Functional ligands for the axonal cell rcognition molecule contactin

The extracellular domain of RPTP.beta. is the ligand for contactin and its binding results in neurite growth and differentiation. The invention encompasses compounds that mimic, enhance, or suppress the effects of the ligand for contactin, assays for the identification of such compounds, and the use of such compounds to treat neurologic diseases including those characterized by insufficient, aberrant, or excessive neurite growth, differentiation or survival.

1. INTRODUCTION 
The present invention relates to the identification and use of compounds 
that bind to contactin, a surface molecule of neural cells, and stimulate 
the growth, differentiation or survival of targeted neural cells. The 
carbonic anhydrase (CAH) domain of the receptor-type tyrosine phosphatase 
RPTP.beta. is identified as the ligand for contactin. The binding of the 
CAH domain of RPTP.beta. to the contactin on neural cells results in 
neurite growth, differentiation and survival. The invention relates to 
compounds that mimic, enhance, or suppress the effects of the RPTB.beta. 
ligand for contactin, including those molecules which act downstream in 
the signal transduction pathway that results from the binding of the 
ligand to contactin. The invention further relates to assays and methods 
for the identification of such compounds. In addition, the invention also 
relates to the use of such compounds to treat neurologic diseases 
including those characterized by insufficient, aberrant, or excessive 
neurite growth, differentiation or survival. 
2. BACKGROUND OF THE INVENTION 
The ability of cells to respond to signals from their microenvironment is a 
fundamental feature of development. In the developing nervous system, 
neurons migrate and extend axons to establish their intricate network of 
synaptic connections (Goodman and Shatz, 1993, Cell/Neuron (Suppl.), 
72/10:77-98). During migration and axonal pathfinding, cells are guided by 
both attractive and repulsive signals (Hynes and Lander, 1992, Cell, 
68:303-322; Keynes and Cook, 1992, Lurr. Opin. Neurobiol., 2:55-59). The 
ability of the neuron to respond to these signals requires cell surface 
molecules that are able to receive the signal and to transmit it to the 
cell interior resulting in specific biological responses. 
It is well established that protein tyrosine phosphorylation is responsible 
for the regulation of many cellular responses to external stimuli crucial 
for cell growth, proliferation and differentiation (Schlessinger and 
Ullrich, 1992, Neuron, 9:383-391). Tyrosine phosphorylation has been 
implicated in several developmental processes in the nervous system. For 
example, receptor tyrosine kinases were shown to effect neuronal survival 
(Chao, 1992, Neuron, 9:583-593), and cell fate determination (Zipursky and 
Rubin, 1994, Annu. Rev. Neurosci., 17:373-397). Non-receptor tyrosine 
kinases have been shown to be downstream elements in signaling via cell 
recognition molecules that play a role in cell guidance and migration 
(Ignelzi et al., 1994, Neuron, 12:873-884; Umemori et al., 1994, Nature, 
367-572-586). 
The transient nature of signaling by phosphorylation requires specific 
phosphatases for control and regulation (Hunter, 1995, Cell, 80:225-236). 
Indeed, many protein tyrosine phosphatases have been shown to be expressed 
in specific regions of the developing brain, including the olfactory 
neuroepithelium (Walton et al., 1993, Neuron, 11:387-400), the cortex 
(Sahin et al., 1995, J. Comp. Neurol., 351:617-631), and in retinal Muller 
glia (Shock et al., 1995, Mol. Brain Res., 28:110-116). Furthermore, 
expression of several tyrosine phosphatases, such as PTP.alpha. (den 
Hertog et al., 1993, EMBO J., 12:3789-3798), PC12-PTP1 (Sharama and 
Lombroso, 1995, J. Biol. Chem., 270:49-53) and several forms of LAR (Zhang 
and Longo, 1995, J. Cell. Biol., 128:415-431) have been found to be 
regulated during neural differentiation of P19 or PC12 cells. 
Receptor-type tyrosine phosphatases (RPTPs) have been subdivided into 
several groups based on structural characteristics of their extracellular 
domains (Charbonneau and Tonks, 1992, Annu. Rev. Cell Biol., 8:463-493; 
Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). RPTP.beta./.zeta. 
and RPTP.gamma. are members of a distinct group of phosphatases, 
characterized by the presence of a carbonic anhydrase-like domains (CAH), 
fibronectin type III repeats (FNIII), and a long cysteine free region 
(spacer domain) in their extracellular domain (Barnea et al., 1993, Mol. 
Cell. Biol., 13:1497-1506; Krueger et al., 1992, Proc. Natl. Acad. Sci. 
USA, 89:7417-7421; Levy et al., 1993, J. Biol. Chem., 268:10573-10581). 
The expression of RPTP.beta. is restricted to the central and peripheral 
nervous system, while RPTP.gamma. is expressed both in the developing 
nervous system, as well as, in a variety of other tissues in adult rat 
(Canoll et al., 1993, Dev. Brain Res., 75:293-298; Barnea et al., 1993, 
Mol. Cell. Biol., 13:1497-1506). RPTP.beta. exists in three forms, one 
secreted form and two membrane bound forms, that differ by the absence of 
860 residues from the spacer domain (Levy et al., 1993, J. Biol. Chem., 
268:1053-10582; Maurel et al., 1994, Proc. Natl. Acad. Sci. USA, 
91:2512-2516). The secreted form has been identified as a chondroitin 
sulfate proteoglycan from rat brain called phosphocan (3F8 proteoglycan) 
(Barnea et al., 1994, Cell, 76:205; Maurel et al., 1994, Proc. Natl. Acad. 
Sci. USA, 91:2512-2516; Shitara et al., 1994, J. Biol. Chem. 
269:20189-20193). The transmembrane form has also been shown to be 
expressed in a form of a chondroitin sulfate proteoglycan (Barnea et al., 
1994, J. Biol. Chem., 269:14349-14352). Purified phosphocan can interact 
in vitro with the extracellular matrix protein tenascin, and with the 
adhesion molecules, N-CAM and Ng-CAM (Barnea et al., 1994, J. Biol. Chem., 
269:14349-14352; Grumet et al., 1993, J. Cell. Biol., 120:815-824; Grumet 
et al., 1994, J. Biol. Chem., 269:12142-12146; Milev et al., 1994, J. 
Cell. Biol., 127:2512-2516). The experiments of the examples described 
infra were conducted to identify functional ligands for RPTP.beta.. 
3. SUMMARY OF THE INVENTION 
The invention relates to the identification and use of compounds to treat 
neurologic diseases including those characterized by insufficient, 
aberrant, or excessive neurite growth, differentiation or survival. The 
invention is based, in part, on the discovery that the CAH domain of 
RPTP.beta. (RPTP.beta.-CAH) is the ligand for contactin and that its 
binding results in neurite growth, differentiation and survival. More 
specifically, the invention relates to the identification and use of 
compounds that mimic, enhance or suppress the effects of RPTP.beta.-CAH on 
neurite growth, differentiation and survival. The invention further 
relates to assays for identifying such compounds. 
In the examples described infra, it is shown that receptor phosphatase 
RPTP.beta., specifically interacts with two ligands, one on the surface of 
glial cells, and the other on the surface of neuronal cells. Using 
expression cloning in COS7 cells and bioaffinity purification, the 
neuronal ligand was identified to be the rat homologue of the cell 
recognition molecule contactin (F11/F3). Using combinations of soluble and 
membrane bound forms of RPTP.beta. and contactin it is demonstrated that 
the reciprocal interaction between the two molecules is mediated by the 
CAH domain of the phosphatase. Moreover, it is found that when used as a 
substrate, the CAH domain of RPTP.beta. induced neurite growth, 
differentiation and survival of primary neurons and IMR-32 neuroblastoma 
cells. Finally, using antibody perturbation experiments, the contactin 
ligand was found to be a neuronal receptor for the CAH domain of 
RPTP.beta.. The data indicate that the interactions between contactin, a 
cell recognition molecule, and RPTB.beta., a transmembrane protein 
tyrosine phosphatase, plays an important role in neuronal development and 
differentiation. 
3.1. DEFINITIONS 
As used herein, the following terms and abbreviations shall have the 
meanings indicated below: 
______________________________________ 
base pair(s) bp 
carbonic anhydrase CAH 
carbonic anhydrase domain of RPTP.beta. 
RPTP.beta. 
complementary DNA cDNA 
counts per minute cpm 
deoxyribonucleic acid DNA 
fibronectin type III FNIII 
glycosyl-phosphatedylinositol 
GPI 
kilobase pairs kb 
kilodation kDa 
micrograms .mu.g 
micrometer .mu.m 
nanograms ng 
nanometer nm 
nucleotide nt 
phospholipdase C PI-PLC 
polyacrylamide gel electrophoresis 
PAGE 
polymerase chain reaction PCR 
receptor type tyrosine phosphstase beta 
RPTP.beta. 
ribonucleic acid RNA 
sodium dodecyl sulfate SDS 
units u 
______________________________________

5. DETAILED DESCRIPTION OF THE INVENTION 
A large group of protein tyrosine phosphatases have structural 
characteristics suggesting that they function as cell surface receptors. 
Receptor type tyrosine phosphatase .beta. (RPTP.beta.) is expressed in the 
developing nervous system and it contains a carbonic anhydrase (CAH) 
domain as well as a fibronectin type III (FNIII) repeat in its 
extracellular domain. A variety of experiments were conducted to search 
for ligands of RPTP.beta.. These experiments led to the surprising 
recognition that the CAH domain of RPTP.beta. is a functional ligand for 
contactin, a GPI-membrane anchored neuronal cell recognition molecule that 
functions as a receptor on neurons. The CAH domain of RPTP.beta. 
(RPTP.beta.-CAH) induces cell adhesion and neurite growth of primary 
tectal neurons, and differentiation of neuroblastoma cells. The assays of 
the invention identify compounds that mimic, enhance, or inhibit the 
contactin mediated effects of RPTP.beta.-CAH on neural cells including, 
but not limited to, agonists and antagonists of RPTP.beta.-CAH. 
Therapeutic uses of compounds so identified are also provided. The 
invention is described in detail in the following subsections and examples 
for purposes of clarity and not by way of limitation. 
5.1. BIOLOGY OF THE INTERACTION BETWEEN CONTACTIN AND THE CAH DOMAIN OF 
RPTP.beta. 
