Nerve growth factor having altered receptor binding specificities

A method of altering the receptor binding properties and the stability of neurotrophic factors is set forth. Mutant neurotrophic factors having altered receptor binding specificities are described. Specific embodiments include neurotrophic factors that bind trk receptors but do not bind to the low affinity NGF receptor.

INTRODUCTION 
The present invention provides mutant neurotrophic factors of the nerve 
growth factor family which have modified receptor binding affinity and 
biological specificity. It is based, in part, on the development of a 
model system which is useful for the rational design of analogues and 
chimeras of neurotrophic factors. 
BACKGROUND OF THE INVENTION 
The control of cell growth and differentiation requires specific factors 
which exert their effects via interaction with receptors on the surface of 
responsive cells. Despite the increasing number of growth and 
differentiation factors that have been discovered and characterized, the 
precise structures involved in binding and biological activity and the 
sequential and causal molecular events underlying the activation of 
multiple receptors are largely unknown. 
Nerve growth factor (NGF)is a 118 amino acid polypeptide which controls the 
survival, development and differentiation of the sympathetic nervous 
system, as well as parts of the sensory and central nervous systems 
(Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980; Whittemore 
and Seiger, 1987; Thoenen et al., 1987). The biologically active form of 
NGF is a dimer of identical subunits each of which is produced from a 
precursor molecule (Angeletti and Bradshaw, 1971; Angeletti et al., 1973). 
A cDNA clone for NGF was first isolated in the mouse (Scott et al., 1983). 
Subsequently, the NGF gene has been characterized in a number of other 
species including several mammals, birds, reptiles and fishes (Schwarz et 
al., 1989; Hallbook et al., 1991). 
NGF belongs to a family of structurally and functionally related molecules, 
collectively known as neurotrophins of the nerve growth factor family, 
which includes at least three other members, brain-derived neurotrophic 
factor (BDNF) (Barde et al., 1982; Leibrock et al., 1989), neurotrophin-3 
(NT-3) (Hohn et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., 
1990; Ernfors et al., 1990) and neurotrophin-4 (NT-4) (Hallbook et al., 
1991; Ip et al., 1992). 
NGF interacts with a low-affinity receptor expressed on a variety of cell 
types of both neuronal and non-neuronal origin (Ernfors et al., 1988; Yan 
and Johnson, 1988; Heuer et al., 1990; Hallbook et al., 1990). The other 
three neurotrophins of the nerve growth factor family can also bind to the 
low-affinity NGF receptor (Rodriguez-Tebar et al., 1990; Ernfors et al., 
1990; Squinto et al., 1991; Hallbook et al., 1991). This receptor is 
represented by a transmembrane glycoprotein of approximately 75,000 
daltons (p75.sup.NGFR) which binds NGF with a Kd of 10-9M (Johnson et al., 
1986; Radeke et al., 1987). However, high affinity binding (Kd=10-.sup.11 
M), restricted to a subpopulation of p75.sup.NGFR -positive cells, is 
necessary to mediate the biological action of NGF. Banerjee et al., 1973; 
Herrup and Shooter, 1973; Sutter et al., 1979; Richardson et al., 1986). 
While the molecular relationship between the two receptor states is not 
entirely clear, several reports have indicated that the cytoplasmic domain 
of p75.sup.NGFR which lacks structural features known to mediate signal 
transduction in other receptors, is required for high-affinity binding and 
signal transduction (Hempstead et al., 1989; Yan et al., 1991; Berg et 
al., 1991). 
It has recently been demonstrated that the proto-oncogene trk encodes a 
functional receptor for NGF (Kaplan et al., 1991a; Klein et al., 1991). 
The product of the trk proto-oncogene is a 140,000 dalton protein 
(p140.sup.trk) which is a member of the tyrosine kinase family of 
transmembrane receptors (Martin-Zanca et al., 1991). Though it has been 
postulated that this protein participates in the primary signal 
transduction mechanism of NGF, there is considerable disagreement 
regarding the equilibrium binding constant of p140.sup.trk for NGF. 
Whereas Klein et al. (1991) reported that p140.sup.trk binds NGF with both 
low and high affinities, Kaplan et al (1991) and Hempstead et al (1991) 
reported that p140.sup.trk binds NGF with an affinity similar to that of 
p75.sup.NGFR and that coexpression for both receptors is required for high 
affinity binding to occur. Recently, the product of the trk proto-oncogene 
has been shown to constitute a functional receptor for NGF (Kaplan et al., 
1991a; Klein et al., 1991). NGF binding to p140.sup.trk results in rapid 
phosphorylation of this molecule and stimulation of its tyrosine kinase 
activity (Kaplan et al., 1991a; Kaplan et al., 1991b; Klein et al., 1991). 
In contrast, the role of p75.sup.NGFR in signal transduction has remained 
elusive. Recently, it was reported that the cytoplasmic domain of this 
receptor is involved in mediating neuronal differentiation (Yan et al., 
1991) and NGF induced tyrosine phosphorylation (Berg et al., 1991) in PC12 
cells. However, other recent studies have shown that polyclonal antibodies 
against p75.sup.NGFR abolish NGF binding to this molecule and some of the 
high-affinity binding but do not inhibit biological responses to NGF 
(Weskamp and Reichardt, 1991). Recent reports using cell lines expressing 
p140.sup.trk have demonstrated that in the presence of NGF this receptor 
molecule can mediate survival and mitotic proliferation of fibroblasts in 
the absence of p75.sup.NGFR (Cordon-Cardo et al., 1991). These studies 
could not rule out the possibility that binding to p75.sup.NGFR could be 
important in mediating NGF responses in neurons and neuron-like cell 
lines. It has also recently been shown that the trk proto-oncogene can 
rescue NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines 
(Loeb et al., 1991). However, these cells still expressed substantial 
levels of p75.sup.NGFR therefore making it difficult to assess whether the 
presence of this molecule was required for the observed functional 
effects. 
A better understanding of the molecular mechanism by which NGF exerts its 
biological effects is provided by the study of structure-function 
relationships and the creation of NGF mutants with altered properties. 
Initial studies along this line have analyzed the functional importance of 
highly conserved amino acid residues in the chicken NGF (Ibanez et al, 
1990). More recently, an analysis of chimeric molecules between NGF and 
BDNF has delineated regions involved in determining the biological 
specificities of these two factors (Ibanez et al 1991a). Comparison of NGF 
genes from different species has revealed clusters of amino acid residues 
which are highly conserved across different groups of vertebrates (see 
FIG. 1, which demonstrates the conservation of amino acid residues 25 to 
36 (single letter code) in NGFs from different species and in the 
homologous region of different neurotrophins. FIG. 1A shows alignment of 
residues 25 to 36 from rat (SEQ ID NO: 1) (Whittemore et al., 1988), mouse 
(SEQ ID NO: 1) (Scott et al., 1983), human (SEQ ID NO: 1) (Ullrich et al., 
1983), bovine (SEQ ID NO: 1) (Meier et al., 1986), guinea pig (SEQ ID NO: 
1) (Schwarz et al., 1989), chicken (SEQ ID NO: 1) (Ebendal et al., 1986; 
Meier et al., 1986), xenopus (SEQ ID NO: 2) (ref) and snake (SEQ ID NO: 3) 
(Selby et al., 1987) NGF. FIG. 1B shows alignment of residues 25 to 36 
from rat NGF (SEQ ID NO: 1) and the homologous residues of rat BDNF (SEQ 
ID NO: 4) (Maisonpierre et al., 1990), rat NT-3 (SEQ ID NO: 5) 
(Maisonpierre et al., 1990; Ernfors et al., 1990) and xenopus NT-4 (SEQ ID 
NO: 6) (Hallbook et al., 1991). 
Among these conserved parts, the region panning residues 25 to 36 is the 
most hydrophilic and therefore likely to be on the surface of the NGF 
molecule (Meier et al., 1986; Ebendal et al., 1989). Synthetic peptides 
designed from this sequence have been shown to inhibit the in vitro 
biological activity of NGF (Longo et al., 1990). 
