TRK tyrosine kinase receptor is the physiological receptor for nerve growth factor

The present invention relates to a complex comprising nerve growth factor (NGF) and trk-proto-oncogene protein. The present invention also relates to methods for detecting the presence of NGF and trk-proto-oncogene receptor. The present invention further relates to methods that may be used in diagnostics and therapeutics for neurodegenerative diseases such as Alzheimer's and Huntington's by detecting NGF-trk receptor pairs.

FIELD OF THE INVENTION 
The present invention relates to a complex comprising the neurotrophic 
factors nerve growth factor (NGF) and trk-proto-oncogene protein. The 
present invention also relates to methods for detecting the presence of 
NGF ligand, and trk-proto-oncogene receptor protein. 
The present invention further relates to methods of diagnosing and treating 
conditions of nerve growth disease and regeneration such as Alzheimer's 
disease and neuroblastoma. In particular the present method involves 
detection of the ligand receptor pairs. 
The present invention further relates to methods for detecting neurotrophic 
factor receptor/ligand complexes on the basis of structural and functional 
relatedness to trk and NGF. 
BACKGROUND OF THE INVENTION 
The development of the vertebrate nervous system is characterized by a 
series of complex events beginning with an apparently homogeneous 
neuroepithelium in the early embryo and leading to formation of diverse, 
highly ordered, and interconnected neural cell types in the adult. 
Considerable descriptive and experimental evidence has been amassed which 
points to the existence of limiting diffusible factors that are required 
for the targeting, survival, and proper synaptic arrangement of neurons 
(R. W. Oppenheim, In: Studies in Developmental Neurobiology. (Cowan, W. M. 
ed.), Oxford Univerity Press, pp. 74-133, 1981; W. D. Snider and E. M. 
Johnson, Ann. Neurol. 26:489-506 (1989)). Functional neuronal circuits are 
sculpted from an initially overabundant production of neurons during 
development. In the mid term embryo, a process of programmed cell death 
eliminates a major proportion of the neuron population, leaving behind the 
appropriate number of neurons required for innervation of target tissues 
(V. Hamburger and R. Levi-Montalcini, J. Exp. Zool. 111:457-502 (1949); 
Y.-A. Barde, Neuron 2:1525-1535 (1989)). 
The discovery of Nerve Growth Factor (NGF) provided the first direct 
evidence for the existence of neurotrophic, polypeptide factors (R. 
Levi-Montalcini and V. Hamburger, J. Exp. Zool. 116:321-362 (1951); R. 
Levi-Montalcini and P. U. Angeletti, Physiol. Rev. 48:534-569(1968)). This 
has been followed by the more recent description of additional 
neurotrophic factors: BDNF, CTNF, and NT-3, (for review see W. D. Snider 
and E. M. Johnson, Ann. Neurol. 26:489-506 (1989); G. Barbin et al., J. 
Neurochem. 43:1468-1478 (1984); P. C. Maisonpierre et al., Science 
247:1446-1451 (1990)). The physiological consequences elicited by NGF in 
vitro and in vivo have been at the center of research in neurobiology for 
several decades. Consequently, considerable information is now available 
about the cell types that respond to NGF in the peripheral and central 
nervous systems. 
NGF is known to play a role in the targeting and survival of sympathetic 
and neural crest-derived sensory neurons as well as in selected 
populations of cholinergic neurons in the brain (L. A. Greene and E. M. 
Shooter, Annu. Rev. Neurosci. 3:353-402 (1980); H. Thoenen and Y.-A. 
Barde, Physiol. Rev. 60:1284-1335 (1980); H. Gnahn et al., Dev. Brain. 
Res. 9:45-52 (1983)). It appears that the NGF dependent cholinergic 
neurons in the basal forebrain correspond to the population of cells that 
undergo attrition of Alzheimer's disease (F. Hefti, Annals of neurology, 
13:109-110 (1983); Hefti and Wemer, 1986; Johnson and Tanuchi, 1987; P. J. 
