. 4A, lane
7, and B, lane 8). Clone 10-125A can also clearly
recognize Rat1-EJ cell-derived NDF at higher NDF sample loading (Fig. 5B, lanes 9 and 10). Based upon this
qualitative comparison of the intensity of blotted bands, we conclude
that natural rat NDF contains both and
isoforms in an
approximately 3:2 ratio. When the presence of
and
isoforms
was quantified by the bioaffinity technique (21) using these
monoclonal antibodies, a 38-43%
isoform was estimated in
the natural rat NDF preparation with sample loading at concentrations
of 100-250 ng/ml.
Figure 5:
Binding of NDF isoforms to T47D human
mammary carcinoma cells. The ability of various NDF proteins to
displace radiolabeled NDF-1
was analyzed
on monolayers of T47D human breast cancer cells. Binding reactions were
carried out with 5 ng of
I-NDF-
1
per ml for 2 h at 4 °C. This was followed by extensive
washing of the cell monolayers and determination of bound
radioactivity. The amount of bound NDF-
1
is expressed relative to ligand binding in the absence of
competitor unlabeled protein. A, the following unlabeled
proteins were used to displace cell-bound human
NDF
1
; bacterially made human
NDF-
1
(
), and two rat NDF isoforms,
2 (
) and
4 (
), expressed in CHO cells. B, the following human NDF isoforms expressed in CHO cells
were used as unlabeled competitors to bacterially made human
NDF-
1
(
):
1 (
),
2
(
),
1 (
), and
2 (
). Averages of duplicate
determinations and the corresponding range (bars) are shown.
Each experiment was repeated three times.
To quantitatively compare receptor binding
characteristics of different NDF isoforms, the purified NDF
preparations were analyzed for their binding to T47D human mammary
carcinoma cells. Ligand displacement analysis was performed with
radiolabeled human NDF-1
. The latter
differs from the corresponding rat protein in only 1 amino acid. This
EGF-like domain displayed an apparent dissociation constant of
200-400 pM (Fig. 5A). Comparative ligand
displacement analysis confirmed that CHO cell-derived rat NDF-
2,
as well as human NDF-
1 and -
2, have lower binding affinities
by a factor of 8-10 (Fig. 5, A and B).
Recombinant CHO rat NDF-
4 displayed ligand displacement activity
that is three times higher than rat NDF-
2 (Fig. 5A), but is less active than human NDF-
1 or
-
2 (Fig. 5B). Human NDF-
1 or -
2 can best
compete with labeled human NDF-
1
EGF
domain in ligand displacement analysis.
We also performed tritiated
thymidine uptake experiments to examine NDF-dependent stimulation of
DNA synthesis in NIH3T3 cells transfected with plasmids carrying human
HER-3 or HER-4 receptor genes. Expression of HER-3 or HER-4 receptor
proteins was examined by immunoprecipitation using specific antibodies
against either receptor molecule (data not shown). As shown in Fig. 6A, CHO cell-derived NDF- isoforms (curve 3:
rat NDF-
4; curve 4, human NDF-
1) can induce more than a
16-fold stimulation of DNA synthesis in cells transfected with HER-3
receptor in a dose-dependent manner. Rat NDF prepared from medium
conditioned by Rat1-EJ cells also exhibited a similar stimulation
profile (Fig. 6A). The 50% effective concentration for
stimulation of DNA synthesis by these samples was approximately
3-4 ng/ml or 70-100 pM. In contrast, the CHO
cell-derived NDF-
isoforms appeared to have little stimulatory
effect on cells expressing HER-3 when compared to the stimulation on
cells without transfection of receptor.