SUMMARY OF THE INVENTION 
The present invention provides mutant neurotrophic molecules of the nerve 
growth factor family which have novel receptor binding affinities and 
specificities as compared to their parent molecules. The invention is 
based, in part, on the use of NGF as a model system to determine the role 
of specific amino acids in the binding of the molecule to both the 
p75.sup.NGFR and the p140.sup.trk receptor. The present invention is based 
on the further discovery, using such model systems, that modifications can 
be made to NGF that result in loss of the ability of the molecule to bind 
to p75.sup.NGFR while maintaining the ability of the molecule to bind to 
p140.sup.trk and to exhibit biological activity comparable to the 
wild-type molecule. In various embodiments, modifications made to NGF as 
well as to corresponding regions in other members of the nerve growth 
factor family result in neurotrophic factors with greater specificity to 
the trk signal transducing receptors.

DETAILED DESCRIPTION OF THE INVENTION 
Nerve growth factor (NGF), like many other growth factors and hormones, 
binds to two different receptor molecules on the membrane of responsive 
cells. The product of the proto-oncogene trk, p140.sup.trk, is a tyrosine 
kinase receptor that has recently been identified as a signal transducing 
receptor for NGF. The role of the low-affinity NGF receptor, p75.sup.NGFR, 
in signal transduction is less clear. The crystal structure of NGF has 
recently been determined, although the structures involved in receptor 
binding and biological activity are still unknown. 
Site-directed mutagenesis combined with binding and biological assays 
provides a valuable tool to assess the functional importance of amino acid 
residues associated with the binding of neurotrophic molecules. Such 
studies, combined with the resolution of the three-dimensional crystal 
structure of NGF (McDonald et al., 1991) enables the rational design of 
neurotrophic molecules with altered receptor binding properties. 
Accordingly, the present invention is directed to novel, mutant 
neurotrophic factors that are created by modifying one or more amino acids 
in the parent or wild-type neurotrophic factor of the nerve growth factor 
family. Such modifications are selected so as to reduce binding of the 
factor to the low affinity NGF receptor while maintaining the ability of 
the factor to bind to a trk receptor. 
As contemplated herein, modification of specific amino acid residues alters 
the specificity of the NGF. Based on the three dimensional structure of 
NGF, it has been determined that these alterations are in amino acid 
residues in a .beta.-hairpin loop exposed on the outside arm of the NGF 
dimer (McDonald et al., 1991). As described herein, residues with a 
positively charged side-chain within this region appear to be responsible 
for the main contact between NGF and p75.sup.NGFR. 
Applicants have discovered that NGF molecules mutated in these positions do 
not bind to the p75.sup.NGFR but retain binding to the trk proto-oncogene 
product and biological activity. These results suggest that p140.sup.trk 
alone is sufficient, at least in culture, to mediate biological activity 
of NGF in neuronal cells. Experiments conducted using mutated NGFs show 
that binding to p75.sup.NGFR is not required for induction of early gene 
expression, such as c-fos, or for neuronal differentiation of PC12 cells. 
Moreover, neither neurite outgrowth nor neuronal survival of cultured 
sympathetic neurons, which express both p75.sup.NGFR mRNA and protein 
(Ernfors et al., 1988; Yan and Johnson, 1988) and trk mRNA (G. Barbany, 
unpublished), was affected by the loss of binding to p75.sup.NGFR. These 
results demonstrate, for the first time, that the trk proto-oncogene 
product alone is sufficient to mediate a response to NGF in cultured 
neuronal cells, thus opening up unique possibilities to unravel the role 
of both p75.sup.NGFR and p140.sup.trk in mediating the biological 
activities of NGF and to create unique neurotrophic molecules with 
improved binding specificities. 
Although it may be possible that, in the mutants described herein, NGF 
binds through a different binding site to a new pocket created by an 
heterodimer of p75.sup.NGFR and p140.sup.trk (Hempstead et al., 1991) or, 
alternatively, free p75.sup.NGFR could still contact complex 
NGF-p140.sup.trk and in this way cooperate in signal transduction, 
p75.sup.NGFR -p140.sup.trk complexes have not been detected in 
cross-linking experiments performed with either PC12 cells or sensory 
neurons under conditions which allowed detection of p75.sup.NGFR or 
p140trk homodimers (Meakin and Shooter, 1991). The fact that the mutant 
molecules of the present invention retain binding to p140trk strongly 
argues for the fact that the observed biological activities were mediated 
by this receptor molecule alone. 
Although not intended to limiting, it is postulated herein that in certain 
neurotrophins of the nerve growth factor family, namely NGF, NT-3 and 
NT-4, the positively charged amino acids in the .beta.-hairpin loop 30 to 
34 play a significant role in the ability of the molecules to bind to 
p75.sup.NGFR. Thus, according to one embodiment of the invention, 
alterations are made in one or several of these amino acids such that the 
overall charge in this region is altered, thereby reducing the ability of 
the molecule to bind to p75.sup.LNGF while maintaining the ability of the 
molecules to bind to their corresponding trk receptors. As used herein, 
amino acid residue 1 is the first amino acid in the mature protein. 
In one such embodiment, the positively charged side chain of Lys32 is 
replaced by, for instance the methyl group of Ala. Such reduction reduces 
the binding of the molecule to p75.sup.NGFR to 5% of the binding seen with 
parent NGF (Table 2 and FIG. 6). In another embodiment, the change of 
Lys34 into Ala reduces binding to A875 cells to 55% of the parent levels. 
In yet another embodiment, the simultaneous replacement of Lys32, Lys34 
and Glu35 by alanine is used to completely abolish the binding of the 
mutant molecule to p75.sup.NGFR. In the case of each of these mutants, 
despite reduced or absent binding to p75.sup.NGFR, the molecules retain 
wild-type biological activity on explants of sympathetic ganglia. 
In an additional embodiment, mutant neurotrophic factors are designed based 
on the discovery that positively charged amino acids around amino acid 95 
in a second .beta.-hairpin loop may interact with Lys32 and Lys34 to form 
a positively charged interface involved in binding to p75.sup.NGFR. 
Simultaneous modification of Lys32 with either of the two other lysines 
(Lys 34 or Lys 95) results in loss of binding to p75.sup.NGFR. Despite the 
lack of binding to p75.sup.NGFR, these mutants bind to p140.sup.trk and 
retain their biological activity, as measured by neuronal differentiation 
of PC12 cells and survival of cultured sympathetic neurons. 
Because the other three known neurotrophins can also bind to the 
low-affinity NGF receptor (Rodriguez-Tebar et al., 1990; Ernfors et al., 
1990; Squinto et al., 1991; Hallbook et al., 1991), comparable changes in 
the corresponding amino acids would be expected to result in comparable 
alterations is binding specificity while not affecting the ability of 
these molecules to bind to their respective signal transducing trk 
receptor. Lys95 is conserved in all four proteins described so far (in 
Xenopus NT-4, the amino acid in position 95 is Lys, whereas in human NT-4 
positions 94 and 96 are the positively charged amino acids glutamine and 
arginine). Further, in NT-3 and NT-4, Lys32 is replaced by Arg. Lys34 is 
also conserved in NT-4. Accordingly, alteration in any of these positively 
charged amino acids (e.g. by replacing the positively charged amino acid 
by a neutral, or negatively charged amino acid) to lower the binding to 
p75.sup.NGFR would be contemplated by the present invention. 
The present invention also includes altered molecules wherein other 
modifications to the .beta.-loop region around Lys95 are made. For 
example, in BDNF, Lys32 and Lys34 are replaced by Ser and Gly, 
respectively. Interestingly, the spatially close loop of residues 93 to 96 
in BDNF has three consecutive positively charged residues (two lysines and 
an arginine) that may compensate for the absence of Lys32 and Lys34. A 
chimeric NGF molecule which has residues 23 to 35 (variable region I) 
replaced by the corresponding residues in BDNF (Ibanez et al., 1991a) 
showed a 10-fold reduction of low affinity binding to PC12 cells. The low 
affinity binding was restored in another chimeric molecule that contains 
both variable region I and residues 94 to 98 (variable region V) from 
BDNF, indicating that the three positively charged residues at positions 
95, 96 and 97 in BDNF can compensate for the lack of Lys32 and Lys34. 
Although both NGF and BDNF appear to equally compete for binding to 
p75.sup.NGFR, this receptor also recognizes differences between the two 
ligands which are reflected, in the case of BDNF, by positive 
cooperativity and slower dissociation kinetics (Rodriguez-Tebar et al., 
1990). It therefore appears that BDNF and NGF are recognized by 
p75.sup.NGFR as similar albeit not identical structures. The results 
described herein provide a structural explanation for the observed 
differences between NGF and BDNF and suggest that other neurotrophins may 
interact with p75.sup.NGFR through the same region. 