Whitehouse et al., Science 215:1237-1239 (1982)). In vivo studies, in 
which NGF was injected in the periphery of the mouse embryo trunk, result 
in enhanced survival of sensory ganglia that are normally targeted for 
cell death (V. Hamburger et al., J. Neurosci. 1:60-71 (1981); I. B. Black 
et al., In: Growth Factors and Development, Current Topics in 
Developmental Biology, vol. 24 (Nilsen-Hamilton, ed.), pp. 161-192 
(1990)). 
Exposure of embryos to NGF antibodies results in reduced survival of dorsal 
root ganglion neurons while injection of NGF antibodies into neonate mice 
has the principal effect of inhibiting the survival of sympathetic neurons 
(R. Levi-Montalcini and B. Booker, Proc. Natl. Acad. Sci. USA, 46:373-384 
or 384-391 (1960); S. Cohen, Proc. Natl. Acad. Sic. USA, 46:302-311 
(1960); E. M. Johnson et al., Science 210:916-918 (1980)). 
In vitro, some tumor cell lines of neural origin respond to the presence of 
NGF by undergoing differentiation along neuronal pathways. PC12 cells, 
derived from a rat pheochromocytoma, are the best characterized of these 
cell lines and represent a widely accepted model for NGF-mediated response 
and for neuronal differentiation (L. A. Greene and A. S. Tischler, Proc. 
Natl. Acad. Sci. USA 73:2424-2428 (1976)). 
Although much is understood about the biology of NGF outside the cell, the 
mechanisms by which NGF elicits neurotrophic effects within the cell have 
not been fully resolved. Interaction of NGF with a cell receptor is a 
requisite step in the transmission of neurotrophic signals within the cell 
(for review see M. V. Chao, In: Handbook of Experimental Pharmacology, 
vol. 95/II Peptide Growth Factors and Their Receptors II (Sporn, M. B. and 
Roberts, A. B. eds.), Springer-Verlag, Heidelberg, pp. 135-165 (1990)). A 
major advance in understanding NGF interactions with the cells was the 
identification and cloning of a 75kDa receptor (75kNGF-R) that binds NGF, 
and is present in NGF-responsive cells. The clones of the gene encoding 
75kNG-R have been characterized from several species (M. V. Chao et al., 
Science 232:418-421 (1986); M. J. Radeke et al., Nature 325:593-597 
(1987)). Unfortunately, the structural and biological properties of 
75kNGF-R have provided limited clues about the nature of the NGF signal 
trandsuction pathway inside the cell. 75kNGF-R displays the binding 
properties of a low affinity NGF receptor (Kd.apprxeq.10.sup.-9 M) when 
expressed in exogenous cell lines and analysis of the intracellular domain 
has not revealed putative domains of catalytic action (M. V. Caho, In: 
Handbook of Experimental Pharmacology, Vol. 95/II Peptide Growth Factors 
and Their Receptors II (Sporn, M. B. and Roberts, A. B. eds.), 
Springer-Verlag, Heidelberg, pp. 135-165 (1990)). 
The biological responsiveness to NGF, however, is widely held to depend 
upon interactions with a high affinity binding component implying that 
other receptor or receptor subunits may be involved in NGF responses. The 
search for potential second messengers that might transmit NGF signals in 
PC12 cells has led to recent evidence indicating that activation of 
tyrosine kinases may represent an early response to the presence of NGF 
(Maher 1988). These data implicate tyrosine kinases as candidates in the 
composition of a high affinity receptor. 