During development of the nervous systems, neurons are guided by secreted 
and cell bound molecules that provide both negative and positive cues. The 
experiments described in the examples of Sections 6.1 and 6.2 show that 
RPTP.beta., a receptor type protein tyrosine phosphatase, may provide such 
a signal by interacting with the axonal recognition molecule contactin. 
RPTP.beta. is a developmentally regulated protein that exists in three 
forms, one secreted and two membrane bound. The extracellular region of 
RPTP.beta. has a multidomain structure consisting of a CAH-like domain, a 
single FNIII repeat, and a long cysteine free spacer region. The complex 
structural nature of its extracellular region may result in a 
multifunctional protein that is able to interact with different proteins. 
As documented by the data shown herein, the CAH and the FNIII domains bind 
to at least two potential ligands present on neurons or glial cells. 
Functional expression cloning in COS7 cells and affinity purification with 
a specific affinity matrix followed by microsequencing enabled unequivocal 
identification of the cell recognition molecule contactin (F3/F11) as a 
neuronal ligand of RPTP.beta.. The interaction between contactin and 
RPTP.beta. is mediated via the CAH domain of the phosphatase, while the 
FNIII domain appears to bind to another molecule expressed on the surface 
of glial cells. It was previously shown that the secreted proteoglycan 
form of RPTP.beta. interacts with tenascin, N-CAM and Ng-CAM (Grumet et 
al., 1994, J. Biol. Chem., 269:12142-12146; Barnea et al., 1994, J. Biol. 
Chem., 269:14349-14352; Grumet et al., 1993, J. Cell. Biol., 120:815-724; 
Milev et al., 1994, J. Cell. Biol., 127:1703-1715). Since N-CAM and Ng-CAM 
do not bind directly to the CAH or the FNIII domain of RPTP.beta., they 
may interact with the large spacer domain of the phosphatase. 
Alternatively, they could interact with RPTP.beta. through a third 
component. Contactin may fulfill this function since it has been shown to 
interact with Ng-CAM, Nr-CAM, and the matrix proteins tenascin and 
restriction (Brummendorf et al., 1993, Neuron, 10:711-727; Morales et al., 
1993, Neuron, 11:1113-1122; Zisch et al., 1992, J. Cell. Biol., 
119:203-213). The various subdomains of the extracellular region of 
RPTP.beta. are able to interact with several distinct proteins that are 
expressed on diverse cell types in the central nervous system. 
In contrast to other cell recognition molecules that are widely expressed 
in the nervous system, members of the contactin subgroup appear to be 
expressed in a restricted manner on specific axons during development 
(Dodd et al., 1988, Neuron, 1:105-116; Faivre-Sarrailh et al., 1992, J. 
Neurosci., 12:257-267). The spatial and temporal expression pattern of 
these proteins indicates they play an important role during development of 
the nervous system. Contactin was found to be exclusively expressed on 
neurons during development in fiber-rich areas of the retina, tectum, 
spinal cord and cerebellum (Ranscht, 1988, J. Cell. Biol, 107:1561-1573). 
It was found to be localized in the postnatal and adult mouse cerebellum 
in axonal extensions of the granule cells in the parallel layer 
(Faivre-Sarrailh et al., 1992, J. Neurosci., 12:257-267). This pattern of 
expression is overlapping with the expression pattern of RPTP.beta.in the 
rat. RPTP.beta.was shown to be expressed in fiber-rich regions such as the 
parallel fibers of the cerebellum and the spinal cord (Canoll et al., 
1993, Dev. Brain Res., 75:293-298; Milev et al., 1994, J. Cell. Biol., 
127:1703-1715). RPTP.beta. is also expressed on glial and radial glial 
cells, and its secreted form is produced by astrocytes. Therefore, both 
forms of RPTP.beta. may modulate neuronal function via interactions with 
contactin. 
The contactin subgroup of glycoproteins all share structural similarity in 
that they are, glycosyl-phosphatidylinositol (GPI)-anchored proteins. They 
also exist in soluble forms generated as a result of membrane release or 
by expression of alternative spliced forms (Brummendorf and Rathjen, 1993, 
J. Neurochem., 61:1207-1219). Differential expression of the 
membrane-bound and soluble forms of contactin was found in the 
hypothalamus-hypophyseal system (Rougon et al., 1994, Braz. J. Med. Biol. 
Res., 2:409-414). RPTP.beta. also exists in either membrane bound or 
secreted forms that are developmentally regulated. Therefore, both 
RPTP.beta.and contactin may act as either a ligand or a receptor for each 
other. Hence, the classical notion of ligand receptor interaction does not 
fully explain this system since both components might switch roles at 
different stages of development. For example, the soluble form of 
RPTP.beta. produced by glial cells may act as a ligand for the membrane 
bound form of contactin expressed on the surface of neuronal cells. 
Conversely, the soluble form of contactin may act as ligand for the 
membrane bound form of RPTP.beta. expressed on the surface of glial cells. 
Moreover, interaction between the membrane bound forms of contactin 
expressed on the surface of neurons with the membrane form of RPTP.beta. 
expressed on the surface of glial cells may lead to bidirectional signals 
between these two cell types. Such complex interactions between the 
various forms of RPTP.beta. and contactin may generate developmentally 
regulated unidirectional and bidirectional signals. 
While not being limited to any theory or explanation of how the invention 
works, the following is hypothesized to explain how the CAH domain of 
RPTB.beta. binds to contactin. Carbonic anhydrases are highly efficient 
enzymes that catalyze the hydration of CO.sub.2. Yet, the CAH domain of 
PTPases were not thought to be endowed with enzymatic activity due to 
substitution of two of the three key histidine residues that are essential 
for enzymatic activity (Barnea et al., 1993, Mol. Cell. Biol., 
13:1497-1506). In contradistinction, the highly packed hydrophobic core as 
well as the hydrophobic residues that are exposed on the surface of 
carbonic anhydrase structure and which are conserved in the CAH domains of 
RPTP.gamma. and .beta. may be involved in protein-protein interaction and 
thus function as a ligand binding domain (Barnea et al., 1993, Mol. Cell. 
Biol., 13:1497-1506). It is of note that Vaccinia virus contains a 
transmembrane protein with a CAH-like domain in its extracellular domain, 
which was thought to be involved in binding of the virion to host proteins 
(Maa et al., 1990, J. Biol. Chem., 265: 1669-1577). Therefore, in theory 
but not by way of limitation, compounds exhibiting effects which mimic, 
enhance, or inhibit the contactin mediated effects of RPTP.beta.-CAH on 
neuronal cells may do so via other members of the contactin family of 
glycoproteins, and may do so even if lacking in CAH activity. 
A number of models may be proposed for how contactin, a GPI-linked protein 
that is inserted into the outer leaflet of the plasma membrane, transmits 
a signal into the cells to promote neurite outgrowth. In theory, and not 
by way of limitation, one possibility is that contactin is able to 
interact with a transmembrane signaling component. The p180 protein that 
was coprecipitated with contactin is a candidate for such a signaling 
protein (FIG. 6C). p180 may be membrane-associated since it may not be 
released by phospholipase C treatment. Another potential signal transducer 
may be L1/Ng-CAM or a related molecule. This transmembrane CAM was shown 
to interact with contactin (Brummendorf et al., 1993, Neuron, 10:711-727), 
and to initiate second messenger cascade via its cytoplasmic domain 
(Doherty and Walsh, 1994, Curr. Opin. Neurobiol., 4:49-55). The best 
characterized GPI linked signaling protein is the ciliary neurotrophic 
factor receptor (CNTF receptor). Following ligand binding, the CNTFR 
interacts with the signal transducer gp130. The gp130 protein that is 
shared by several lymphokines and cytokines such as IL-6, LIF and 
Oncostatin, undergoes dimerization followed by recruitment of the 
cytoplasmic Jak protein tyrosine kinases. Stimulation of the Jak kinases 
leads to activation of both the Ras/MAP kinase and the Stat signaling 
pathways that relay signals from the cell surface to the nucleus. A 
contactin associated protein such as p180 may have a function similar to 
the function of gp130. 
As demonstrated by the examples infra, the binding of the CAH domain of 
RPTP.beta. to contactin leads to cell adhesion and neurite outgrowth. It 
seems unlikely that the induction of neurite growth is a default response 
resulting from cell adhesion per se. Neurons were found to adhere to 
extracellular matrix proteins such as tenascin and restriction in short 
term binding assays, but these substrates did not promote further neurite 
extension (Schachner et al., 1994, Perspect. Dev. Neurobiol., 1:33-41). It 
was recently reported that the FNIII domain of contactin is responsible 
for adhesion, while the neurite promoting activity was attributed to the 
Ig domains (Durbec et al., 1994, Eur. J. Neuro., 6:461-472). Another study 
demonstrated that contactin can mediate the repulsion of neurons by 
restriction (Pesheva et al., 1993, Neuron, 10:69-82). Again, this effect 
was proposed to occur in a stepwise manner, first an adhesion step that 
was followed by a signal that was transduced to the cells leading to 
retraction. Therefore, in light of the results presented herein, it may be 
that in response to different stimuli, the same molecule can transmit 
opposite signals depending on the context or milieu. Whatever the 
mechanism, the results presented here demonstrate that a receptor type 
tyrosine phosphatase serves as a functional ligand for a GPI-anchored cell 
adhesion molecule. 
Contactin may also serve as a functional ligand for RPTP.beta.. Modulation 
of phosphatase activity by neuronal contactin may result in signaling to 
glial cells. If this does occur, this kind of bidirectional flow of 
information should allow the interacting cells to respond quickly to local 
environmental changes during development. Two other receptor type tyrosine 
phosphatases RPTP.mu. and RPTP.kappa. were shown to mediate cell-cell 
interaction in a hemophilic manner (Brady-Kalany et al., 1993, J. Cell. 
Biol., 122:961-972; Gebbink et al., 1993, J. Biol. Chem., 268:16101-16104; 
Sap et al., 1994, Mol. Cell. Biol., 14:1-9). However, changes in catalytic 
activity as a result of these interactions could not be detected. These 
phosphatases are joining a growing family of proteins that are involved in 
cellular recognition that contain intrinsic enzymatic activities, 
including kinases (Dtrk; Pulido et al., 1992), EMBO J., 11:391-404, .beta. 
subunit of Na.sup.+, K.sup.+ -ATPase (AMOG; Gloor et al., 1990, J. Cell. 