Also embodied herein are modifications of parent neurotrophic factors to 
enhance their stability. As described herein, selective modification of 
one or more of the amino acid residues occurring between amino acid 25 and 
36 of neurotrophic factors of the nerve growth factor family appears to 
alter the stability of the factors. Accordingly, the invention 
contemplates alteration of such amino acids, followed by measuring the 
stability and biological activity of the altered molecule to select those 
factors that retain their biological activity but have enhanced stability 
as compared to the parent molecule. 
The present invention, which relates to second generation factors derived 
from neurotrophic factors such as NGF, BDNF, NT-3 and NT-4, may be 
utilized to treat diseases in essentially the same way as the parent 
factors are utilized. For instance, they may be utilized to treat diseases 
and disorders of the nervous system which may be associated with 
alterations in the pattern of neurotrophic factor expression or which may 
benefit from exposure to the neurotrophic factor. 
With respect to factors produced according to the present invention that do 
not bind to p75.sup.NGFR, such molecules may have increased specificity 
toward target cells and therefore be effective in lower dosages. Further, 
such molecules may produce less side effects than the parent molecules, 
which bind to a more widely distributed array of neuronal cells. 
Retrograde transport, presumed to be mediated by p75.sup.NGFR, could be 
prevented by the use of mutated neurotrophins thereby allowing a local 
effect of mutated neurotrophins in defined areas of the brain where their 
high-affinity receptors are expressed (such as in the hippocampus, 
following brain insults). 
Materials and Methods 
The following experimental procedures were used throughout the experiments 
described herein. 
DNA cloning and site-directed mutagenesis 
A 770 base pair EcoRI fragment containing the pre-proNGF coding sequence 
from the rat NGF gene (Whittemore et al., 1988) was cloned into pBS KS+ 
(Stratagene). Single stranded DNA from this plasmid was used as template 
for oligonucleotide based site-directed mutagenesis as described by Kunkel 
et al. (1985) and detailed in Ibanez et al. (1990). The replacements were 
confirmed by nucleotide sequence analysis by the chain-termination method 
(Sanger et al., 1977). For protein expression, DNA inserts containing the 
desired replacements were then subcloned in pXM (Yang et al., 1986). 
Production and quantitation of recombinant proteins 
COS cells grown to about 70% confluency were transfected with 25 .mu.g 
plasmid DNA per 100 mm dish using the DEAE dextranchloroquine protocol 
(Luthman and Magnusson, 1983). To correct for differences in the amounts 
of recombinant protein produced by the different constructs, 35 mm dishes 
transfected in parallel were grown in the presence of 100 .mu.Ci/ml 
.sup.35 S-cysteine (Amersham). Aliquots of conditioned media were then 
analyzed by SDS/PAGE and the amounts of recombinant protein in the 
different samples were equilibrated after densitometer scanning of the 
corresponding autoradiograms as previously described (Ibanez et al., 
1991b). The absolute amount of parent NGF protein was assessed by 
quantitative immunoblotting of conditioned media and by measurement of 
biological activity in cultured sympathetic ganglia using standards of 
purified mouse NGF (Ibanez et al., 1990; Ibanez et al., 1991b). The data 
obtained from these analysis were then used to determine the protein 
concentration in the samples containing mutant proteins. 
Pulse-chase and immunoprecipitation 
Forty eight hours after transfection cells were incubated in cysteine-free 
media for 4 hours. The cells were then pulse-labeled with 1 mCi/ml of 
.sup.35 S-cysteine during 15 min. The chase was performed by replacing the 
labeling media with complete medium fortified with 2 mg/ml of cold 
cysteine. Parallel wells were harvested at different times and cell 
extracts and conditioned media were immunoprecipitated with a polyclonal 
rabbit antiserum (rabbit no. 30) against mouse NGF (Ebendal et al., 1989) 
and analyzed by SDS/PAGE under reducing conditions as previously described 
(Ibanez et al., 1990; Ibanez et al., 1991b). 
Binding assays 
Mouse NGF was labeled with .sup.125 I by the chloramine-T method to an 
average specific activity of 3.times.107 cpm/.mu.g. Rat PC12 cells (Greene 
and Tischler, 1976), human A875 cells (Buxser et al., 1983) and mouse 
rtrk-3T3 cells (Kaplan et al., 1991a) were used at 2 to 10.times.10.sup.6 
cells/ml. Steady state binding was measured in competition assays 
performed at 37.degree. C. using 1.5.times.10-.sup.9 M .sup.125 I-NGF and 
serial dilutions of conditioned media containing equivalent amounts of 
parent or mutated NGF protein. All components were added at the same time 
and the cells were collected by centrifugation after equilibrium was 
reached (1-2 hours incubation). Control experiments using medium from mock 
transfected COS cells showed that other proteins present in the 
conditioned medium had no effect on the binding of .sup.125 I-NGF to the 
cells. Nonspecific binding was measured in a parallel incubation to which 
at least a 1000-fold molar excess of unlabelled NGF was added. All results 
were corrected for this nonspecific binding, which was always less than 
10% of total binding. The concentration of each mutant and wild type NGF 
that gave 50% binding (IC.sub.50) was determined, and relative binding was 
calculated using the relationship: (mutant IC.sub.50 /wild type 
IC.sub.50).times.100. 
Biological assays 
Serial dilutions of conditioned media containing equivalent amounts of 
recombinant protein (in the range of 0.2 to 20 ng/ml) were assayed for 
biological activity on explanted chick embryonic day 9 sympathetic ganglia 
as previously described (Ebendal, 1984; Ebendal, 1989). Fibre outgrowth 
was scored on a semiquantitative scale in biological units (BU) by 
comparison to standards obtained with purified mouse NGF, for which 1 BU 
is equivalent to approximately 5 ng/ml. The concentration of each NGF 
protein that gave 0.5 BU in this scale was determined, and used to 
calculate the relative activity compared to that obtained with parent NGF. 
PC12 cells plated in 35 mm wells coated with poly-D-lysine were incubated 
with serial dilutions of conditioned media containing equivalent amounts 
of recombinant protein. At different time intervals, the percentage of 
cells bearing fibers longer than two cell diameter was determined 
microscopically. 
Induction of c-fos mRNA was measured by quantitative Northern blot analysis 
of total mRNA from PC12 cells treated with dilutions of conditioned media 
containing equivalent amounts of recombinant parent and mutant NGF. Total 
RNA was extracted as previously described (Ibanez et al., 1990). Ten .mu.g 
of total RNA was electrophoresed in a 1% agarose gel containing 0.7% 
formaldehyde and transferred to nitrocellulose membranes. The filters were 
then hybridized with a .alpha.-.sup.32 P-dCTP radiolabelled rat c-fos gene 
fragment (Curran et al., 1987) and washed at high stringency. The amount 
of c-fos mRNA was determined by densitometer scanning of autoradiograms. 
Dissociated neurons of the superior cervical ganglion from post-natal day 1 
rats were cultured in 35 mm wells coated with poly-D-lysine at a density 
of 30,000 cells/well. Serial dilutions of conditioned media containing 
equivalent amounts of recombinant protein were added at the time of 
plating and neuronal survival was determined after 72 hours by phase 
contrast microscopy. 
EXAMPLE 1 
Amino acid residues in the .beta.-hairpin loop 30-34 involved in receptor 
binding to PC12 cells 
Experiments and Results 
Conditioned media containing equal amounts of mutant NGF proteins was used 
to displace .sup.125 I-NGF from its receptors on the NGF-responsive 
pheochromocytoma cell line PC12. Competitive binding assays were performed 
using concentrations of .sup.125 I-NGF (.about.1.5 nM) at which 80% of the 
radiolabelled ligand associated with the cells is bound to low-affinity 
NGF receptors (Sutter et al., 1979). Concentrations of parent and mutant 
proteins required to displace 50% of the .sup.125 I-NGF from the PC12 
cells (IC.sub.50) were calculated (Table 1). The conservative replacement 
of Lys25 for Arg or the replacement of either of the three residues (26, 
27 and 29) for Ala did not affect the affinity of the protein for 
receptors on PC12 cells (Table 1). However, a 3 to 4-fold reduction in 
binding affinity was observed when Asp30 or Ile31 were modified (See 
Example 2, Table 1, and FIG. 4A). The importance of Ile 31 was further 
tested by replacement with Met (the residue that occurs at this position 
in BDNF) (Leibrock et al., 1989) and with Val. Interestingly, only the 
most conservative change (I31V) allowed a binding affinity similar to 
parent NGF (Table 1). A marked reduction of receptor binding was seen 
after replacement of Lys32 with Ala, in which case the affinity was 
reduced approximately 6-fold compared to parent NGF (Table 1 and FIG. 4A). 