The trk proto-oncogene encodes a tyrosine kinase (TK) receptor with a 
tightly regulated neural pattern of expression during murine development 
(D. Martin-Zanca et al., Genes Dev. 4:683-694 (1990); D. Martin-Zanca et 
al., In: The Avian Model in Developmental Biology: From Organism to Genes, 
Editions du CNRS - 1990, pp. 291-302 (1990)). In vivo, transcripts for 
this gene were observed only in neural crest-derived sensory neurons of 
the peripheral nervous system through E17 of mouse development. Several 
lines of evidence have led applicants to investigate the possible 
involvement of trk in pC12 cell NGF-mediated events. 
The need exists in the field to determine whether trk proto-oncogene 
tyrosine kinase receptor is activated via direct interaction with NGF. The 
present invention provides a complex comprising NGF ligand and 
trk-proto-oncogene receptor. The direct binding of NGF to the trk receptor 
leads to tyrosine phosphorylation and tyrosine kinase activity in response 
to NGF exposure in trk expressing cells. Knowledge of the trk 
physiological receptor and cognate NGF complex may allow a detailed study 
of nerve growth and regeneration. Furthermore, the demonstration of 
NGF-trk receptor complexes demonstrates methods for identifying related 
tyrosine kinase receptors providing additional neurotropic-factor pairs. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a complex comprising a 
nerve growth factor (NGF) ligand and trk-proto-oncogee protein receptor 
and methods of utilizing the complex. 
In one embodiment the present invention relates to a complex of NGF and 
trk-proto-oncogene receptor protein wherein said complex is free of 
protein with which it is naturally associated. 
In another embodiment the present invention relates to a complex comprising 
a NGF ligand and trk-proto-oncogene receptor protein wherein one member of 
said complex is bound to a solid support. 
In yet another embodiment the present invention relates to a method of 
detecting the NGF:trk-proto-oncogene receptor protein complex in a sample 
comprising reacting said sample with an antibody that binds specifically 
with either NGF or trk-proto-oncogene receptor protein on the complex. A 
positive immunological reaction is indicative of the presence of the 
complex in the sample. 
In a further embodiment, the present invention relates to a method of 
diagnosing degenerative neuronal diseases in a patient suspected of having 
the disease comprising reacting a biological sample from the patient with 
an antibody that binds with NGF:trk-proto-oncogene receptor protein 
complex. 
In yet another embodiment, the present invention relates to a method of 
diagnosing a tissue undergoing neuronal regeneration in a patient 
comprising reacting a biological sample from the patient with an antibody 
that binds to a NGF:trk-proto-oncogene receptor protein complex. 
A further embodiment of the present invention relates to a method of 
diagnosing a disease state in the patient suspected of having the stated 
disease comprising reacting a biological sample from the patient with an 
antibody that binds to a trk NGF:trk-proto-oncogene receptor protein 
complex. 
In another embodiment, the present invention relates to a method for 
detecting NGF in a sample comprising contacting the sample with 
trk-proto-oncogene receptor protein under conditions such that binding of 
NGF present in the sample to the receptor is effected and detecting the 
presence of bound NGF. 
In a further embodiment the present invention relates to a method for 
detecting trk-proto-oncogene receptor protein in a sample comprising the 
steps of contacting the same with NGF under conditions such that binding 
of said receptor present in the sample to NGF is effected and detecting 
the presence of bound receptor. 
Various other objects and advantages of the present invention will become 
apparent from the drawings and the following description of the invention. 
The entire contents of all publications mentioned herein are incorporated 
by reference.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a complex comprising nerve growth factor 
(NGF) and trk-proto-oncogene protein. The present invention further 
relates to methods of utilizing the complex. 
One embodiment of the present invention relates to a complex formed by the 
interaction of NGF with trk-proto-oncogene protein that is free of protein 
with which it is naturally associated. The trk-proto-oncogene product is a 
140kDa glycoprotein tyrosine kinase and a component of a high affinity NGF 
receptor. 
The present invention relates to detection and quantitation methods that 
may be used in diagnostics and therapeutics in identifying NGF (ligand), 
trk-proto-oncogene protein receptor or the ligand-receotor complex. 