Biol., 109:755-788), and .beta. subunit of prolyl 4-hydroxylase (cognin; 
Rao and Hausman, 1993, Proc. Natl. Acad. Sci. USA, 90:2950-2954). 
In summary, the experiments and data described herein demonstrate that 
RPTP.beta. is a functional ligand for the GPI-anchored cell recognition 
molecule contactin. The interactions between these two proteins is 
mediated by the CAH domain of the phosphatase. In addition, the FNIII of 
RPTP.beta. repeat is required for interaction with glia cells, 
demonstrating that the multidomain structure of RPTP.beta. enables 
interactions with different proteins, and indicates that other potential 
ligands may modulate these interactions. 
5.2. SCREENING METHODS AND ASSAYS 
The methods and assays of the invention can be used to identify compounds 
that enhance, mimic, or inhibit the contactin mediated effects of 
RPTP.beta.-CAH on neuronal cells. Such compounds include those which act 
directly at the site of interaction between the CAH domain of RPTP.beta. 
and contactin, and those which act downstream in the intracellular signal 
transduction pathway which stems from that interaction. In addition, such 
compounds include peptides and polypeptides as well as small organic and 
inorganic molecules. 
The methods and assays of the invention involve exposing contactin 
expressing neural cells to RPTP.beta.-CAH in the presence and absence of a 
test compound. It is shown that the exposure of such cells to 
RPTP.beta.-CAH alone will induce contactin mediated cell adhesion, 
outgrowth, differentiation, survival and neurite extension. Comparison of 
the results obtained when the cells are exposed to RPTP.beta.-CAH alone 
with the results obtained when exposed to RPTP.beta.-CAH and the test 
compound reveals those test compounds which enhance, mimic, or inhibit the 
contactin mediated neural survival, differentiation and growth. Enhancer 
compounds will cause the effects of RPTP.beta.-CAH to increase while 
inhibitor compounds will cause the effects to decrease. Compounds which 
mimic RPTP.beta.-CAH will cause the same effects both in the presence and 
absence of RPTP.beta.-CAH. 
Covalent cross linking will reveal those compounds which are acting at the 
site of interaction between RPTP.beta.-CAH and contactin. If such 
molecules are proteins, they can be isolated and further characterized by 
expression cloning, affinity purification, and microsequencing. 
Treatment of the neural cells with PI-PLC or exposing them to antibodies 
against contactin will prevent contactin mediated effects. Therefore, 
those compounds which are acting downstream of the contactin step of the 
signal transduction pathway are identified by exerting their effects even 
in the presence of antibodies against contactin or where the cells were 
subjected to prior treatment with PI-PLC. 
A specific embodiment of the methods and assays of the invention is 
provided below to further illustrate the invention. The scope of the 
invention is not, however, meant to be limited to the specific details of 
the embodiment. The materials and methods of this embodiment are set forth 
more fully in the examples infra. 
To analyze the effects of a test compound in accordance with the invention, 
chick fetal cells, which express contactin on their cell surface are 
plated on dishes coated with the test compound alone, RPTP.beta.-CAH and 
the test compound or RPTP.beta.-CAH alone, or Ng-CAM as two controls. 
Those plated on NG-CAM should show no attachment or extension. Those 
plated on RPTP.beta.-CAH will show the attachment and neurite extension as 
in the examples infra. If those plated on RPTP.beta.-CAH and the test 
compound, show enhanced or decreased growth and extension as compared with 
those on RPTP.beta.-CAH alone, then the test compound enhances or inhibits 
the contactin mediated effects of RPTP.beta.-CAH. If those plated on the 
test compound alone show similar growth to those plated with 
RPTP.beta.-CAH alone, then the test compound mimics the contactin mediated 
effects of RPTP.beta.-CAH. A similar comparison can be made using the 
human neuroblastoma cell line IMR-32. 
5.3. PEPTIDES THAT MIMIC, ENHANCE, OR INHIBIT THE CONTACTIN MEDIATED 
EFFECTS OF RPTP.beta.-CAH ON NEURONAL CELLS 
The following peptides corresponding to the CAH domain of RPTP.beta. and 
homologues and derivatives thereof may be used in accordance with the 
invention to mimic, enhance, or inhibit the contactin mediated effects of 
RPTP.beta.-CAH on neuronal cells. As used herein, the word modulate shall 
have its usual meaning, but shall also encompass the meanings of the words 
enhance, inhibit and mimic. The peptides of the invention correspond to 
amino acid residues 31 to 300 of RPTP.beta. (RPTP.beta.-CAH) and have 
amino acid sequence (reading from amino to carboxy terminus): 
##STR1## 
Wherein: "n" is an integer from 0 to 500; 
"X" may represent an amino group, a hydrophobic group, including but not 
limited to carbobenzoxyl, dansyl, or T-butyloxycarbonyl; an acetyl group; 
a 9-fluorenylmethoxycarbonyl (FMOC) group; a macromolecular carrier group 
including but not limited to lipid-fatty acid conjugates, polyethylene 
glycol, or carbohydrates; and 
"Z" may represent a carboxyl group; an amido group; a T-butyloxycarbonyl 
group; a macromolecular carrier group including but not limited to 
lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates. 
In addition to the full-length sequence set forth above, the peptides of 
the invention may include truncations thereof which mimic, enhance, or 
inhibit the contactin mediated effects of RPTP.beta.-CAH on neuronal 
cells. Such truncated peptides may comprise peptides having at least 3 
amino acid residues and which demonstrate the ability to mimic, enhance, 
or inhibit the contactin mediated effects of RPTP.beta.-CAH as measured by 
the methods and assays of the invention as described in Section 5.2. 
The peptides of the invention also include analogs of RPTP.beta.-CAH and of 
RPTP.beta.-CAH truncations which may include, but are not limited to, 
peptides comprising the RPTP.beta.-CAH sequence, or truncated sequence, 
containing one or more amino acid substitutions, insertions and/or 
deletions. Analogs of RPTP.beta.-CAH homologs, described below, are also 
within the scope of the invention. The analogs of the invention mimic, 
enhance, or inhibit the contactin mediated effects of RPTP.beta.-CAH on 
neuronal cells, and may, further, possess additional advantageous 
features, such as, for example, increased bioavailability, and/or 
stability, or reduced host immune recognition. One possible class of amino 
acid substitutions would include those amino acid changes which are 
predicted to stabilize the structure of the peptides of the invention. 
Amino acid substitutions may be of a conserved or non-conserved nature. 
Conserved amino acid substitutions consist of replacing one or more amino 
acids of the RPTP.beta.-CAH peptide sequence with amino acids of similar 
charge, size, and/or hydrophobicity characteristics, such as, for example, 
a glutamic acid (E) to aspartic acid (D) amino acid substitution. When 
only conserved substitutions are made, the resulting peptide is 
functionally equivalent to RPTP.beta.-CAH or the RPTP.beta.-CAH peptide 
from which it is derived. Non-conserved substitutions consist of replacing 
one or more amino acids of the RPTP.beta.-CAH peptide sequence with amino 
acids possessing dissimilar charge, size, and/or hydrophobicity 
characteristics, such as, for example, a glutamic acid (E) to valine (V) 
substitution. 
Amino acid insertions may consist of single amino acid residues or 
stretches of residues ranging from 2 to 500 amino acids in length. One or 
more insertions may be introduced into RPTP.beta.-CAH or into fragments, 
analogs or homologs thereof (described below). 
Deletions of RPTP.beta.-CAH, RPTP.beta.-CAH fragments, analogs, and/or 
RPTP.beta.-CAH homologs are also within the scope of the invention. Such 
deletions consist of the removal of one or more amino acids from the 
RPTP.beta.-CAH or RPTP.beta.-CAH-like peptide sequence, with the lower 
limit length of the resulting peptide sequence being 4 to 6 amino acids. 
Such deletions may involve a single contiguous or greater than one 
discrete portion of the peptide sequences. 
The peptides of the invention may further include homologs of 
RPTP.beta.-CAH and/or RPTP.beta.-CAH truncations which mimic, enhance, or 
inhibit the contactin mediated effects of RPTP.beta.-CAH on neuronal 
cells. Such RPTP.beta.-CAH homologs are peptides whose amino acid 
sequences are comprised of the amino acid sequences of peptide regions of 
other biological species that correspond to RPTP.beta.-CAH. 
The peptides of the invention may be made by any of the many known methods 
of synthesis in the chemical or biological arts. By way of example but not 
by way of limitation, the following methods may be used. Chemical 
synthesis of polypeptides from amino acid stocks may be used and is 
described in detail in Creighton, 1984, Proteins, W. H. Freeman and 
Company, N. Y., especially Chapter 1, which is incorporated by reference 
herein in its entirety. Biological methods for producing the peptides or 
polypeptides of the invention include eucaryotic and procaryotic 
expression systems which have been transfected with nucleic acid encoding 
the peptides or polypeptides of the invention. These expression systems 
include, but are not limited to, those employing COS-7 cells as set forth 
in the examples infra. Preferred expression systems and other biological 
methods for producing the peptides and polypeptides of the invention are 
described in detail in Sambrook et al., 1987, Molecular Cloning a 
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., Chapters 16-18, 
which are incorporated by reference herein in their entirety. Preferred 
expression systems also include Fab expression libraries, as described in 
Huse et al., 1989, Science, 246:1275-1281, and phage display libraries, as 
described in Clarkson and Wells, 1994, TIBTEK, 12:173-184, which are both 
incorporated by reference herein in their entirety. 
While not being limited to any theory or explanation of how the invention 
works, the following is hypothesized to explain which peptides 
corresponding to the CAH domain of RPTP.beta. may mimic, enhance or 
inhibit the contactin mediated effects of RPTP.beta.-CAH on neuronal 
cells. The peptides of the invention may exhibit no CAH activity, as is 
hypothesized to be the case with RPTB.beta. and y (Barnea et al., 1993, 
Mol. Cell. Biol., 13:1497-1506). The peptides of the invention may 
comprise those moieties of RPTP.beta.-CAH which are necessary to result in 
a conformational epitope having the ability to mimic, enhance or inhibit 
the contact mediated effects of RPTP.beta.-CAH on neuronal cells. These 
moieties may include the highly packed hydrophobic core and hydrophobic 
exposed residues which are conserved among the CAH domains of other 
proteins (Barnea et al., 1993, Mol. Cell. Biol., 13:1497-1506). Because 
RPTB.beta. does not bind contactin, the peptides of the invention may 
comprise those amino acids at the amino-terminal region of RPTP.beta. 
which diverge from RPTP.beta. and the CAH domains of other proteins. The 
peptides of the invention may also include those moieties of 
RPTP.beta.-CAH produced using random expression libraries as described 
above and then selected by the methods and assays of the invention. 