Replacement of Lys34 with Ala and Val36 with Leu reduced binding to 50 and 
45% of the wt, respectively (Table 1 and FIG. 4A). Surprisingly, the 
incompletely processed E35A mutant showed close to parent binding affinity 
(Table 1 and FIG. 4A), indicating that intermediates in NGF biosynthesis 
can bind to NGF receptors as efficiently as the mature protein. 
Discussion 
The crystal structure of the NGF dimer (McDonald et al., 1991) reveals a 
novel structure consisting of three antiparallel pairs of strands and four 
loop regions which contain almost all the variable residues observed 
between different NGF-related molecules. One of these loops corresponds to 
the residues analyzed in the present study and includes a .beta.-hairpin 
turn (residues 30 to 34). Our results show that residues in region 25-36 
are important for stability, receptor binding and biological activity of 
the NGF molecule (FIG. 9A). 
Lys25 was shown to play an important structural role since only the closely 
related Arg, but not Ala or Gln, could replace Lys at this position to 
form a stable protein. In agreement with this, the crystal structure 
revealed that Lys25 makes a side-chain hydrogen bond to Glu55 which is 
important for the correct folding of the NGF protein (McDonald et al., 
1991). Deletion of Ala28 prevented the accumulation of NGF protein in the 
conditioned media indicating a structural role for this position. 
Replacement of Glu 35 for Ala resulted in the production of incompletely 
processed polypeptides in the range of 23 to 34K which were shown to have 
similar receptor binding affinity and biological activity as the fully 
processed, parent molecule. The fact that an in vitro synthesized 
full-length NGF precursor of 35K was previously shown to have very low 
levels of biological activity suggests that removal of some amino terminal 
sequences may be important for the activation of the NGF precursor 
(Edwards et al., 1988). Our results also demonstrate that, in addition to 
conserved domains in the NGF propeptide (Suter et al., 1991), residues in 
the mature molecule also play a role in the biosynthesis of fully 
processed, mature NGF. 
Replacement of the non polar side chain at Val36 with Leu was also shown to 
affect receptor binding to PC12 cells. In contrast to Ile31, Val36 is 
deeply buried in the NGF monomer and it appears to be involved in the 
formation of the hydrophobic core of the NGF subunit (McDonald et al., 
1991). The fact that Leu, but not Ala, could replace Val at this position 
indicates the importance of the hydrophobic contribution of Val36 to the 
core of the molecule and suggest that the reduced binding of the V36L 
mutant probably reflects structural rearrangements required to accommodate 
the larger Leu side-chain at this position. 
EXAMPLE 2 
Modification of Asp30 and 1le31 
Experiments and Results 
The specific biological activity of the mutant NGF proteins was first 
studied by assaying their ability to stimulate neurite outgrowth from E9 
chick sympathetic ganglia (Levi-Montalcini and Angeletti, 1968; Ebendal, 
1984; Ebendal, 1989). In agreement with their ability to displace .sup.125 
I-NGF from PC12 cells, the biological activities of the mutants K25R, 
T26A, T27A and T29A were all similar to the activity of parent NGF (Table 
1). To test the possibility that the Thr residues could compensate for 
their modification when changed individually, a triple mutant was 
generated where the three Thr residues were simultaneously replaced by 
Ala. However, this mutant failed to accumulate in the medium of 
transfected cells at detectable levels (Table 1). 
A 4-fold reduction of biological activity was seen with the D30N and I31A 
mutants (Table 1 and FIG. 4B) which correlated with their respective 
receptor binding affinities (Table 1). To eliminate the possibility that 
the decreased activity was due to the reduced stability of these mutant 
molecules (FIG. 2C), induction of c-fos mRNA was tested in PC12 cells. It 
is well documented that maximal induction of c-fos mRNA in these cells 
takes place within 30-45 min after exposure to NGF (Milbrandt, 1986; 
Gizango-Ginsberg and Ziff, 1990), a time period that is 20 to 30 times 
shorter than the estimated half-lives of these molecules. A peak in c-fos 
mRNA was detected after 30 min exposure of PC12 cells to parent NGF (FIG. 
4C). Both the D30N and the I31A mutants induced maximal c-fos mRNA levels 
after 30 min which, however, were 3-4 fold lower than the maximal level 
obtained with parent NGF (FIG. 4C). 
Interestingly, four mutants with reduced binding affinities to PC12 cells 
(I31M, K32A, K34A and V36L) showed parent levels of biological activity 
(Table 1 and FIG. 4B). Thus, for the K32A mutant, the 6-fold reduction in 
binding did not affect its biological activity in the sympathetic ganglia 
(compare FIGS. 4A and B). In agreement with the receptor binding data, the 
E35A mutant displayed parent levels of biological activity, despite the 
fact that it contained only approximately 5% of a correctly processed, 
mature protein (Table 1 and FIG. 4B). 
Discussion 
Several important hydrogen bonding side-chains are buried in the NGF 
subunit, including Asp30 (McDonald et al., 1991). These results showed 
that the half life of the NGF molecule is reduced about 20 times when 
Asp30 is replaced by Ala, a residue that would prevent the proposed 
hydrogen bond from the sidechain of Asp30 to the main chain at Lys34. The 
reduced recovery and half-life of the D30N mutant show that Ash can work 
at this position albeit at a lower efficiency. On the other hand, 
elimination or alanine replacement of Gly33 resulted in loss of recovery 
of NGF protein probably due to a reduced stability of the molecule. 
Glycine at this position allows the formation of a turn by having 
main-chain torsion angles outside the allowed range for amino acids with a 
side chain (Sibanda et al., 1989). Taken together, the results with the 
Asp30 and Gly33 mutants suggest that these residues play a structural role 
in the stabilization of the .beta.-hairpin loop 30-34 and that their 
modification may have functional effects through changes in the 
conformation of the loop (FIG. 9A). The high conservation of these 
positions in other members of the NGF family suggest that these residues 
could play a similar role in the other three neurotrophins. 
As a result of the turn at 30 to 34, the hydrophobic Ile31 becomes exposed 
on the surface of the NGF molecule. Replacement of this residue by Ala 
reduced both receptor binding in PC12 cells and biological activity. 
Interestingly, only biological activity but not receptor binding was 
rescued after replacement into Met, whereas wild type binding and 
biological activity were seen after change into Val. In addition, 
preliminary results showed a 5-fold reduction in binding to p140.sup.trk 
in the I31A mutant but parent levels in I31M. Taken together these results 
suggest a role for the non polar side-chain of Ile31 in both biological 
activity which correlates with binding to p140.sup.trk and low affinity 
binding (FIG. 9A). 
EXAMPLE 3 
Simultaneous replacement of Lys32, Lys34 and Glu35 by Ala 
The three charged residues Lys32, Lys34 and Glu35, where an individual 
mutation had no effect on the biological activity, were simultaneously 
replaced by Ala, thereby eliminating the charged side chains at these 
positions. Interestingly, this mutant protein was completely recovered as 
a fully processed protein in spite of the fact that it contained the E35A 
mutation (FIG. 3A). The triple mutation reduced binding of this protein to 
PC12 cells to less than 1% of that seen with the parent molecule (Table 1 
and FIG. 4A). The same result was obtained when the cells were 
preincubated with the mutant protein for 2 hours prior to the addition of 
.sup.125 I-NGF (not shown). However, the biological activity of the triple 
mutant in sympathetic ganglia was close to parent NGF activity (Table 1 
and FIG. 4B). 
Neurite outgrowth was assayed in PC12 cells to test if the loss of binding 
correlated with the biological activity in these cells (FIG. 5A). The 
individual change of Lys32, Lys34 and Glu 35 to Ala did not significantly 
changed the ability of the proteins to stimulate neurite outgrowth in 
spite of their different affinities to NGF receptors on these cells. 
Moreover, the triple mutant (K32A+K34A+E35A) also elicited parent activity 
despite its greatly reduced low affinity binding to PC12 cells (FIG. 5A). 