Neurons of the central and peripheral nervous system are dependent on NGF 
for their continued survival. To date, NGF-dependent neurons that have 
been identified are sensory neural crest-derived (trigeminal, superior, 
juglar and dorsal rout ganglia neurons), sympathetic neurons and 
cholinergic neurons of the basal, media septal and diagonal septal band 
nuclei of the brain. This last neuronal type are found to be degenerative 
in Alzheimer's and Huntington's disease. 
The knowledge and understanding of NGF-mediated response as occurring via a 
complex with the trk tyrosine kinase has broad implications for the study 
of nerve survival, regeneration and accurate diagnosis and potential 
therapy for neurodegenerative diseases that affect NGF-dependent neurons. 
Since NGF-dependent neurons respond via the NGF-trk proto-oncogene tyrosine 
kinase complex, the methods described herein provide a means for 
identifying other neuronal types other than those described above which 
may lead to the identification of other neuronal disorders. In this 
regard, applicants have recently identified trk expression (and therefore 
NGF-responsive neurons) in the trigeminal mesencephalic nucleus. These 
neurons mediate many important sensory functions throughout the brain and 
may be affected in as yet unidentified neuronal disorders. 
The methods of the present invention may also aid in the understanding of 
the role of the interaction between NGF and its receptor, the 
trk-proto-oncogene product as a transducer of NGF signals. Considerable 
expertise and information is available from the past study of tyrosine 
kinases in other biological systems (i.e., oncogenesis and cell growth) 
that indicate existing biochemical cascades within the cell that are the 
signal transducing pathways to the nucleus. Thus NGF binding to trk 
initiates a signal cascade inside the cell that is amendable to 
identification, study, and perhaps ultimately, to manipulation, utilizing 
skills and methodologies that are already in existence. 
The present invention further relates to a method of detecting and 
quantitating trk-protooncogene receptor in a biological sample using 
labeled NGF as a probe. Suitable labels include, for example, radiolabels 
such as .sup.125 I, and fluorescein. 
Using standard methodologies well known in the art and described herein, a 
biological sample can be extracted with a non-ionic detergent and 
incubated with labeled NGF in the presence or absence of unlabeled NGF. 
The resulting complex can be separated from the uncomplexed (or unbound) 
labeled material, for example, by immunoprecipitating the complex with a 
specific polyclonal antibody, for example, the 43-4 or 4.7 rabbit anti-trk 
antisera and, in parallel, monoclonal phosphotyrosine antibody, such as 
Ptyr 4G10, for example, that recognizes the trk-proto-oncogene receptor 
protein or the NGF-trk proto oncogene receptor complex. The overall signal 
resulting from the presence of label associated with the resulting complex 
is compared with the signal from a mock sample. The mock sample is 
prepared using purified trk-proto-oncogene receptor protein in a known 
quantity treated the same way as the biological sample. 
Alternatively, the complex may be separated from uncomplexed material by 
precipitating with polyethylene glycol. In both methodologies, the amount 
of label that is immunoprecipitated or precipitated is directly related to 
the amount of complex in the biological sample. 
The present invention also relates to a method for detecting and 
quantitating NGF in a biological sample using labeled trk-proto-oncogene 
receptor as a probe. The method is carried out as a reciprocal binding 
assay following the methodology described above except substituting as 
antibody, one that specifically recognizes NGF or the 
NGF-trk-proton-cogene receptor complex. Antibodies against NGF are well 
known in the art. 
The present invention also relates to further methods of detecting and 
quantitating NGF-trk-proto-oncogene protein receptor complexes in a 
sample. In one aspect, complexes are detected and quantitated using 
antibodies directed against NGF, trk-proto-oncogene receptor protein or 
the NGF-receptor complex. Antibodies can be either polyclonal or 
monoclonal; examples of both are described above and below in the Example 
Section. A sample an be extracted with non-ionic detergent and incubated 
with label NGF or trk-proto-oncogene receptor protein. After incubation, 
the sample is covalently cross-linked with a lipophilic photoaffinity 
cross-linking agent for example, HSAB. Chemical crosslinking agents, such 
as disuccinimidil suberate (DSS) may also be used in this procedure. The 
sample is immunoprecipitated with specific antibody or precipitated with 
polyethylene glycol. Quantitation requires chromatographic separation by, 
for example, gel electrophoresis, followed by autoradiography. 