Any of the peptides of the invention may be identified as having the 
ability to mimic, enhance, or inhibit the contactin mediated effects of 
RPTP.beta.-CAH on neuronal cells by any of the methods and assays of the 
invention as described more fully in section 5.2 above. 
5.4. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION 
Any of the identified compounds can be administered to an animal host, 
including a human patient, by itself, or in pharmaceutical compositions 
where it is mixed with suitable carriers or excipient(s) at doses 
therapeutically effective to treat or ameliorate a variety of disorders, 
including those characterized by insufficient, aberrant, or excessive 
neurite growth, differentiation or survival, including but not limited to: 
ALS; general ataxia; Parkinson's disease; Alzheimer's disease; 
Huntington's disease; general neropathy; cerebral palsy; neurologic 
trauma; and mental retardation. A therapeutically effective dose further 
refers to that amount of the compound sufficient to result in amelioration 
of symptoms associated with such disorders. Techniques for formulation and 
administration of the compounds of the instant application may be found in 
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 
latest edition. 
5.4.1. EFFECTIVE DOSAGE 
Pharmaceutical compositions suitable for use in the present invention 
include compositions wherein the active ingredients are contained in an 
effective amount to achieve its intended purpose. More specifically, a 
therapeutically effective amount means an amount effective to prevent 
development of or to alleviate the existing symptoms of the subject being 
treated. Determination of the effective amounts is well within the 
capability of those skilled in the art, especially in light of the 
detailed disclosure provided herein. 
For any compound used in the method of the invention, the therapeutically 
effective dose can be estimated initially from cell culture assays. For 
example, a dose can be formulated in animal models to achieve a 
circulating concentration range that includes the IC50 as determined in 
cell culture. Such information can be used to more accurately determine 
useful doses in humans. 
A therapeutically effective dose refers to that amount of the compound that 
results in amelioration of symptoms or a prolongation of survival in a 
patient. Toxicity and therapeutic efficacy of such compounds can be 
determined by standard pharmaceutical procedures in cell cultures or 
experimental animals, e.g., for determining the LD50 (the dose lethal to 
50% of the population) and the ED50 (the dose therapeutically effective in 
50% of the population). The dose ratio between toxic and therapeutic 
effects is the therapeutic index and it can be expressed as the ratio 
between LD50 and ED50. Compounds which exhibit high therapeutic indices 
are preferred. The data obtained from these cell culture assays and animal 
studies can be used in formulating a range of dosage for use in human. The 
dosage of such compounds lies preferably within a range of circulating 
concentrations that include the ED50 with little or no toxicity. The 
dosage may vary within this range depending upon the dosage form employed 
and the route of administration utilized. The exact formulation, route of 
administration and dosage can be chosen by the individual physician in 
view of the patient's condition. (See e.g. Fingl et al., 1975, in "The 
Pharmacological Basis of Therapeutics", Ch. 1 p1). 
Dosage amount and interval may be adjusted individually to provide plasma 
levels of the active moiety which are sufficient to maintain the desired 
effects. For examples with respect to the therapeutic peptides and 
polypeptides that can be used in accordance with the invention, usual 
patient dosages for systemic administration range from 1-2000 mg/day, 
commonly from 1-250 mg/day, and typically from 10-150 mg/day. Stated in 
terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, 
commonly from 0.02-3 mg/kg/day, typically from 0.2-1.5 mg/kg/day. Stated 
in terms of patient body surface areas, usual dosages range from 0.5-1200 
mg/m.sup.2 /day, commonly from 0.5-150 mg/m.sup.2 /day, typically from 
5-100 mg/m.sup.2 /day. Dosage amount and interval may be adjusted 
individually to provide plasma levels of the active moiety which are 
sufficient to maintain the desired effects. Usual average plasma levels 
should be maintained within 50-5000 .mu.g/ml, commonly 50-1000 .mu.g/ml, 
and typically 100-500 .mu.g/ml. 
In cases of local administration or selective uptake, the effective local 
concentration of the drug may not be related to plasma concentration. 
The amount of composition administered will, of course, be dependent on the 
subject being treated, on the subject's weight, the severity of the 
affliction, the manner of administration and the judgment of the 
prescribing physician. 
5.4.2. COMPOSITION AND FORMULATION 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that is itself known, e.g., by means of 
conventional mixing, dissolving, granulating, dragee-making, levigating, 
emulsifying, encapsulating, entrapping or lyophilizing processes. 
Pharmaceutical compositions for use in accordance with the present 
invention thus may be formulated in conventional manner using one or more 
physiologically acceptable carriers comprising excipients and auxiliaries 
which facilitate processing of the active compounds into preparations 
which can be used pharmaceutically. Proper formulation is dependent upon 
the route of administration chosen. 
For injection, the agents of the invention may be formulated in aqueous 
solutions, preferably in physiologically compatible buffers such as 
Hanks's solution, Ringer's solution, or physiological saline buffer. For 
transmucosal administration, penetrants appropriate to the barrier to be 
permeated are used in the formulation. Such penetrants are generally known 
in the art. 
For oral administration, the compounds can be formulated readily by 
combining the active compounds with pharmaceutically acceptable carriers 
well known in the art. Such carriers enable the compounds of the invention 
to be formulated as tablets, pills, dragees, capsules, liquids, gels, 
syrups, slurries, suspensions and the like, for oral ingestion by a 
patient to be treated. Pharmaceutical preparations for oral use can be 
obtained solid excipient, optionally grinding a resulting mixture, and 
processing the mixture of granules, after adding suitable auxiliaries, if 
desired, to obtain tablets or dragee cores. Suitable excipients are, in 
particular, fillers such as sugars, including lactose, sucrose, mannitol, 
or sorbitol; cellulose preparations such as, for example, maize starch, 
wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl 
cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, 
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may 
be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic 
acid or a salt thereof such as sodium alginate. 
Dragee cores are provided with suitable coatings. For this purpose, 
concentrated sugar solutions may be used, which may optionally contain gum 
arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, 
and/or titanium dioxide, lacquer solutions, and suitable organic solvents 
or solvent mixtures. Dyestuffs or pigments may be added to the tablets or 
dragee coatings for identification or to characterize different 
combinations of active compound doses. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can 
contain the active ingredients in admixture with filler such as lactose, 
binders such as starches, and/or lubricants such as talc or magnesium 
stearate and, optionally, stabilizers. In soft capsules, the active 
compounds may be dissolved or suspended in suitable liquids, such as fatty 
oils, liquid paraffin, or liquid polyethylene glycols. In addition, 
stabilizers may be added. All formulations for oral administration should 
be in dosages suitable for such administration. 
For buccal administration,the compositions may take the form of tablets or 
lozenges formulated in conventional manner. 
For administration by inhalation, the compounds for use according to the 
present invention are conveniently delivered in the form of an aerosol 
spray presentation from pressurized packs or a nebulizer, with the use of 
a suitable propellant, e.g., dichlorodifluoromethane, 
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other 
suitable gas. In the case of a pressurized aerosol the dosage unit may be 
determined by providing a valve to deliver a metered amount. Capsules and 
cartridges of e.g. gelatin for use in an inhaler or insufflator may be 
formulated containing a powder mix of the compound and a suitable powder 
base such as lactose or starch. 
The compounds may be formulated for parenteral administration by injection, 
e.g., by bolus injection or continuous infusion. Formulations for 
injection may be presented in unit dosage form, e.g., in ampoules or in 
multi-dose containers, with an added preservative. The compositions may 
take such forms as suspensions, solutions or emulsions in oily or aqueous 
vehicles, and may contain formulatory agents such as suspending, 
stabilizing and/or dispersing agents. 
Pharmaceutical formulations for parenteral administration include aqueous 
solutions of the active compounds in water-soluble form. Additionally, 
suspensions of the active compounds may be prepared as appropriate oily 
injection suspensions. Suitable lipophilic solvents or vehicles include 
fatty oils such as sesame oil, or synthetic fatty acid esters, such as 
ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions 
may contain substances which increase the viscosity of the suspension, 
such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, 
the suspension may also contain suitable stabilizers or agents which 
increase the solubility of the compounds to allow for the preparation of 
highly concentrated solutions. 
Alternatively, the active ingredient may be in powder form for constitution 
with a suitable vehicle, e.g., sterile pyrogen-free water, before use. 
The compounds may also be formulated in rectal compositions such as 
suppositories or retention enemas, e.g., containing conventional 
suppository bases such as cocoa butter or other glycerides. 
In addition to the formulations described previously, the compounds may 
also be formulated as a depot preparation. Such long acting formulations 
may be administered by implantation (for example subcutaneously or 
intramuscularly) or by intramuscular injection. Thus, for example, the 
compounds may be formulated with suitable polymeric or hydrophobic 
materials (for example as an emulsion in an acceptable oil) or ion 
exchange resins, or as sparingly soluble derivatives, for example, as a 
sparingly soluble salt. 
A pharmaceutical carrier for the hydrophobic compounds of the invention is 
a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a 
water-miscible organic polymer, and an aqueous phase. The cosolvent system 
may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl 
alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v 
polyethylene glycol 300, made up to volume in absolute ethanol. The VPD 
co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose 
in water solution. This co-solvent system dissolves hydrophobic compounds 
well, and itself produces low toxicity upon systemic administration. 
Naturally, the proportions of a co-solvent system may be varied 
considerably without destroying its solubility and toxicity 
characteristics. Furthermore, the identity of the co-solvent components 
may be varied: for example, other low-toxicity nonpolar surfactants may be 
used instead of polysorbate 80; the fraction size of polyethylene glycol 
may be varied; other biocompatible polymers may replace polyethylene 
glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides 
may substitute for dextrose. 
Alternatively, other delivery systems for hydrophobic pharmaceutical 
compounds may be employed. Liposomes and emulsions are well known examples 
of delivery vehicles or carriers for hydrophobic drugs. Certain organic 
solvents such as dimethylsulfoxide also may be employed, although usually 
at the cost of greater toxicity. Additionally, the compounds may be 
delivered using a sustained-release system, such as semipermeable matrices 
of solid hydrophobic polymers containing the therapeutic agent. Various of 
sustained-release materials have been established and are well known by 
those skilled in the art. Sustained-release capsules may, depending on 
their chemical nature, release the compounds for a few weeks up to over 
100 days. Depending on the chemical nature and the biological stability of 
the therapeutic reagent, additional strategies for protein stabilization 
may be employed. 