The possibility that the apparent discrepancy observed between binding and 
biological activity was due to a slower receptor-mediated degradation was 
also examined. As seen with other peptide hormones which undergo receptor 
mediated endocytosis (i.e. insulin), a reduced binding affinity may not 
always translate into a reduced biological activity when examined over a 
longer period of time. As a consequence of the reduced binding, mutant 
molecules may have a lower rate of receptor-mediated degradation which 
results in a slower but prolonged biological activity that can reach 
parent levels when integrated over a period of time. To investigate this 
possibility, the kinetics of both an early (c-fos mRNA induction) and a 
delayed (stimulation of neurite outgrowth) response in PC12 cells were 
studied. Despite their reduced binding affinities, both the K32A and the 
triple mutant induced c-fos mRNA and neurite outgrowth with the same time 
course and intensity as the parent molecule (FIGS. 5B and C). 
EXAMPLE 4 
Affect of Mutation of Lys32 and Lys34 on NGF binding 
Receptor binding assays to PC12 cells were performed using high 
concentrations of .sup.125 I-NGF at which most of the observed binding is 
of the low-affinity type (Sutter et al., 1979). However, since PC12 cells 
express both p75.sup.NGFR and p140.sup.trk receptors (Herrup and Thoenen, 
1979; Hosang and Shooter, 1985; Kaplan et at., 1991b) these results can 
not clearly discriminate between the binding of the mutant NGFs to either 
one of these two molecules. Therefore, the binding affinities were 
compared of the mutants K32A, K34A, E35A and the triple mutant 
K32A+K34A+E35A to A875 cells, a human melanoma cell line which expresses 
high amounts of only p75.sup.NGFR (Buxser et al., 1983) and to rtrk-3T3 
cells, a fibroblast cell line that expresses only rat p140.sup.trk (Kaplan 
et al., 1991a). 
The replacement of the positively charged side chain of Lys32 by the methyl 
group of Ala reduced the binding of the molecule to p75.sup.NGFR to 5% of 
the binding seen with parent NGF (Table 2 and FIG. 6). The change of Lys34 
into Ala reduced binding to A875 cells to 55% of the parent levels. 
However, the simultaneous replacement of Lys32, Lys34 and Glu35 completely 
abolished the binding of the mutant molecule to p75.sup.NGFR (Table 2 and 
FIG. 6). The individual change of Glu35 into Ala had no effect on the 
binding affinity to A875 cells (Table 2 and FIG. 6), indicating that the 
loss of binding seen with the triple mutant was due to the modification of 
the positively charged residues Lys32 and Lys34. 
Despite its 20-fold reduction in binding to p75.sup.NGFR, the K32A mutant 
was indistinguishable from parent NGF in binding to p140.sup.trk expressed 
on rtrk-3T3 cells (Table 2 and FIG. 6). Similarly, also the K34A and E35A 
mutants showed parent affinity to p140.sup.trk (Table 2 and FIG. 6). 
Interestingly, the triple mutant, which failed to displace .sup.125 I-NGF 
from p75.sup.NGFR, retained significant binding to p140.sup.trk, at about 
55% of the level seen with parent NGF (Table 2 and FIG. 6). Furthermore, 
in an additional embodiment involving the NGF molecule, Lys32, Lys34 and 
Lys95 form a positively charged interface involved in binding to 
p75.sup.NGFR. Simultaneous modification of Lys32 with either of the two 
other lysines results in loss of binding to p75.sup.NGFR. Despite the lack 
of binding to p75.sup.NGFR, these mutants retain binding to p140.sup.trk 
as well as biological activity, as measured by neuronal differentiation of 
PC12 cells and survival of cultured sympathetic neurons. 
EXAMPLE 5 
Modification of residues in the 25-36 region alters the stability of the 
NGF molecule. 
Alanine-scanning mutagenesis (Cunningham and Wells, 1989) was applied to 
map structurally and functionally important residues in the region between 
amino acid residues 25 and 36 of rat NGF. This region is highly conserved 
among different species of vertebrates (FIG. 1A) and shows 50-60% 
conservation in other members of the NGF family (FIG. 1B). Mutant proteins 
were transiently expressed in COS cells. The yield of mutant protein 
production was assessed by SDS-PAGE of metabolically labeled polypeptides 
in conditioned media of transfected cells in order to standardize for the 
amount of mutant protein used for receptor binding and biological assays. 
As shown in Table 1, the levels of mutant NGF proteins varied over a 
10-fold range. Five of the mutant NGF proteins (K25A, A28.DELTA., D30A, 
G33.DELTA. and V36A) did not accumulate in the medium at detectable 
levels. Interestingly, these residues correspond to the five positions 
from this domain that are strictly conserved among the different members 
of the NGF family (FIG. 1B). No protein was detected either after Lys25 or 
Gly 33 were changed into the more similar amino acid residues Gln and 
Ala, respectively (Table 1). In contrast, the D30A and V36A mutants could 
be rescued by replacement into Asn and Leu, respectively, though at lower 
levels than those seen with the wild type (wt) protein (Table 1). Lys25 
was also changed into Arg, the most conservative replacement possible at 
this position. This mutation allowed the detection of NGF protein at about 
50% of the levels of the parent protein (Table 1). 
The variations observed in the amounts of mutant protein may reflect 
differences in protein synthesis, stability or secretion of individual 
polypeptides in COS cells. To discriminate between these possibilities, 
pulse-chase experiments were carried out followed by immunoprecipitation 
and SDS-PAGE. After a 15 min pulse, a predominating 23K parent NGF 
precursor protein could be immunoprecipitated from cellular extracts (FIG. 
2A). Fully processed, mature 13K NGF was detected after 30 min of chase 
and almost all of the intracellular NGF disappeared after three hours. The 
disappearance of intracellular NGF correlated with the appearance of NGF 
in the media which peaked 6 hours after the chase and remained at this 
level for at least 14 more hours (FIG. 2B). The I31A mutant, which was 
produced 3 to 4 times lower than parent NGF (Table 1), accumulated in the 
transfected cells to a similar extent as the parent protein (FIG. 2A). 
However, lower levels of the I31A mutant were detected in the media, and a 
drop of 50% was seen in the last 17 hours after the chase (FIG. 2B), 
indicating a reduced stability of the I31A protein. Similarly, the amount 
of the D30N mutant protein, produced at 10-fold lower levels than parent 
NGF (Table 1), decreased significantly after 3 hours of chase (FIG. 2C). 
In addition, very low levels of the D30A mutant protein could be seen 
after 3 hours of chase, although they dropped to undetectable levels in 
the following 12 hours (FIG. 2C). The reduced half-lives of the I31A, D30N 
and D30A mutant proteins, estimated to be 18, 12 and 3 hours, 
respectively, indicated that the reduced yields of these mutants were due 
to lower stability of these proteins in the conditioned media. The greatly 
reduced peak levels seen after 3 hours of chase in the D30N and D30A 
mutants suggested that in this case protein synthesis could also be 
affected (FIG. 2C). 
EXAMPLE 6 
Replacement of Glu 35 for Ala 
Fully processed, mature E35A mutant protein was detected in the conditioned 
media at a level corresponding to 5% of parent NGF (Table 1). However, 
after immunoprecipitation, several higher molecular weight polypeptides 
(in the range of 23 to 34K) were seen which were only very weakly detected 
in the parent NGF sample (FIG. 3A). Pretreatment of the conditioned media 
at 70.degree. C. in the presence of 1% SDS and 1.5M NaCl prior to 
immunoprecipitation did not affect the polypeptide pattern 
immunoprecipitated from the E35A mutant (not shown), indicating that the 
higher molecular weight polypeptides did not represent unrelated proteins 
that coprecipitated with the E35A mutant. Instead, the size of these 
polypeptides suggests that they represent incompletely processed 
intermediates in the biosynthesis of the E35A protein. Pulse-chase 
experiments using this mutant revealed that both the incompletely 
processed and mature forms of this protein were very stable in the 
conditioned media (FIGS. 2C and 3B). 