The present invention also relates to diagnostic methodology using the 
methods described above. The disorders which are diagnosed by the methods 
of the present invention include, for example, neurodegenerative diseases 
that affect NGF-dependent neurons such as Alzheimer's and Huntington's 
diseases. The present diagnostic methods can also be used to measure 
neuronal disorders in tissue derived from neuronal cell types described 
previously, which may lead to diagnostics of as yet unidentified neuronal 
disorders. 
The present invention further relates to methods of detecting other trk 
related receptor and NGF related neurotrophic factor complex using similar 
methods as those utilized above for detecting the trk/NGF complex. The trk 
gene is a member of a structurally related gene family of which at present 
at least three members have been identified (trk, trkb, and trkc). 
Likewise a growing number of neurotrophic factors are emerging on the 
basis of similar structure and function to NGF such as BDNF and NT-3 for 
example. It is very likely that methods used to identify the trk/NGF 
complex will lead to parallel discoveries with the additional trk and 
NGF-related genes. The strategies used to identify, characterize and study 
these trk-related/NGF-related complexes (ie.: trkb/BDNF) will be based on 
the discovery herein described. Any implications at the practical or 
therapeutic levels will apply to these neurotrophic factors. The knowledge 
of trk-related/NGF-related complexes, for example, Trkb/BDNF, will provide 
insight into the survial capacities of a different subset of nerve cells 
to those dependant on NGF. Similar assays and strategies previously 
described to those conceived or devised for detecting the trk/NGF complex 
would apply to the detection of the related complex for example, use of 
phosphotyrosine and trkb antibodies for immunoprecipitating 
trk-related/NGF-related complexes. 
The present invention further relates to therapeutic methodologies and the 
development of detection kits or pharmacological agents that enhance 
NGF-mediated nerve regeneration or survival. This will depend on the use 
of trk antibodies and phosphotyrosine antibodies to assay for the quality 
of the procedure. Most obvious in the area of potential therapeutic value 
is the development of drugs that either enhance or inhibit tyrosine 
phosphorylation. Since trk mediates signalling via phosphorylation on 
tyrosine of messenger molecules, its signalling could be altered as 
required in cells. These studies would initially be developed and assessed 
in tissues or cell culture systems prior to any potential application. 
Drugs would be added to trk-expressing tissue culture cells together with 
or in the absence of NGF and the state of trk activation, as measured by 
tyrosine phosphorylation, could be assessed. Progress in developing these 
drugs would be most effectively monitored with antibodies that recognize 
trk and/or phosphorylated tryosine. Thus development of any useful 
therapies in this area will depend on the ability to identify the 
activation state of trk and/or any of its downstream substrates. Next, 
animal models (rat or mouse) will be used in which specific nerve 
connections are disrupted, the promising pharmaceuticals administered, and 
finally analysis of the sacrificed animals will be performed to assess the 
regeneration of nerves using trk/ngf or trk-related, NGF-related anitbody 
assays as described. 
The present invention also relates to other therapeutic methods for 
designing pharmaceuticals that enhance the stimulation of degenerative 
nerves in diseases such as Alzheimer's and Huntington's. 
trk and low affinity NGF receptor 75/cNGF-R are required together for high 
affinity response to NGF. Methods could be devised that would enhance 
detection of NGF using the high affinity complex. Knowledge of the 
existence for a trk/NGF complex could lead to the development of modified 
NGF molecules that hyperstimulate trk activation. These NGF derivatives 
might be of importance in the stimulation of degenerating nerves stemming 
from diseases, for example, Alzheimer's and Huntington's, or from injury. 