The pharmaceutical compositions also may comprise suitable solid or gel 
phase carriers or excipients. Examples of such carriers or excipients 
include but are not limited to calcium carbonate, calcium phosphate, 
various sugars, starches, cellulose derivatives, gelatin, and polymers 
such as polyethylene glycols. 
Many of the compounds of the invention may be provided as salts with 
pharmaceutically compatible counterions. Pharmaceutically compatible salts 
may be formed with many acids, including but not limited to hydrochloric, 
sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be 
more soluble in aqueous or other protonic solvents that are the 
corresponding free base forms. 
5.4.3. ROUTES OF ADMINISTRATION 
Suitable routes of administration may, for example, include oral, rectal, 
transmucosal, or intestinal administration; parenteral delivery, including 
intramuscular, subcutaneous, intramedullary injections, as well as 
intrathecal, direct intraventricular, intravenous, intraperitoneal, 
intranasal, or intraocular injections. 
Alternately, one may administer the compound in a local rather than 
systemic manner, for example, via injection of the compound directly into 
an affected area, often in a depot or sustained release formulation. 
Furthermore, one may administer the drug in a targeted drug delivery 
system, for example, in a liposome coated with an antibody specific for 
affected cells. The liposomes will be targeted to and taken up selectively 
by the cells. 
5.4.4. KAGING 
The compositions may, if desired, be presented in a pack or dispenser 
device which may contain one or more unit dosage forms containing the 
active ingredient. The pack may for example comprise metal or plastic 
foil, such as a blister pack. The pack or dispenser device may be 
accompanied by instructions for administration. Compositions comprising a 
compound of the invention formulated in a compatible pharmaceutical 
carrier may also be prepared, placed in an appropriate container, and 
labelled for treatment of an indicated condition. Suitable conditions 
indicated on the label may include treatment of a neurologic disease such 
as one characterized by insufficient, aberrant, or excessive neurite 
growth, differentiation or survival. 
RPTP.beta./.zeta. is a receptor-type tyrosine phosphatase that is expressed 
predominantly in the developing nervous system. Its extracellular domain 
consists of a carbonic anhydrase-like motif (CAH), a fibronectin type III 
repeat (FNIII) and a long spacer region. In the examples described infra, 
fusion proteins containing these different subdomains fused to the Fc 
portion of human IgG were used to screen a panel of neuronal cells to 
search for ligands for RPTP.beta.. This screen identified two potential 
cell associated ligands that are expressed either in neurons or in glial 
cells. The CAH domain construct (.beta.C-Fc) bound to neuronal cells lines 
while the addition of the FNIII (.beta.CF-Fc) enabled the interaction of 
RPTP.beta. with glial cell lines. Cross-linking experiments identified 
that the CAH domain bound to a protein of 140 kDa in IMR-32 neuroblastoma 
and GH3 anterior pituitary cell lines. Expression cloning in COS7 cells 
revealed that the gene encoding this ligand was the rat homologue of the 
neural cell recognition molecule contactin (F11/F3). This protein consists 
of six C2 type Ig domains, four fibronectin type III repeats and a 
hydrophobic region that mediates its attachment to the membrane by a GPI 
anchor. Transfection of COS7 cells with rat or human contactin cDNA 
resulted in binding of RPTP.beta., and treatment with phospholipase C 
completely abolished the binding of .beta.CF-FC to the cells. In addition, 
soluble contactin-Fc fusion bound to COS cells expressing a chimeric 
receptor that contained the extracellular region of RPTP.beta.. Finally, 
it was found that the CAH domain of RPTP.beta. induced cell adhesion and 
neurite growth of primary tectal neurons, and differentiation of IMR-32 
neuroblastoma cells. This response could be completely blocked with 
antibodies against contactin, demonstrating that contactin is the neuronal 
receptor for RPTP.beta.. The ability of a receptor-type tyrosine 
phosphatase to serve as a ligand for a GPI-linked cell recognition 
molecule illustrates the potential bidirectional nature of information 
flow during neural growth and development. 
6. EXAMPLE: THE INTERACTION BETWEEN CONTACTIN AND THE CAH DOMAIN OF 
RPTP.beta. 
The subsections below describe the biological interaction between contactin 
and the CAH domain of RPTP.beta.. The data demonstrate that ligands for 
RPTP.beta. are differentially expressed in neuronal and glial cell lines. 
In addition, it is shown that a 140 kDa protein from these cell lines 
interacts with the CAH domain of RPTP.beta., and that this 140 kDa protein 
is contactin. The data also demonstrate that RPTP.beta.interacts with both 
membrane-bound and soluble contactin. 
6.1. MATERIALS AND METHODS 
6.1.1. CELL CULTURE 
SF763T and SF767T human astrocytoma cell lines were grown in athymic nu/nu 
mice to create a tumor derived cell line. The parental lines (SF763 and 
SF767) were generously provided by Dr. Michael E. Bernes (The Barrow 
Neurological Institute, Phoenix, Ariz.). All other cell line used were 
supplied by the American Type Culture Collection (Rockville, Md.). For 
culturing of rat sensory neuron, spinal sensory ganglia were dissected 
from newborn rat pups and dissociated by incubation with trypsin (0.05% 
for 10 minutes). The ganglia were washed several times in L15+10% fetal 
calf serum, and triturated with a pasteur pipette. The resulting single 
cell suspension was not subjected to preplating. The cells were plated at 
15,000 cells per well in an eight-well chamber slide (Nunc) precoated with 
10 mg/ml laminin in PBS. The medium was L15/CO.sub.2 with supplements as 
described (Hawrot and Patterson, 1979, Meth. Enzymol., 58:547-584), and 
nerve growth factor was added at 50 ng/ml. The cells were cultured for two 
days prior to staining. 
6.1.2. GENERATION AND PRODUCTION OF FC-FUSIONS 
To construct the Fc-fusion molecule, different subdomains of 
RPTP.beta.extracellular region were amplified using pfu (Stratagene, La 
Jolla, Calif.) and cloned into a unique BamHI site upstream from the hinge 
region of human IgG1-Fc. For the construction of .beta.C and .beta.CF 
fusions a DNA fragment was amplified from position -20, within the 
Bluescript sequence to position 939 and 1245 respectively (.beta.C-Fc aa 
1-313, .beta.CF-Fc aa 1-415) (Levy et al., 1993, J. Biol. Chem., 
268:10573-10581). In frame fusion was made by creating a BamHI site in the 
3' primer maintaining the original amino acids sequence in the fusion 
junction. These fragments were further cloned into HindIII-BamHI 
linearized pC.gamma.1 vector, a modified version of pIG1 that contained a 
cDNA form instead of the genomic fragment of human IgG (Simmons, 1993, in 
Cellular Interactions in Development. A Practical Approach, Hartley (ed.), 
IRL Press). The same strategy was used to construct human contactin-Fc 
(Hcon-Fc) fusion molecule. Briefly, total RNA was prepared from Y79 
retinoblastoma cells and converted to single strand cDNA using SuperScript 
II reveres transcriptase (Gibco-BRL) following the suppliers protocol. 
This cDNA was use as a template to clone human contactin by three 
overlapping PCR reactions into EcoRI-BamHI sites of pC.gamma.1 vector. In 
order to use these sites, the EcoRI site at position 3173 (Reid et al., 
1994, Brain Res. Mol. Brain Res., 21:1-8), was eliminated by changing a 
single base during the PCR reaction. The final construct contained amino 
acids 1-1020 of human contactin fused to the IgG region. To construct 
.beta.F-Fc the region between nucleotides 901 to 1242 was amplified with a 
set of primers that introduced SacII and BamHI sites in the ends of the 
fragment. This fragment was cloned into pCN.gamma.1 between the globulin 
gene and a sequence encoding a signal peptide derived from TGF.beta. gene 
(Plowman et al., 1992, J. Biol. Chem., 267:13073-13078). The integrity of 
all the above constructs was checked by complete nucleotide sequence 
determination or by restriction enzyme analysis. Fusion proteins were 
produced transiently in COS7 cells or by cotransfection with pN1012-Neo 
into 293 cells and selecting for individual G418 resistant clones as 
described (Peles et al., 1991, EMBO J., 10:2077-2086). Purification of 
fusion proteins was achieved by affinity chromatography on Protein-A 
Sepharose CL 4B (Pharmacia). Bound proteins were eluted with 100 mM sodium 
citrate PH 2.5, 1M MgCl.sub.2, followed by buffer exchange on a PD-10 
desalting column (Pharmacia). The proteins were analyzed by gel 
electrophoresis followed by silver staining (ICN, Costa Mesa, Calif.). 
Concentration of the purified proteins was determined by bradford reagent 
(BioRad, Richmond, Calif.), and by an ELISA assay using peroxidase coupled 
antibody against human IgG (Pierce, Roxford, Ill.). The same antibody was 
used to detect the fusion proteins by western blotting followed by 
chemiluminescence reagent (ECL; Amersham) as described previously (Peles 
et al., 1992, Cell, 69:205-216). 
6.1.3. EXPRESSION CLONING IN COS CELLS 
Total cellular RNA was prepared from GH3 cells using acid guanidinium 
thiocyanate extraction (Chomczynski and Sacchi, 1987, Anal. Biochem., 
162:156-159), and Poly(A) RNA was isolated by two passages over an oligo 
dT cellulose column (Pharmacia). cDNA was synthesized using the 
Superscript kit (Gibco BRL, Bethesda, Md.) by priming with a random primer 
that contained a HindIII site. Following the addition of EcoRI adaptors 
the double-stranded cDNA was size selected on agarose gel. cDNAs larger 
then 2 kb were ligated into a EcoRl and HindIII-digested pcMP1 plasmid 
vector, a derivative of the pCMV-1 vector (Lammers et al., 1993, J. Biol. 
Chem., 168:24456-22462). E. coli DH1OB cells (GIBCO BRL) were transformed 
by electroporation REF. This procedure generated a cDNA library with 
2.times.10.sup.6 independent clones. Pools of 3000 bacterial clones were 
grown for 24 hours and scraped from plates using, LB containing 15% 
glycerol. Twenty percent of the cultures were saved as glycerol stocks at 
-70.degree. C. and plasmid DNA was prepared from the rest using the Wizard 
plasmid purification kit (Promega). 