EXAMPLE 7 
Effect of Alterations in Lys95 on p75.sup.NGFR binding 
Experiments and Results 
The results with the Lys32, Lys34 and the triple mutant suggest that these 
two positively charged residues form contact points between NGF and the 
p75.sup.NGFR molecule. Examination of the NGF crystal structure with 
computer graphics revealed that another positively charged residue, Lys95, 
is spatially close to Lys32 and Lys34. As in the case of the other two 
residues, Lys95 is also fully exposed and does not participate in 
secondary interactions. To test the possibility that Lys95 could also take 
part in the contact to the p75.sup.NGFR molecule, this residue was 
replaced by Ala. A double mutant K32A+K95A and a quadruple mutant 
K32A+K34A+E35A+K95A were also generated. The K95A mutant showed 65% 
binding to PC12 cells compared to parent NGF (FIG. 7). However, 
combination of K95A with K32A or with K32A+K34A+E35A drastically reduced 
binding to PC12 cells to 0.7% of parent levels (FIG. 7). The reduction of 
low-affinity binding to PC12 cells correlated with loss of binding to 
p75.sup.NGFR expressed on A875 cells (Table 2 and FIG. 7). In the case of 
the quadruple mutant, no IC.sub.50 could be calculated. However, despite 
their inability to bind to p75.sup.NGFR, these mutants retained the 
ability to displace .sup.125 I-NGF from p140.sup.trk expressed on 
fibroblasts (Table 2 and FIG. 7) and promoted neurite outgrowth from 
sympathetic neurons (FIG. 8A) at significant levels. 
The triple mutant K32A+K34A+E35A and the double mutant K32A+K95A offer a 
possibility to examine the role of p75.sup.NGFR in neuronal survival. 
Dissociated sympathetic neurons from the rat superior cervical ganglion 
were tested for survival after 3 days in culture. Less than 5% of the 
cells survived in the presence of media from mock transfected cells or in 
normal media when compared to parent NGF or purified mouse NGF (FIG. 8B). 
However, in cultures treated with the mutant NGFs the extent of neuronal 
survival was identical to what was seen with the parent protein (FIG. 8B). 
Discussion 
The crystal structure of NGF revealed a cluster of exposed positively 
charged side chains close to and around the .beta.-hairpin loop 30-34 
(FIGS. 9B and C) (McDonald et al., 1991). It is possible that the high 
overall negative charge observed for the p75.sup.NGFR (an estimated pI of 
4.4 (Radeke et al., 1987) may require a complementary ionic interaction 
from the highly basic NGF dimer (pI 9.3) in this region. The results 
presented here provide strong support to the notion that these positively 
charged amino acid residues serve as the main points of contact between 
NGF and p75.sup.NGFR Several lines of evidence support this hypothesis: 
First, as revealed by the crystal structure, Lys32, Lys34 and Lys95 are 
highly exposed (50-70% side-chain solvent accessibility) and their 
side-chains do not have a structural role in the molecule (McDonald et 
al., 1991). Second, as shown here, replacement of Lys32 for Ala reduced by 
6-fold the affinity of the mutant to receptors on PC1'2 cells under 
low-affinity binding conditions. Third, the simultaneous replacement of 
Lys32, Lys34 and Glu 35 further reduced low affinity binding to PC12 cells 
to less than 1% of that seen with parent NGF. This was not due to the E35A 
mutation since replacement of Glu35 for Ala did not change the affinity of 
binding. Fourth, replacement of Lys95 had a synergistic effect when 
combined with K32A, reducing the binding to PC12 cells to almost 
undetectable levels. Fifth, in all cases, the loss of low-affinity binding 
to PC12 cells correlated with loss of binding to p75.sup.NGFR expressed on 
A875 cells. In the case of the triple mutant K32A+K34A+E35A and the double 
mutant K32A+K95A binding to p75.sup.NGFR was completely abolished or 
reduced 150-fold, respectively. And sixth, despite the loss of binding to 
p75.sup.NGFR, all mutant NGFs retained binding to p140 trk and biological 
activity, further demonstrating that the loss of low-affinity binding was 
not due to drastic alterations in conformation of the mutant proteins. 
The synergistic effects observed with the multiple lysine mutants indicate 
that these positively p75.sup.NGFR residues cooperate in the formation of 
an interface for binding to p75.sup.NGFR (FIGS. 9B and C). Lys32 appears 
to be making the strongest contact followed by Lys34 and Lys95 which are 
probably responsible for the residual binding observed in the K32A mutant. 
Additional positively charged residues, like the previously studied Arg100 
and Arg103 (Ibanez et al., 1990) and perhaps Lys88, may also contribute to 
the binding interface (FIGS. 9B and C). The loss of binding to 
p75.sup.NGFR in the K32A+K34A+E35A and the K32A+K95A mutants suggests that 
a minimal number of positive charges are required on the surface of the 
NGF molecule to provide a stable contact with p75.sup.NGFR. This model 
does not rule out the possibility that other type of contacts like, for 
example, the hydrophobic residue Ile31, may also contribute to stabilize 
the association between NGF and p75.sup.NGFR. 
The other three known neurotrophins can also bind to the low-affinity NGF 
receptor (Rodriguez-Tebar et al., 1990; Ernfors et al., 1990; Squinto et 
al., 1991; Hallbook et al., 1991). Lys95 is conserved in all four proteins 
described so far and, in NT-3 and NT4, Lys32 is replaced by Arg, another 
positively charged amino acid residue. Lys34 is also conserved in NT-4. 
However, in BDNF, Lys32 and Lys34 are replaced by Ser and Gly, 
respectively. Interestingly, the spatially close loop of residues 93 to 96 
in BDNF has three consecutive positively charged residues that may 
compensate for the absence of Lys32 and Lys34. In support for this 
hypothesis, a chimeric NGF molecule which has residues 23 to 35 (variable 
region I) replaced by the corresponding residues in BDNF (Ibanez et al., 
1991a) showed a 10-fold reduction of low affinity binding to PC12 cells. 
The low affinity binding was restored in another chimeric molecule that 
contains both variable region land residues 94 to 98 (variable region V) 
from BDNF, indicating that the three positively charged residues at 
positions 95, 96 and 97 in BDNF can indeed compensate for the lack of 
Lys32 and Lys34. Although both NGF and BDNF appear to equally compete for 
binding to p75.sup.NGFR, this receptor also recognizes differences between 
the two ligands which are reflected, in the case of BDNF, by positive 
cooperativity and slower dissociation kinetics (Rodriguez-Tebar et al., 
1990). It therefore appears that BDNF and NGF are recognized by 
p75.sup.NGFR as similar albeit not identical structures. These results 
offer a structural explanation for the observed differences between NGF 
and BDNF and suggest that other neurotrophins may interact with 
p75.sup.NGFR through the same region. 
The present invention is not to be limited in scope by the specific 
embodiments described herein. Indeed, various modifications of the 
invention in addition to those described herein will become apparent to 
those skilled in the art from the foregoing description and accompanying 
figures. Such modifications are intended to fall within the scope of the 
appended claims. 
TABLE 1 
______________________________________ 
Relative yield, receptor binding to PC12 cells and specific 
biological activity of wild type and mutant NGF proteins 
Receptor 
Mutant Binding.sup.c 
Biological 
Protein.sup.a Yield.sup.b 
% of wild type 
Activity.sup.c 
______________________________________ 
wild type 100 100 100 
K25A -- -- -- 
K25Q -- -- -- 
K25R 50 130 100 
T26A 40 100 100 
T27A 46 120 63 
A28.DELTA. -- -- -- 
T29A 18 71 74 
T26A + T27A + T29A 
-- -- -- 
D30A -- -- -- 
D30N 11 25 23 
I31A 28 30 25 
I31M 50 35 100 
I31V 34 130 100 
K32A 76 16 100 
G33.DELTA. -- -- -- 
G33A -- -- -- 
K34A 53 50 100 
E35A 5.sup.d 
.sup. 85.sup.e 
85.sup.e 
K32A + K34A + E35A 
65 &lt;1 65 
V36A -- -- -- 
V36L 51 33 90 
______________________________________ 
.sup.a Mutants are abbreviated by the wild type (wt) residue (single amin 
acid designation), followed by its codon number and the mutant residue. 
.DELTA.indicates that the corresponding residue was deleted. 
.sup.b Steadystate levels calculated after SDSPAGE of metabolically 
labeled conditioned media. The short line indicates that the level of 
mutant protein was below detection (&lt;2% of wt NGF). 
.sup.c Data from two doseresponse experiments varied by .+-.10% of the 
average values reported here. 
.sup.d Data based on the fully processed form (see text for details). 
.sup.e Data based on both processed and unprocessed forms (see text for 
details). 