Many substrates of tryosine kinases have been identified. Identification of 
trk-specific substrates could lead to discovery of an intermediate 
molecule in the NGF pathway that can be manipulated pharmalogically to 
enhance or inhibit NGF-mediated signals. 
EXAMPLES 
Example 1 
Tyrosine phosphorylation of p140.sup.prototrk in response to NGF 
The stimulation of p140.sup.prototrk tryosine phosphorylation in response 
to NGF addition to PC12 cells is rapid, specific and occurs in the 
presence of physiological amounts of NGF. 
This previous study utilized immunoblotting analysis with phosphotyrosine 
(P-tyr) antibodies to detect tyrosine phosporylation of p140.sup.prototrk. 
To determine if enhancement of serine or threonine phosphorylation of 
p140.sup.prototrk are induced by NGF, and to compare the relative amounts 
of tyrosine, serine, and threonine phosphorylation, PC12 cells were 
labeled with .sup.32 P-orthophosphate prior to NGF treatment and 
immunoprecipitation with antibodies to p140.sup.prototrk. 
p140.sup.prototrk was phosphorylated predominately on serine residues in 
immunoprecipitates from untreated cells and cells treated with 50 ng/ml 
NGF for 5 min. The presence of NGF, however stimulated thr tyrosine 
phosphorylation of p140.sup.prototrk 20-fold, although this represented 
less than 5% of the newly incorporated phosphate residues. In contrast, 
p140.sup.prototrk was labeled predominantly on tyrosine in immune complex 
kinase assays from NGF-treated PC12 cells or in .sup.32 P-labeled NIH-3T3 
cells transfected with the rat trk gene (rtrk-3T3) (FIG. 1a). The tyrosine 
phosphorylation of p140.sup.prototrk expressed in NIH-3T3 cells was 
constitutive, apparently due to autocrine stimulation by NGF produced by 
these cells. treatment of rtrk-3T3 cells with suramin, a polyanionic 
compound which inhibits and reverses the binding of some growth factors to 
their receptors (M. Hosang et al., J. Cell. Biochem. 29:265-273 (1985)), 
markedly reduced the tyrosine phosphorylation of p140.sup.prototrk in vivo 
and in immune complex kinase assays (FIG. 1b). when NGF was added to the 
suramin treated cells for 10 min, tyrosine phosphorylation of 
p140.sup.prototrk observed in vivo and invitro was stimulated at least 
10-fold (FIG. 1b). 
trk tyrosine phosphorylation occured within one minute of NGF treatment 
cells, reached maximum levels after five minutes, and declined thereafter 
(FIG. 2a). Residual phosphorylation was detected after two days of 
treatment with NGF when the cell population was fully differentiated. trk 
tyrosine phosphorylation was also specific to NGF. Other peptide growth 
factors that elicit tyrosine phosphorylation in PC12 cells were tested in 
our assay (V. Hamburger and R. Levi-Montalcini, J. Exp. Zool. 111:457-502 
(1949) I. B. Black et al., Growth Factors and Development, Current Topics 
in Developmental Biology, vol. 24: (ed. Nilsen-Hamilton, M.) 161-192 
1990)). EGF, basic FGF, insulin, and the phorbol ester PMA failed to 
induce trk was seen in cells treated with both basic FGF and NGF (FIG. 
2b). It has been previously shown that these agents produce similar 
patterns of early responses in PC12 cells, including transcriptional 
activation of c-fos and cmyc (R. Levi-Montalcini and B. Booker, Proc. 
Natl. Acad. Sic. USA 46:384-391 (1960)). However, of these factors, only 
NGF and basic FGF stimulate neurite outgrowth. 
To determine the minimal concentration of NGF capable of eliciting trk 
tyrosine phosphorylation, a dose response experiment was performed. 