Figure 6:
Effect of NDF on stimulation of thymidine
uptake in HER-3 or HER-4 transfected NIH3T3. A, HER-3
transfected cells: CHO cell-derived human NDF-1 (
), rat
NDF-
2 (
), rat NDF-
4 (
), human NDF-
1
(
), and rat1-EJ cell-derived NDF (
). B, HER-4
transfected cells: CHO cell-derived human NDF-
1 (
), rat
NDF-
2 (
), human NDF-
1 (
), rat NDF-
4
(
), rat1-EJ cell-derived NDF (
), and control with no addition
of NDF (
). C, effect of E. coli-derived NDFs
at 2 µg/ml concentration.
The stimulatory effect on
cells transfected with HER-4 receptor is also shown in Fig. 6B. Both CHO cell-derived NDF- and -
isoforms exhibit very strong stimulation of thymidine uptake by
transfected NIH3T3 cells, ranging from approximately 8-16-fold
stimulation (Fig. 6B), as compared to the control with
no addition of NDF. The concentration required for half-maximal
stimulation is approximately 15-100 pM for different
isoform preparations. NDF-
isoforms exhibited weaker stimulation
effect than the
isoforms. Rat1-EJ cell-derived NDF also displays
strong stimulation.
Similar experiments were also performed by using E. coli-derived NDF isoforms including full-length human and
rat NDFs, as well as NDF EGF domains. Consistent with CHO cell-derived
isoforms, all human and rat NDF-
isoforms and EGF domain
isoform exhibited strong stimulation on cells transfected with HER-3 or
HER-4, while
isoforms only display weaker stimulation on HER-4
transfected cells (Fig. 6C).
By gel filtration analysis, the determined molecular weight of NDFs appears to be greater than 100,000 in all CHO cell-derived NDF isoforms and is also greater than 50,000 in E. coli-derived NDF isoforms. However, the molecular weights of NDFs determined by gel filtration in conjunction with light scattering are consistent with a monomeric NDF molecule predicted from their primary sequences (Table 1). These data suggest that NDFs are monomeric, but have an atypical shape in solution.
The molecular properties of NDFs were
further confirmed by sedimentation velocity experiments in which the
measured s is consistent with a
hydrated prolate ellipsoid with an axial ratio
of 8, i.e. a rod-like shape. Most of this asymmetry appears to be due to the
polypeptide rather than the carbohydrate because a similar axial ratio
is found for nonglycosylated NDF molecules derived from E. coli. Other experiments by sedimentation equilibrium indicated that
glycosylated NDF isoforms have a carbohydrate content of 35-39%.
The unique rod-shaped structure deduced from physical analysis is
consistent with the proposed NDF domain structures (4, 10) where the Ig-like homology unit and the
glycosylated spacer region may be very extended and separated away from
EGF domain.
Since multiple pro-NDF transcripts do exist in Rat1-EJ
cells, it is reasonable to predict that rat1-EJ cells may express
different isoforms (14) . However, we were unable to obtain
enough quantities of Rat1-EJ cell-derived NDF for comparative
C-terminal determination and for verification of isoforms. In this
report, Western blot analyses using specific monoclonal antibodies
against human NDF- and -
isoforms confirm that the natural
rat NDF preparation contains a mixture of both isoforms at a ratio of 3
to 2. When the amino acid composition of rat 1-EJ NDF was used to fit
the amino acid composition data for the mixture (see the preceding
paper, (10) ), there is sufficient agreement in the data to
suggest that Rat1-EJ NDF may share identical C-terminal processing
sites with CHO cell-derived rat NDF-
2 and -
4. Other evidence
to support a similar processing is that the Rat1-EJ NDF shows a similar
40-44-kDa molecular size upon SDS-polyacrylamide gel
electrophoresis.
Glycosylated and nonglycosylated NDF- and
-
isoforms share similar secondary structures which are rich in
-sheet structure with no detectable helical structure. We believe
that the
-sheet structures may spread over the whole molecule
including the Ig domain, spacer region, and EGF domain in order to
account for the high proportion of
structure (see Table 3).
The EGF domain alone, either
or
isoform, also contains
approximately 50%
-sheet structure, which is consistent with that
observed for the EGF and TGF-
molecules(27, 28) .