TABLE 2 
______________________________________ 
Relative receptor binding to A875 cells and rtrk-NIH3T3 
cells of wild type and mutant NGF proteins. 
binding to binding to 
Mutant A875 cells rtrk-NIH3T3 cells 
Protein % of wild type 
______________________________________ 
wild type 100 100 
K32A 5 100 
K34A 5 90 
E35A 100 100 
K32A + K34A + E35A 
no IC.sub.50 
55 
K95A 55 80 
K32A + K95A &lt;A 40 
K32A + K34A + E35A + 
no IC.sub.50 
40 
K95A 
______________________________________ 
Data from three independent experiments varied by .+-.10% of the average 
values reported here. 
REFERENCES 
Angeletti, R. H. and Bradshaw, R. A. (1971). Nerve growth factor from mouse 
submaxillary gland: amino acid sequence. Proc. Natl. Acad. Sci. USA 68, 
2417-20. 
Angeletti, R. H., Hermodson, M. A. and Bradshaw, R. A. (1973). Amino acid 
sequences of 2.5S nerve growth factor. II Isolation and characterization 
of the thermolytic and peptic peptides and the complete covalent 
structure. Biochemistry 12, 100-15. 
Banerjee, S. P., Snyder, S. H., Cuatrecasas, P. and Greene, L. A. (1973). 
Binding of nerve growth factor in superior cervical ganglia. Proc. Natl. 
Acad. Sci. USA 79, 2519-2523. 
Barde, Y. -A., Edgar, D. and Thoenen, H. (1982). Purification of a new 
neurotrophic factor from mammalian brain. Embo J. 1, 549-553. 
Berg, M., Sternberg, D., Hempstead, B. and Chao, M. (1991). The 
low-affinity p75 nerve growth factor (NGF) receptor mediates NGF-induced 
tyrosine phosphorylation. Proc. Natl. Acad. Sci. USA 88, 7106-7110. 
Buxser, S. E., Watson, L., Kelleher, D. J. and Johnson, G. L. (1983). 
Purification of the receptor for nerve growth factor from A875 melanoma 
cells by affinity chromatography. J Biol Chem 258, 3370-5. 
Cordon-Cardo, C., Tapley, P., Jing, S., Nanduri, V., O'Rourke, E., 
Lambelle, F., Kovary, K., Klein, R., Jones, K., Reichardt, L. and 
Barbacid, M. (1991). The trk tyrosine kinase mediates the mitogenic 
properties of nerve growth factor and neurotrophin-3. Cell 66, 173-183. 
Cunningham, B. C. and Wells, J. A. (1989). High-resolution epitope mapping 
of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244, 
10811085. 
Curran, T., Gordon, M. B., Rubino, K. L. and Sambucetti, L. C. (1987) 
Isolation and characterization of the c-fos (rat) cDNA and analysis of 
post-translational modification in vitro. Oncogene 2, 79-84. 
Ebendal, T. (1984). In Organizing Principles of Neural Development, S. 
Sharma, eds. (New York: Plenum Press), pp. 93-107. 
Ebendal, T. (1989). Use of collagen gels to bioassay nerve growth factor 
activity. In Nerve Growth Factors, R. A. Rush, eds. (Chichester: John 
Wiley & Sons), pp. 81-2 4 
Ebendal, T., Larhammar, D. and Persson, H. (1986). Structure and expression 
of the chicken.about.nerve growth factor. EMBO J. 5, 1483-7. 
Ebendal, T., Persson, H., Larhammar, D., Lundstromer, K. and Olson, L. 
(1989). Characterization of antibodies to synthetic nerve growth factor 
(NGF) and proNGF peptides. J Neurosci Res 22, 223-240. 
Edwards, R. H., Selby, M. J., Garcia, P. D. and Rutter, W. J. (1988). 
Processing of the native nerve growth factor precursor to form 
biologically active nerve growth factor. J. Biol. Chem. 263, 6810-5. 
Ernfors, P., Hallbook, F., Ebendal, T., Shooter, E., Radeke, M. J., Misko, 
T. P. and Persson, H. (1988). Developmental and regional expression 
of.about.-Nerve Growth Factor receptor mRNA in the chick and rat. Neuron 
1, 983-996. 
Ernfors, P., Ibariez, C. F., Ebendal, T., Olson, L. and Persson, H. (1990). 
Molecular cloning and neurotrophic activities of a protein with structural 
similarities to.about.nerve growth factor: developmental and topographical 
expression in the brain. Proc. Natl. Acad. Sci. USA 87, 5454-5458. 
Gizang-Ginsberg, E. and Ziff, E. (1990). Nerve growth factor regulates 
tyrosine hydroxylase gene transcription through a nucleoprotein complex 
that contains c-Fos. Genes Dev 4, 477-491. 
Greene, L .A. and Tischler, A. S. (1976). Establishment of a noradrenergic 
clonal line of rat adrenal pheochromocytoma cells which respond to nerve 
growth factor. Proc. Natl. Acad. Sci. USA 73, 2424-8. 
Hallook, F., Ayer.LeLievre, C., Ebendal, T. and Persson, H. (1990). 
Expression of nerve growth factor receptor mRNA during early development 
of the chicken embryo: emphasis on cranial ganglia. Development 10.about., 
693-704. 
Hallbook, F., Ibanez, C. F. and Persson, H. (1991). Evolutionary studies of 
the nerve growth factor family reveal a novel member abundantly expressed 
in Xenopus ovary. Neuron 6, 845-858. 
Hempstead, B., Martin-ZAnca, D., Kaplan, D., Parada, L. and Chao, M. 
(1991). High-affinity NGF binding requires coexpression of the trk 
proto-oncogene an.about.1 the low-affinity NGF receptor. Nature 350, 
678-683. 
Hempstead, B. L., Schleifer, L. S. and Chao, M. V. (1989). Expression of 
functional nerve growth factor receptors after gene transfer. Science 243, 
373-375. 
Herrup, K. and Shooter, E. M. (1973). Properties of the .beta.-nerve growth 
factor receptor of avian dorsal root ganglia. Proc. Natl Acad. Sci. USA 
70, 3884-88. 
Herrup, K. and Thoenen, H. (1979). Properties of the nerve growth factor 
receptor of a clonal line of rat pheochromocytoma (PC12) cells. Exp Cell 
Res 121, 71-8. 
Heuer, J. G., S., F.-N., Wheeler, E. F. and Bothwell, M. (1990). Structure 
and developmental expression of the chicken NGF receptor. Dev Biol 137, 
287-304. 
Hohn, A., Leibrock, J., Bailey, K. and Barde, Y.-A. (1990). Identification 
and characterization of a novel member of the nerve growth 
factor/brain-derived neurotrophic factor family. Nature 344, 339-341. 
Hosang, M. and Shooter, E. M. (1985). Molecular characteristics of nerve 
growth factor receptors on PC12 cells. J Biol Chem 260, 655-62. 
Ibanez, C. F., Hallbook, F., Ebendal, T. and Persson, H. (1990). 
Structure-function studies of nerve growth factor: functional importance 
of highly conserved amino acid residues. EMBO J. 9,1477-1483. 
Ibanez, C. F., Ebendal, T. and Persson, H. (1991a). Chimeric molecules with 
multiple neurotrophic activities reveal structural elements determining 
the specificities of NGF and BDNF. EMBO J. 10, 2105-2110. 
Ibanez, C. F., Hallook, F., Soderstrom, S., Ebendal, T. and Persson, H. 
(1991b). Biological and immunological properties of recombinant human, rat 
and chicken nerve growth factors: a comparative study. J. Neurochem. 57, 
1033-1041. 
Ip, N. Y., Ibanez, C. F., Nye, S. H., McClain, J., Jones, P. F., Gies, D. 
R., Belluscio, L., Le Beau, M. M., Espinosa III, R., Squinto, S. P., 
Persson, H. and Yancopoulos, G. (1992). Mammalian neurotrophin-4: 
structure, chromosomal localization, tissue distribution and receptor 
specificity. Proc Natl Acad Sci U S A, in press. 
Johnson, D., Lanahan, A., Buck, C. R., Sehgal, A., Morgan, C., Mercer, E., 
Bothwell, M. and Chao, M. (1986). Expression and structure of the human 
NGF receptor. Cell 47, 545-554. 
Kaplan, D., Hempstead, B., Martin-Zanca, D., Chao, M. and Parada, L. 
(1991a). The trk proto-oncogene product: a signal transducing receptor for 
nerve growth factor. Science 252, 554-558. 
Kaplan, D., Martin-Zanca, D. and Parada, L. (1991b). Tyrosine 
phosphorylation and tyrosine kinase activity of the trk proto-oncogene 
product induced by NGF. Nature 350, 158-160. 