Tyrosine phosphorylation was half maximal at 0.1 ng/ml NGF (50 pM) (FIG. 
2c) indicating the trk phosphorylation occurs at physiologically relevant 
concentrations of NGF (S. Cohen, Proc. Natl. Acad. Sci. USA 46:302-311 
(1960)). 
Example 2 
Expression of trk gene in embryonic sensory neural crest-derived neurons 
The trk gene is expressed in embryonic sensor neural crest-derived neurons 
including dorsal root ganglia (DRG) (FIG. 3 and Martin-Zanca et al. 1990). 
This expression is confined to neurons (note that the darkly staining 
glial cells are devoid of silver grains) and maintained in the adult. To 
determine wther the trk protein in embryonic neurons was responsive to 
NGF, DRG from E13.5 and E14.5 mouse embryos were explanted, maintained in 
50 ng/ml NGF on ice.gtoreq.10 mm., lysed, and subjected to trk antibody 
precipitation and anti-ptyr immunoblotting analysis. As shown in FIG. 3A, 
phosphorylation of the p145.sup.prototrk was detectable in 14.5 day DRG 
but not in two independent preparations of 13.5 day DRG. Tyrosine 
phosphorylated trk protein was not detectable in the absence of 
exogenously administered NGF. 
Dissection of DRG provides primarily the cell bodies and eliminates the 
axons, therefore the significance of these data with regard to timing and 
degree of p145.sup.prototrk activation should be interpreted with caution. 
The results in 14.5 DRG, however, determine that freshly dissected 
embryonic DRG neurons contain trk protein which is phosphorylated in 
response to NGF. 
Example 3 
NGF stimulates p140.sup.prototrk tyrosine phosphorylation in several 
trk-expressing cell types 
To determine whether phosphorylation of p140.sup.prototrk in response to 
NGF was unique to rat PC12 cells or occurred in other NGF responsive cell 
lines, the state of the trk protein in additional neuroblastoma cell lines 
from different species was assayed. It was observed that p140.sup.prototrk 
tyrosine phosphorylation was also enhanced by NGF in the human 
neuroblastoma cell line LA-N-5 and in the murine cell line SY5Y (FIG. 4b). 
LA-N-5 and SY5Y cells express 4-fold less trk mRNA than PC12 cells, 
accounting for the lower amounts of tyrosine phosphorylated trk observed 
in these cell lines compared to PC12 cells. 
Derivatives of the PC12 cell line have been generated by mutagenesis that 
have lost high affinity response to NGF (Bothwell et al. 1981). One such 
line, NR18, lacks 75kNGF-R. Introduction of 75kNGF-R into these cells 
resulted in the reconstitution of biphasic scatchard profile and at least 
partial function reconstitution (Hempstead et al. J. Biol. Chem. 
265:9595-9598 (1990)). NR18 cells express the trk proto-oncogene at 
greatly reduced levels (see Hempstead et al. 1991). 
The Applicants next analyzed the phosphorylation state of the trk receptor 
on the NR18 cell line that has greatly reduced responsiveness to NGF 
(Bothwell et al., Cell 21:857-866 (1980)). Consistent with RNA expression 
data (see Hempstead et al. 1991) no phosphorylation of p140.sup.prototrk 
in response to NGF was observed in these cells (FIG. 4b). Thus, in NR18 
cells, the tyrosine phosphorylation of p140.sup.prototrk correlates with 
the reduced ability of NGF to elicit a biological response. 
Example 4 
Trk receptor directly binds to NGF 
The above results, demonstrating the rapid phosphorylation of 
p140.sup.prototrk in several trk-expressing cell lines treated with NGF, 
suggested that the trk receptor might directly bind NGF. To determine if 
NGF was capable of binding to p140.sup.prototrk, several cell lines were 
analyzed for the ability of trk-specific antisera to precipitate 
receptor-ligand complexes in affinity crosslinking experiments (FIG. 5). 