Plasmid DNA (10 .mu.g) was transfected into COS7 cell grown on chamber 
slides (Nunc) with lipofectamin (GIBCO BRL). After 72 hours cells were 
incubated for one hour with medium containing 0.5 .mu.g/ml .beta.CF-Fc. 
Unbound Fc-fusion proteins were removed by three washes with cold DMEM/F12 
and the cells were fixed with 4% paraformaldehyd in PBS. Immunostaining 
was performed with ABC staining system (Vector Lab), using biotinylated 
anti-human IgG antibodies (Fc specific; Jackson Labs, West Grove, Pa.) 
following by streptavidin alkaline phosphatase and NBT/BCIP as substrate 
according to the protocol provided by the manufacturer. One positive pool 
(#54) was subdivided and rescreened until a single clone (F8) was 
isolated. 
DNA sequence determination was carried out using the dideoxy-chain 
termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci., USA 
74:5463), with Sequenase 2.0 (United States Biochen-Lical Corporation, 
Cleveland, Ohio). Sequencing was performed on both strands by priming with 
synthetic oligonucleotides. 
6.1.4. CONSTRUCTION OF RPTP.beta./EGF-RECEPTOR CHIMERAS 
To generate a plasmid for the expression of .beta.CF/EK chimeras, a portion 
of the extracellular domain of RPTP.beta.containing the CAH and the FINIII 
domains (.beta.CF, aa 1-418) was fused to the human EGF receptor at 
position 634, twelve amino acids after the transmembrane domain in its 
extracellular region. These fragments were amplified using pfu 
(Stratagene, La Jolla, Calif.) with a specific set of primers that 
introduce a BstBI site at the junction between the two genes. The 
resulting fragments were ligated into Bluescript (Stratagene, La Jolla, 
Calif.). Proper fusion between the two molecules was verified by 
nucleotide sequence analysis. This chimeric gene was then subcloned into a 
NotI site in the reteroviral vector SR.alpha.-SL and viral stocks where 
prepared by cotransfecting COS-7 cells with this vector along with a 
helper virus plasmid (Muller et al., 1991, Mol. Cell. Biol., 
11:1785-1792). These viruses where used to infect NIH 3T3 (clone 2.2), 
which lack endogenous EGF-receptor. Following infection, cells where 
selected in a medium containing 1 mg/ml G418 (Gibco-BRL) and resistant 
colonies were individually grown and assayed for the expression of the 
chimeric receptor by Western blotting with antibodies against the carboxyl 
terminus of the EGF-R (Kris et al., 1985, Cell, 40:619-625) as described 
previously (Peles et al., 1992, Cell, 69:205-216). 
6.1.5. BINDING OF FC-FUSION PROTEINS 
Confluent monolayer of cells were incubated for one hour with conditioned 
medium containing 0.25-0.5 mg/ml Fc-fusion protein. The unbound proteins 
were removed by three washes with binding medium (0.1% BSA, 0.2% none fat 
dry milk in DMEM/F12) and the cells were further incubated with 1 ng/ml 
.sup.125 I!-Protein A (Amersham), for 30 minutes at 4.degree. C. Plates 
were washed three times with cold binding medium and cell bound 
radioactivity was determined as described previously (Peles et al., 1993, 
EMBO J., 12:961-971). Cellular staining using the Fc-fusion proteins was 
done using the procedure described above for expression cloning. 
15 6.1.6. CHEMICAL CROSS-LINKING EXPERIMENTS 
Cells were incubated for four hours with medium containing, the different 
Fc-fusion proteins. Following three washes with cold PBS/Ca (1 mM 
CaCl.sub.2 in PBS), the cells were incubated for additional 30 minutes 
with PBS/Ca containing 1 mM DTSSP 
(3,3'-Dithiobissulfosuccinimidyl-propionate!, Pierce, Rockford, Ill.). 
Free cross-linker was removed by additional PBS wash followed by quenching 
with 100 mM glycine in TBS for 10 minutes at 4.degree. C. Cell lysates 
were made in SBN lysis buffer (Peles et al., 1991, EMBO J., 10:2077-2086), 
and Sepharose-protein A was added to the cleared lysates. Following two 
hours incubation at 4.degree. C., the beads were washed three times with 
HNTG buffer (Peles et al., 1991, EMBO J., 10:2077-2086), and the bound 
proteins were eluted by adding SDS PAGE sample buffer containing 5% 
.beta.-mercaptoethanol and further incubation for 10 minutes at 95.degree. 
C. 
6.1.7. PROTEIN PURIFICATION AND SEQUENCING 
Cellular membranes were prepared from 5.times.10.sup.8 GH3 cells by 
homogenization in hypotonic buffer that included 10 mM Hepes pH 7.5, 1 mM 
EGTA, 1 mM MgCl.sub.2, 10 .mu.g/ml aprotinin, 10 .mu.g/ml leupeptin and 2 
mM PMSF. Nuclei and unbroken cells were removed by low speed 
centrifugation (1000 g.times.10 minutes at 4.degree. C.), and the 
supernatant was then subjected to high speed centrifugation at 40000 g (30 
minutes at 4.degree. C.). The membrane pellet was resuspended in SML 
solubilization buffer (2% Sodium monolaurate, 2 mM MgCl2, 2 mM PMSF in 
PBS). After one hour incubation on ice the detergent-insoluble materials 
was removed by centrifugation, and the sample was diluted tenfold with PBS 
containing 2 mM MgCl.sub.2. This sample was loaded on a column of 
.beta.CF-FC bound to Sepharose Protein A (200 .mu.g .beta.CF-Fc/ml beads) 
at 40.degree. C. The column was washed with SML buffer containing 0.15% 
detergent and the bound proteins were eluted by adding SDS sample buffer 
and heating to 95.degree. C. Proteins were separated on 7.5% gel and 
electroblotted in CAPS buffer (100 mM CAPS, 10% MeOH) to ProBlott membrane 
(Applied Biosystems). The membrane was stained with coomassie R-250 and 
the 140 kDa band was excised and subjected to direct microsequencing 
analysis. Microsequencing was performed with an Applied Biosystems Model 
494 sequencer, run using standard reagents and programs from the 
manufacturer. 
To obtain internal peptide sequence the blotted band was moistened with 
neat acetonitrile, then reduced by the addition of 200 ul of 0.1M Tris pH 
8.5, 10 mM dithiothreitol, 10% acetonitrile. After incubation at 
55.degree. C. for 30' the sample was cooled to room temperature and 20 ul 
of 0.25M 4-vinylpyridine in acetonitrile added. After 30 minutes at room 
temperature the blots were washed 5 times with 10% acetonitrile. Digestion 
was performed for 16 hours with 1 ug modified trypsin (Promega) in 50 ul 
of 0.1M Tris pH 8.0, 10% acetonitrile, 1% octylglucoside. Digestion was 
stopped by the addition of 2 ul of neat trifluoroacetic acid (TFA). 
Peptides were separated on a 1 mm.times.200 mm Reliasil C-18 reverse phase 
column on a Michrom UMA HPLC run at 50 ul per minute. Solvents used were 
0.1% TFA in water and 0.085% TFA in 95% acetonitrile/5% water. A linear 
gradient of 5 to 65% B was run over 60 minutes. Absorbance was monitored 
at 214 nm and peaks were collected manually into a 96 well polyethylene 
microtitre plate. Purified peptides were sequenced as described above. 
6.1.8. TREATMENT WITH PI-PLC 
Cells grown to confluency in 90 mm dishes were metabolically labeled with 
100 .mu.g Ci/ml .sup.35 S!-methionine and cysteine mix (NEN, Boston, 
Mass.) for four hours at 37.degree. C. Labeled cells were washed three 
times with MEM and incubated with 250 mU of phosphatidylinositol specific 
phospholipase C (PI-PLC, Boehringer Mannheim or a kind gift from Dr. J. 
Salzer) for 50 minutes at 37.degree. C. The supernatant was collected and 
cleared by centrifugation (1000 g), membranes were prepared from the cells 
and further solubilized in SML buffer as described above. .beta.CF-Fc 
bound to Sepharose-protein A beads was added to the supernatant and the 
membrane fractions for one hour at 4.degree. C. The beads were washed 
twice with 0.15% sodium monolaurate in PBS and once in PBS before the 
addition of SDS sample buffer. The precipitated proteins were separated on 
7.5% cell and subjected to autoradiography. 
For binding experiments, cell were treated with different amounts of PI-PLC 
(as indicated in the legend to the figures) in MEM containing 0.5% BSA for 
30-60 minutes at 37.degree. C. Cells were briefly washed and binding of 
.beta.CF-Fc was performed as described above. 
6.2. RESULTS 
6.2.1. THE CAH DOMAIN OF RPTP.beta. MEDIATES AN INTERACTION WITH NEURONS 
To identify cellular ligands for RPTP.beta., fusion proteins were 
constructed between different subdomains of RPTP.beta. and the Fc portion 
of human IgG. Three chimeric constructs were made, one containing both the 
carbonic anhydrase and the fibronectin domains (.beta.CF-Fc) and two 
others carrying each domain by itself (.beta.C-FC or .beta.F-FC; FIG. 1A 
and 1B). 
Initially, .beta.CF-Fc was used to screen for a membrane bound ligand on 
the surface of different neuronal and glial cell lines. As shown in FIG. 
1C, several cell lines that bind this fusion protein were identified. 
These were the IMR-32 neuroblastoma cells, the two closely related 
neuroendocrine derived cell lines GH3 and GH1, and five different 
glioblastoma cell lines. 
The fact that these positive cell lines were derived from glial and 
neuronal origins raised the possibility that RPTP.beta. may interact with 
two different membrane-associated ligands. Alternatively, a single ligand 
may exist which is expressed by both neurons and glia cells. To explore 
these two possibilities it was examined whether a fusion protein that 
contained only the CAH domain of RPTP.beta. (.beta.C-Fc) will retain the 
same cell specificity observed with .beta.CF-FC. It was reasoned that in a 
multidomain receptor like RPTP.beta., each domain might function as an 
independent unit in terms of its interaction with a specific ligand. Thus, 
the use of a single domain in binding experiments might allow the 
identification of a cell type specific ligand. As depicted in FIG. 2A, 
this fusion protein, indeed, binds to the same neuronal and neuroendocrine 
cell lines. In contrast, none of the glioblastomas were positive, 
suggesting that there are at least two ligands for RPTP.beta. that are 
differentially expressed on neuronal or glial cells. This result also 
implied that the CAH domain mediates the interaction of RPTP.beta. with a 
specific ligand present in neurons but not in glia cells. 