Klein, R., Jing, S., Nanduri, V., O'Rourke, E. and Barbacid, M. (1991). The 
trk proto-oncogene encodes a receptor for nerve growth factor. Cell 
65,189-197. 
Leibrock, J., Lottspeich, A. H., Hofer, M., Hengerer, B., Masiakowski, P., 
Thoenen, H. and Barde, Y.-A. (1989). Molecular cloning and expression of 
brain-derived neurotrophic factor. Nature 341,149-52. 
Levi-Montalcini, R. and Angeletti, P. (1968). Nerve growth factor. Physiol. 
Rev. 48, 534-569. 
Loeb, D., Maragos, J., Martin-Zanca, D., Chao, M., Parada, L. and Greene, 
L. (1991). The trk proto-oncogene rescues NGF responsiveness in mutant NGF 
nonresponsive PC12 cell lines. Cell 66, 961-966. 
Longo, F., Vu, T.-K. H. and Mobley, W. (1990). The in vitro biological 
effect of nerve growth factor is inhibited by synthetic peptides. Cell 
Reg. 1,189-195. 
Luthman, H. and Magnusson, G. (1983). High efficiency polyoma DNA 
transfection of chloroquine treated cells. Nucl. Acids Res. 11, 1295-1305. 
Maisonpierre, P. C., Belluscio, L., S, S., Ip, N.Y., Furth, M. E., Lindsay, 
R. M. and Yancopoulos, G. D. (1990). Neurotrophin-3: A neurotrophic factor 
related to NGF and BDNF. Science 247,1446-1451. 
Martin-Zanca, D., Oskam, R., Mitra, G., Copeland, T. and Barbacid, M. 
(1991). Molecular and biochemical characterization of the human .about.rk 
proto-oncogene. Mol Cell Biol 9, 24-33. 
McDonald, N. Q., Lapatto, R., Murray-Rust, J., Gunning, J., Wlodawer, A. 
and Blundell, T. L. (1991). New protein fold revealed by a 2.3-A 
resolution crystal structure of nerve growth factor. Nature 354, 411-414. 
Meakin, S. and Shooter, E. (1991). Molecular investigations on the 
high-affinity nerve growth factor receptor. Neuron 6,153-163. 
Meier, R., Becker-Andre, M., Gotz, R., Heumann, R., Shaw, A. and Thoenen, 
H. (1986). Molecular cloning of bovine and chick nerve growth factor 
(NGF): delineation of conserved and unconserved domains and their 
relationship to the biological activity and antigenicity of NGF. EMBO J. 
5,1489-93. 
Milbrandt, J. (1986). Nerve growth factor rapidly induces c-fos mRNA in 
PC12 rat pheochromocytoma cells. Proc Natl Acad Sci U S A 83, 4789-93. 
Radeke, M. J., Misko, T. P., Hsu, C., Herzenberg, L. A. and Shooter, E. M. 
(1987). Gene transfer and molecular cloning of the rat nerve growth factor 
receptor. Nature 325, 593-597. 
Richardson, P. M., Verge Issa, V. M. K. and Riopelle, R. J. (1986). 
Distribution of neuronal receptors for nerve growth factor in the rat. 
Neurosci. 6, 2312-2321. 
Rodriguez-Tebar, A., Dechant, G. and Barde, Y.-A. (1990). Binding of brain 
derived neurotrophic factor to the nerve growth factor receptor. Neuron 4, 
487492. 
Rosenthal, A., Goeddel, D. V., Nguyen, T., Lewis, M., Shih, A., Laramee, G. 
R., Nikolics, K. and Winslow, J. W. (1990). Primary structure and 
biological activity of a novel human neurotrophic factor. Neuron 4, 
767-773. 
Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with 
chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-7. 
Sauve, K., Nachman, M., Spence, C., Bailon, P., Campbell, E., Tsien, W.-H., 
Kondas, J., Hakimi, J. and Ju, G. (1991). Localization in human 
interleukin 2 of the binding site to the (x chain (p55) of the interleukin 
2 receptor. Proc Natl Acad Sci USA 88, 4636-4640. 
Schwarz, M. A., Fisher, D., Bradshaw, R. A. and Isackson, P. J. (1989). 
Isolation and sequence of a cDNA clone of beta-nerve growth factor from 
the guinea pig prostate gland. J. Neurochem. 52, 1203-9. 
Scott, J., Selby, M., Urdea, M., Quiroga, M., Bell, G. I. and Rutter, W. 
(1983). Isolation and nucleotide sequence of a cDNA encoding the precursor 
of mouse nerve growth factor. Nature 302, 538-40. 
Selby, M. J., Edwards, R. H. and Rutter, W. J. (1987). Cobra nerve growth 
factor: structure and evolutionary comparison. J. Neurosci. Res. 18, 29314 
8. 
Sibanda, L., Blundell, T. and Thornton, J. (1989). Conformation of 
.beta.-hairpins in protein structures. J Mol Biol 206, 759-777. 
Squinto, S. P., Stitt, T. N., Aldrich, T. H., Davis, S., Bianco, S. M., 
Radziejewski, C., Glass, D. J., Masiakowski, P., Furth, M. E., Valenzuela, 
D. M., DiStefano, P. S., Yancopoulos, G. D. (1991). trkB encodes a 
functional receptor for brain-derived neurotrophic factor and 
neurotrophin-3 but not nerve growth factor. Cell 65, 885-993. 
Suter, U., Heymach Jr, J. and Shooter, E. (1991). Two conserved domains in 
the NGF propeptide are necessary and sufficient for the biosynthesis of 
correctly processed and biologically active NGF. EMBO J 10, 2395-2400. 
Sutter, A., Riopelle, R. J., Harris-Warrick, R. M. and Shooter, E. M. 
(1979). Nerve growth factor receptors: characterization of two distinct 
classes of binding sites on chick embryo sensory ganglia cells. J. Biol. 
Chem. 254, 5972-82. 
Thoenen, H., Bandtlow, C. and Heumann, R. (1987). The physiological 
function of nerve growth factor in the central nervous system: comparison 
with the periphery. Rev. Physiol. Biochem. Pharmacol. 109,145-78. 
Thoenen, H. and Barde, Y. A. (1980). Physiology of nerve growth factor. 
Physiol. Rev. 60, 1284-1325. 
Ullrich, A., Gray, A., Berman, C. and Dull, T. J. (1983). Human 
.beta.-nerve growth factor gene highly homologous to that of mouse. Nature 
303, 821-25. 
Weskamp, G. and Reichardt, L. (1991). Evidence that biological activity of 
NGF is mediated through a novel subclass of high affinity receptors. 
Neuron 6, 649-663. 
Whittemore, S. R., Friedman, P. L., Larhammar, D., Persson, H., Gonzalez, 
C. M. and Holets, V. R. (1988). Rat beta-nerve growth factor sequence and 
site of synthesis in the adult hippocampus. J. Neurosci. Res. 20, 403-10. 
Whittemore, S. R. and Seiger, A. (1987). The expression, localization and 
functional significance of beta-nerve growth factor in the central nervous 
system. Brain Res. 434, 439-64. 
Yan, H., Schlessinger, J. and Chao, M. (1991). Chimeric NGF-EGF receptors 
define domains responsible for neuronal differentiation. Science 252, 
561-564. 
Yan, Q. and Johnson, E. (1988). An immunohistochemical study of the nerve 
growth factor receptor in developing rats. J Neurosci 8, 3481-98. 
Yang, Y. C., Ciarlette, A. B., Temple, P. A., Chung, M. P., Kovacic, S., 
WitekGianotti, J. S., Leary, A. C., Kritz, R., Donahue, R. E., Wong, G. G. 
and Clark, S. C. (1986). Human IL-3 (multi-CSF): identification by 
expression cloning of a novel hematopoietic growth factor related to 
murine IL-3. Cell 47, 3-10. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
Lys ThrThrAlaThrAspIleLysGlyLysGluVal 
1510 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
LysThrLysAlaThrAspIleLysGlyLysGluVal 
1510 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LysThrThrAlaThrAspIleLysGlyAsnThrVal 
1510 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LysLysThrAlaValAspMetSerGlyGlyThrVal 
1510 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
LysThrSerAlaIleAspIleArgGlyHisGlnVal 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
LysArgThrAlaValAspAspArgGlyLysIleVal 
15 10