The cell lines assayed were rat PC12, human LA-N-5, mouse SY5Y, mouse 
NIH-3T3, mounse rtrk-3T3, and human AB75 cells. NGF induces the tyrosine 
phosphorylation of p140.sup.prototrk in PC12, LA-N-5, SY5Y, and rtrk-3T3, 
but not in AB75-melanoma or NIH-3T3 cells which express no detectable trk 
messenger RNA. .sup.125 I-labeled NGF was crosslinked to cells using the 
lipophilic photoaffinity agent HSAB. Previous studies with this 
crosslinking agent have shown that in PC12 cells and sympathetic neurons, 
two NGF containing species of 100 kDa and 150-160 kDa are observed (J. 
Massague et al., J. Biol. Chem. 256:9419-9424 (1981); Hempstead, et al. 
1990; S. O. Meaking and E. M. Shooter, Neuron 6:153-163 (1991)). The 100 
kDa species represents .sup.125 I-NGF bound to 75kNGF-R (M. Hosang and E. 
M. Shooter, J. Biol. Chem. 260:655-662 (1985)). Following crosslinking, 
the cells were washed to remove unbound .sup.125 I-NGF, lysed in 
detergent, and the lysates incubated with antibodies (FIG. 4). It was 
observed that the 160 kDa species in anti-NGF or anti-p140.sup.prototrk 
immunoprecipitates from PC12 and rtrk-3T3 cells, and not in A875 or IH-3T3 
cells. The immunoprecipitation of the 160 kDa species was blocked by 
addition of a trk-derived peptide used to generate the antibody, and was 
not seen if excess unlabeled NGF was added to the .sup.125 I-NGF treated 
cells prior to crosslinking. A 160 kDa crosslinked product was also 
observed in LA-N-5 and SY5Y cells. The crosslinked 100 kDa species were 
present in PC12 and A875 cells and not in the 3T3 cell lines, reflecting 
the absence of expression of the 75kNGR-R in NIH-3T3 cells. The above 
experiments establish that NGF binds to p140.sup.prototrk and that this 
binding is seen only in cell lines which show p140.sup.prototrk tyrosine 
phosphorylation in response to NGF. 
Of equal importance to the demonstration of binding, it is essential to 
determine whether the affinity of binding reflects physiologically 
relevant conditions. Scatchard plot analysis was carried out to determine 
that affinity of NGF for p140.sup.prototrk expressed in NIH-3T3 cells. 
Crude membranes were prepared from cells and assays by binding to .sup.125 
I-NGF. Membranes obtained from rtrk-3T3 cells displayed a linear Scatchard 
plot with a Kd of approximately 10.sup.-9 M (FIG. 6). By this analysis, 
the number of receptors was approximately 200,000-500,000/cell. 
Example 5 
Expression of trk or trk-related messenger RNA in several cell types 
The trk gene is a member of a gene family of TK receptors that includes the 
related gene trkb. To determine if trk is transcribed in PC12 cells, the 
PG,29 expression of trk transcripts was assayed by Northern transfer 
analysis with a full-length trk cDNA probe (R. Klein et al., Development 
4:845-850 (1990). PC12 cells contained trk transcripts (FIG. 7). The level 
of trk transcripts was not affected by the addition of NGF. To determine 
whether additional trk-related genes were transcribed in PC12 cells, mRNA 
was hybridized at low stringency with the highly conserved trk Tk domain. 
trk transcripts have been found in LA-N-5 cells, Sy5y cells and DRG from 
13.5 day or 14.5 day embryonic mice. trkb expressing cell lines, as 
determined by mRNA analysis will help determine the next steps in 
interactions with trkb ligand (BDNF). 
While the foregoing invention has been described in some detail for 
purposes of clarity and understanding, it will be appreciated by one 
skilled in the art from a reading of this disclosure that various changes 
in form and detail can be made without departing from the true scope of 
the invention.