Accordingly, if the binding of .beta.C-FC to neuronal ligand reflects the 
interactions occurring in vivo, one would expect to see similar binding 
specificity on cultures of primary neurons. The binding of the different 
fusion proteins to cultured dorsal root ganglion cells (DRG), followed by 
detection of the bound proteins by immunostaining (FIG. 2B), was analyzed. 
.beta.C-FC and .beta.CF-FC bound to GH3 cells, as well as to the primary 
neurons. A fusion protein containing the fibronectin domain alone 
(.beta.F-Fc) failed to bind to either GH3 cells or DRG neurons (FIG. 2B). 
In other experiments, binding of .beta.F-FC to several glial cell lines 
was detected, but no binding of this domain to neuronal derived cell lines 
or neurons derived from rat DRGs and chick cortex was detected. In 
addition, it was examined whether the binding specificity observed with 
the CAH domain of RPTP.beta. is unique to this receptor by comparing it 
with the related phosphatase RPTP.gamma. (Barnea et al., 1993, Mol. Cell. 
Biol., 13:1497-1506). A fusion protein made with the CAH domain of this 
highly homologous family member did not bind to GH3 cells or to primary 
neurons (FIG. 2C). 
Altogether these results suggests that specific ligands for RPTP.beta. 
exist on the surface of cells from neuronal and glial origin. Different 
subdomains of the receptor mediate its interaction with those distinct 
ligands. The CAH mediates an interaction with neurons while the FNIII 
enables the interaction of RPTP.beta. with glia cells. In the work 
presented here, the identification and molecular characterization of the 
ligand for the CAH domain is described. 
6.2.2. COVALENT CROSS LINKING EXPERIMENTS REVEAL A 140 KDA PROTEIN THAT 
INTERACTS WITH THE CAH DOMAIN OF RPTP.beta. 
To characterize ligands for RPTP.beta., a reversible cross-linker (DSSTP) 
was used, and proteins were sought that specifically bound to .beta.C-Fc. 
Two of the cell lines that bound .beta.C-Fc (IMR32 and GH3), as well as 
COS7 cells as a control, were allowed to react with the fusion proteins 
containing the FNIII or the CAH domains followed by cross-linking and 
precipitation of the complexes. As shown in FIG. 3, a protein of about 140 
kilodalton specifically reacted with .beta.C-Fc in the rat GH3 and human 
IMR-32 cells. No reactivity was detected in control cells or in cells 
incubated with .beta.F-Fc. The cross-linker (DSSTP) used, undergoes 
cleavage in the reducing SDS PAGE conditions and, therefore, permits the 
identification of the true molecular weight of the putative ligand. This 
result suggested that the same ligand is expressed in the rat GH3 and the 
human IMR-32 lines. 
6.2.3. MOLECULAR CLONING OF A CANDIDATE LIGAND FOR RPTP.beta. FROM RAT GH3 
CELLS REVEALS THE RAT HOMOLOGUE OF CONTACTIN 
An expression cloning strategy was employed in an effort to clone the gene 
that encodes the 140 kDa candidate ligand. we have employed. Plasmid pools 
made from a GH3-cDNA library were transfected into COS7 cells and the 
cells were screened for their ability to bind .beta.CF-Fc. Positive cells 
were detected by immunostaining with biotinylated anti-human IgG 
antibodies and streptavidin alkaline phosphatase. One positive pool was 
identified that when transfected yielded several stained cells on the 
slide (FIG. 4A). This pool was subdivided and rescreened four times until 
a single clone (F8) was isolated. Transfection of COS7 cells with this 
plasmid resulted in positive staining of approximately 25%-50% of the 
cells, a number that correlates well with the maximum transfection 
efficiency in our system. DNA sequence analyses of clone F8 showed that it 
contained a 3.9 kb insert and a single long open reading frame of 3063 
nucleotides. The deduced 1021 amino acid sequence encoded by this clone is 
presented in FIG. 4B. Data bank search with this sequence showed that it 
shares 95% and 99% identity at the amino acid level with human and mouse 
contactin respectively (Berglund and Ranscht, 1994, Genomics, 21:571-582; 
Gennarini et al., 1989, J. Cell. Biol., 109:755-788; Reid et al., 1994, 
Brain Res. Mol. Brain Res., 21:1-8). It was therefore concluded that the 
ligand for RPTP.beta. cloned from GH3 cells is the rat homologue of 
contactin. Structurally, this protein consists of six C2 type Ig domains, 
four fibronectin type III repeats and an hydrophobic region that mediates 
its attachment to the membrane by a GPI linkage (FIG. 4B., and Gennarini 
et al., 1989, J. Cell. Biol., 109:755-788; Reid et al., 1994, Brain Res. 
Mol. Brain Res., 21:1-8). Functionally, it is a neural cell adhesion 
molecule that has been suggested to play a morphogenic role during the 
development of the nervous system (Rathjen et al., 1987, J. Cell. Biol., 
104:343-353; Walsh and Doherty, 1991, Cell. Biol. Int. Rep., 
15:1151-1166). 
In parallel to the expression cloning strategy, and as a complementary 
approach, a biochemical procedure was employed that utilized the CAH 
domain as an affinity reagent for protein purification. p140 was purified 
from solubilized membranes prepared from GH3 cells on a column of 
.beta.CF-Fc (FIG. 4C). After resolving the eluted protein on SDS/PAGE, the 
140 kDa species was subject directly to N-terminal sequencing, or was 
digested with trypsin. Two peptide sequences obtained, one from the 
N-terminus and the other from an internal peptide after tryptic digest. 
Both sequences matched the translated F8 sequence and confirmed that 
contactin is indeed a ligand for the CAH domain of RPTP.beta.. 
6.2.4. BINDING ANALYSIS OF RPTPB AND CONTACTIN 
The binding specificity of different subdomains of RPTP.beta. towards 
contactin was examined. COS7 cells were transfected with rat contactin 
(clone F8) and analyzed for their ability to bind fusion proteins 
containing the CAH, FNIII or both domains (FIG. 5A). As expected, 
expression of contactin enabled the binding of the CAH domain of 
RPTP.beta. to the cells. The FNIII domain alone did not bind to contactin 
expressing cells. In addition, similar results were obtained with a fusion 
protein that carries most of the extracellular region of the short form of 
RPTP.beta. (aa 1-644; data not shown). 
The reciprocal interaction, namely, whether soluble contactin molecules are 
able to bind specifically to cells expressing RPTP.beta., was explored 
next. In these experiments, COS7 cells were transfected with chimeric 
receptor constructs that consist of the entire extracellular region of the 
short form of RPTP.beta. (.beta.CFS/EK), the CAH domain plus the FNIII 
repeat (.beta.CF/EK), or the CAH domain alone (.beta.C/EK) fused to the 
transmembrane and intracellular domains of the EGF receptor. A chimeric 
receptor was used instead of the wild type phosphatase because the wild 
type phosphatase was not able to be expressed in heterologous cells. The 
experiment presented in FIG. 5B, shows that human contactin-Fc fusion 
protein binds to cells transfected with these chimeric receptors but not 
to control cells. Taken together, these results demonstrate that 
expression of contactin is both necessary and sufficient for binding to 
the CAH domain RPTP.beta.. 
6.2.5. SOLUBLE CONTACTIN RELEASED FROM THE MEMBRANE BY PHOSPHOLIPASE C 
TREATMENT INTERACTS WITH RPTP.beta. 
Contactin belongs to a family of recognition molecules that TAG-1 and 
BIG-1, all of which are anchored to the plasma membrane via a 
glycosyl-phosphatidylinositol (GPI). Therefore, it was of interest to see 
how phospholipase C (PI-PLC) treatment would effect the interaction 
between contactin and RPTP.beta.. When incubated with COS7 cells 
expressing contactin (clone F8), PI-PLC completely abolished the binding 
of .beta.CF-Fc to the cells (FIG. 6A). Similar results were obtained also 
with GH3 cells (FIG. 6B). 
It has been demonstrated that members of this family and other GPI-linked 
proteins may exist either in a membrane bound or a secreted soluble form 
that is released from the cell surface (Furley et al., 1990, Cell, 
61:157-170; Theveniau et al., 1992, J. Cell. Biochem., 48:61-72). Hence, 
it was examined whether the different forms of contactin, including those 
released after PI-PLC treatment, could interact with RPTP.beta.. To this 
aim, GPI-linked proteins were released from metabolically labeled GH3 
cells with the enzyme and purified contactin by bioaffinity precipitation 
from membrane extracts of the cells or the cell supernatants (FIG. 6C). 
Without PI-PLC treatment, two proteins p140 and p180 from the membrane 
fraction could specifically associate with .beta.C-Fc. These proteins were 
not present in the supernatant and they were not detected with .beta.F-Fc. 
However, after PI-PLC treatment, p140/contactin could be precipitated from 
the medium of the cells, indicating that the soluble form produced by 
phospholipase treatment interacts with RPTP.beta.. This result may suggest 
that, in addition to the interaction between the membrane bound forms of 
these proteins, soluble contactin could potentially interact in vivo with 
RPTP.beta.. As shown in FIG. 6C, .beta.C-Fc could precipitate the 180 
kilodaltons protein only from membrane fraction and not from the cell 
supernatant. PI-PLC treatment did not release this protein from the cells 
suggesting that it is either an integral membrane protein or a 
cytoskeletal protein associated with contactin complexes. Since contactin 
by itself is sufficient to mediate the interaction with RPTP.beta., the 
180 kDa protein may be associated with contactin in the cells and 
coprecipitated with it during the bioaffinity procedure. One intriguing 
possibility is that p 180 is a signaling unit used by contactin on the 
surface of neurons (see below). 
7. EXAMPLE: THE CAH DOMAIN OF RPTP.beta. INDUCES CONTACTIN MEDIATED NEURITE 
OUTGROWTH 
The subsections below describe the induction, by the CAH domain of 
RPTP.beta., of contactin mediated neurite outgrowth. It is shown that the 
CAH domain of RPTP.beta. is a permissive substrate for neuronal adhesion 
and neurite growth. In addition, it is also shown that the neurite growth, 
differentiation and survival induced by the carbonic anhydrase-like domain 
of RPTP.beta. is mediated by neuronal contactin. 