This observation is also consistent with the solution structure of
heregulin-
EGF domain (29) and
NDF-
1
EGF domain (
)elucidated
by two-dimensional NMR analysis.
Secondary structure prediction (30) suggests that Ig and EGF domains in NDF- or -
isoform contain moderate amounts of
-sheet structures. The
observation of
-sheet structures in the Ig domain suggests that
the structure of the Ig domain in NDF is homologous to Ig domains found
in a number of proteins, including CD2 and CD8(31) . The Ig
unit of NDF belongs to the C2 set of immunoglobulin homology units (2) which is shared by the non-immunological members of this
gene superfamily. Included in this family are membrane receptors for
antibodies, receptors for growth factors (e.g. platelet-derived growth factor), and lymphokines (e.g. interleukin-1) and cell adhesion molecules (e.g. neural
cell adhesion molecule)(32) . It is clear that the Ig unit in
NDF does not directly participate in receptor recognition. Instead, its
structural folding implies that its functional role may be similar to
that attributed to other immunoglobulin units, namely, stabilizing
homophilic protein-protein interaction(2) .
Glycosylated
human and rat NDF- and -
isoforms also share similar basic
tertiary structural folding. However, the human and rat NDF-
isoforms contain slightly stronger and more defined near UV CD bands (Fig. 1A). This phenomenon may be attributed to the Tyr
residue that occurs at the C terminus in the NDF-
isoforms that is
absent in the NDF-
isoforms. A similar observation was also found
for the nonglycosylated NDF-
isoforms which contain two extra
tyrosines (Fig. 2A). The tertiary structural folding of
the NDF EGF domain is apparently distinct from the other longer NDF
isoforms (Fig. 2A). The full-length NDF molecule
contains three different domain structures; two of them (Ig and spacer
domains) have been removed from the NDF EGF domain.
Despite their
structural similarity, it is clear that biological functions of
NDF- and -
isoforms are distinct. In binding competition
experiments, glycosylated NDF-
isoform showed much weaker
competition with NDF-
EGF domain than the glycosylated
isoform. The data also suggests that NDF-
isoforms have higher
affinity to the receptor on the cell surface.
The higher affinity of
NDF- isoforms may indicate that the
isoform can better
stimulate DNA synthesis or cell growth. We have used HER-3 and
HER-4-transfected NIH3T3 cells to evaluate the stimulatory effects by
NDF-
and NDF-
isoforms, and EGF domains. NDF-
isoforms,
glycosylated or nonglycosylated or the EGF domain only, did not exert
significant stimulation of DNA synthesis of HER-3-transfected cells but
could stimulate a moderate amount of mitogenic effect on
HER-4-transfected cells. In contrast, all NDF-
isoforms and the
NDF-
EGF domain exhibited strong stimulation of DNA synthesis of
both HER-3 and HER-4 transfected cells. Taken together, these data
suggest that NDF-
and NDF-
seem to have different biological
functions, which must be related to the unique sequence (i.e. structural) difference in the last disulfide loop in the EGF
domain and the C-terminal tail (6 amino acids in length). Earlier
studies have shown that the solution structures of EGF and transforming
growth factor-
analyzed by NMR seems to be homologous and rich in
-sheets,
-turns, and loops(9) . These structures are
shared by other known sequences that bind the EGF receptor.
Significantly, the
and
isoforms of NDF EGF domains have a
main chain structural fold similar to the EGF molecule (see above and (27) ), yet do not bind to the EGF receptor. These data imply
that the receptor binding domain of NDF molecules resides at the
C-terminal region of the EGF domain and is distinct from the EGF
molecule in overall structural folding. NMR analysis has revealed
distinct structural features of heregulin-
(29) and
NDF
(
) on the molecular surface
for possible receptor recognition. Future mutational analysis,
synthesis of chimeric molecules, and further NMR studies should prove
useful in unraveling the molecular basis of NDF receptor binding
specificity.