7.1. MATERIALS AND METHODS 
The materials and methods for this example were the same as those set forth 
in the example described in section 6.1 above, except as supplemented or 
amended below. 
7.1.1. NEURITE OUTGROWTH ASSAYS 
Neurite outgrowth assays using IMR 32 cells were performed as described 
previously (Friedlander et al., 1994, J. Cell. Biol., 125:669-680) using 
35 mm petri dishes coated with different proteins adsorbed on the 
substrate. After blocking the dishes with 1% BSA/PBS, the blocking 
solution was replaced with 3.times.10.sup.4 cells suspended in 140 .mu.l 
of DMEM/F12/ITS. Following incubation for 3 hrs at 37.degree. C. during 
which time most of the cells adhered to the dish, the medium was removed 
and replaced with DMEM/FI2/ITS medium containing antibodies (Ig fraction 
purified by ammonium sulfate precipitation and DE52 chromatography). 
Dishes were incubated for 48 hrs and fixed with Hanks/0.3% sucrose 2.5% 
paraformaldehyde. For PI-PLC treatment, primary tectal neurons 
(5.times.10.sup.4 cells/250 ml) were prepared from E9 chick embryos 
(Grumet et al., 1984, Proc. Natl. Acad. Sci. USA, 81:267-271) and 
incubated with 0.25 .mu.l of PIPLC (1.7 U/ml) in DMEM/F12/ITS+ at 
37.degree. C. for 30 min. The cell suspension was then incubated on dishes 
coated with different substrates without changing the medium. 
7.2. RESULTS 
7.2.1. NEURITE OUTGROWTH INDUCED BY THE CAH DOMAIN OF RPTP.beta. IS 
MEDIATED THROUGH CONTACTIN 
Contactin has been shown to be involved in both positive and negative 
responses of neurons to various stimuli (Brummendorf and Rathjen, 1993, J. 
Neurochem., 61:127-1219). When presented as a ligand to neurons, either as 
a membrane-bound or a soluble form, contactin induces axonal growth 
(Brummendorf et al., 1993, Neuron, 10:711-727; Clarke et al., 1993, Eur. 
J. Cell. Biol., 61:108-115; Durbec et al., 1992, J. Cell. Biol., 
117:877-887; Gennarini et al., 1989, J. Cell. Biol., 109:755-788). Its 
neural receptor has been identified as the recognition molecule Nr-CAM 
(Morales et al., 1993, Neuron 11:1113-1122). On the other hand, contactin 
itself is a receptor present on neurons and mediates their repulsion by 
the extracellular matrix protein janusin (Pesheva et al., 1993, Neuron, 
10:69-82). The results described in the example of Section 6.1 indicate 
that the CAH domain of RPTP.beta. can bind to contactin on cells. To 
analyze effects of this binding on neurons, chick tectal cells, known to 
express contactin, were plated on dishes previously coated with 
.beta.CF-Fc fusion protein or with Ng-CAM or laminin as controls. Cells 
attached and grow processes on both of these substrates (FIG. 7A). 
Treatment of the cells with PI-PLC prior to plating completely abolished 
cell attachment and neurite extension on RPTP.beta.. In contrast, PI-PLC 
did not have a dramatic effect on cells growing on Ng-CAM or laminin as 
substrate (FIG. 7A). Thus, it was concluded that the CAH domain of 
RPTP.beta.is a permissive substrate for neuronal adhesion and neurite 
growth. Moreover, the cell adhesion and axonal elongation induced by 
RPTP.beta. is mediated through a GPI-anchored receptor. 
Next it was investigated whether contactin could be the neuronal receptor 
for the CAH domain of RPTP.beta.. To this aim, a human neuroblastoma cell 
line IMR-32 was used that has the capacity to differentiate and to 
elaborate neurites in response to different stimuli (Ludecke and 
Unnsicker, 1990, Cancer, 65:2270-2278). These cells have fibroblastic 
morphology when crown on petri dishes coated with fibronectin, but on 
laminin substrates they assume a neuronal phenotype and extend processes 
with growth cones (FIG. 7B). A similar morphologic differentiation was 
seen after plating the cells on the CAH domain of RPTP.beta.. In contrast, 
the CAH domain of RPTP.gamma. had no effect on cell adhesion and 
differentiation. These results show that IMR-32 cells respond specifically 
to the carbonic anhydrase domain of RPTP.beta.. To determine whether 
contactin could be acting as a receptor on the IMR-32 cells for 
RPTP.beta., the effects of antibodies against contactin on the growth of 
cells on different substrates were tested. Antibodies against contactin 
inhibited the growth of processes on .beta.C-Fc and .beta.CF-Fc but not on 
laminin (FIG. 7B). In the presence of these antibodies, the IMR-32 cells 
also retracted their processes and many cells lifted off the dish yielding 
fewer cells after 2 days of incubation. No effect was observed with 
control antibodies. Thus, the neurite growth, differentiation and survival 
induced by the carbonic anhydrase-like domain of RPTP.beta. is mediated by 
contactin present in the neurons. 
The present invention is not to be limited in scope by the exemplified 
embodiments which are intended as illustration of single aspects of the 
invention, and any clones, DNA or amino acid sequences which are 
functional equivalent are within the scope of the invention. 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 drawings. Such modifications are intended to 
fall within the scope of the appended claims. 
All references cited herein are hereby incorporated by reference in their 
entirety. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 270 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LysLeuValGluGluIleGlyTrpSerTyrThrGlyAlaLeuAsnGln 
151015 
LysAsnTrpGlyLysLysTyrProThrCysAsnSerProLysGlnSer 
202530 
ProIleAsnIleAspGluAspLeuThrGlnValAsnValAsnLeuLys 
354045 
LysLeuLysPheGlnGlyTrpAspLysThrSerLeuGluAsnThrPhe 
505560 
IleHisAsnThrGlyLysThrValGluIleAsnLeuThrAsnAspTyr 
65707580 
ArgValSerGlyGlyValSerGluMetValPheLysAlaSerLysIle 
859095 
ThrPheHisTrpGlyLysCysAsnMetSerSerAspGlySerGluHis 
100105110 
SerLeuGluGlyGlnLysPheProLeuGluMetGlnIleTyrCysPhe 
115120125 
AspAlaAspArgPheSerSerPheGluGluAlaValLysGlyLysGly 
130135140 
LysLeuArgAlaLeuSerIleLeuPheGluValGlyThrGluGluAsn 
145150155160 
LeuAspPheLysAlaIleIleAspGlyValGluSerValSerArgPhe 
165170175 
GlyLysGlnAlaAlaLeuAspProPheIleLeuLeuAsnLeuLeuPro 
180185190 
AsnSerThrAspLysTyrTyrIleTyrAsnGlySerLeuThrSerPro 
195200205 
ProCysThrAspThrValAspTrpIleValPheLysAspThrValSer 
210215220 
IleSerGluSerGlnLeuAlaValPheCysGluValLeuThrMetGln 
225230235240 
GlnSerGlyTyrValMetLeuMetAspTyrLeuGlnAsnAsnPheArg 
245250255 
GluGlnGlnTyrPheSerArgGlnValPheSerSerTyrThr 
260265270 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1018 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetLysThrProLeuLeuValSerHisLeuLeuLeuIleSerLeuThr 
151015 
SerCysLeuGlyGluPheThrTrpHisArgArgTyrGlyHisGlyVal 
202530 
SerGluGluAspLysGlyPheGlyProIlePheGluGluGlnProIle 
354045 
AsnThrIleTyrProGluGluSerLeuGluGlyLysValSerLeuAsn 
505560 
CysArgAlaArgAlaSerProPheProValTyrLysTrpArgMetAsn 
65707580 
AsnGlyAspValAspLeuThrAsnAspArgTyrSerMetValGlyGly 
859095 
AsnLeuValIleAsnAsnProAspLysGlnLysAspAlaGlyIleTyr 
100105110 
TyrCysLeuAlaSerAsnAsnTyrGlyMetValArgSerThrGluAla 
115120125 
ThrLeuSerPheGlyTyrLeuAspProPheProProGluAspArgPro 
130135140 
GluValLysValLysGluGlyLysGlyMetValLeuLeuCysAspPro 
145150155160 
ProTyrHisPheProAspAspLeuSerTyrArgTrpLeuAsnGluPhe 
165170175 
ProValPheIleThrMetAspLysArgArgPheValSerGlnThrAsn 
180185190 
GlyAsnLeuTyrIleAlaAsnValGluSerSerAspArgGlyAsnTyr 
195200205 
SerCysPheValSerSerProSerIleThrLysSerValPheSerLys 
210215220 
PheIleProLeuIleProIleProGluArgThrThrLysProTyrPro 
225230235240 
AlaAspIleValValGlnPheLysAspIleTyrThrMetMetGlyGln 
245250255 
AsnValThrLeuGluCysPheAlaLeuGlyAsnProValProAspIle 
260265270 
ArgTrpArgLysValLeuGluProMetProThrThrAlaGluIleSer 
275280285 
ThrSerGlyAlaValLeuLysIlePheAsnIleGlnLeuGluAspGlu 
290295300 
GlyLeuTyrGluCysGluAlaGluAsnIleArgGlyLysAspLysHis 
305310315320 
GlnAlaArgIleTyrValGlnAlaPheProGluTrpValGluHisIle 
325330335 
AsnAspThrGluValAspIleGlySerAspLeuTyrTrpProCysVal 
340345350 
AlaThrGlyLysProIleProThrIleArgTrpLeuLysAsnGlyTyr 
355360365 
AlaTyrHisLysGlyGluLeuArgLeuTyrAspValThrPheGluAsn 
370375380 
AlaGlyMetTyrGlnCysIleAlaGluAsnAlaTyrGlyThrIleTyr 
385390395400 
AlaAsnAlaGluLeuLysIleLeuAlaLeuAlaProThrPheGluMet 
405410415 
AsnProMetLysLysLysIleLeuAlaAlaLysGlyGlyArgValIle 
420425430 
IleGluCysLysProLysAlaAlaProLysProLysPheSerTrpSer 
435440445 
LysGlyThrGluTrpLeuValAsnSerSerArgIleLeuIleTrpGlu 
450455460 
AspGlySerLeuGluIleAsnAsnIleThrArgAsnAspGlyGlyIle 
465470475480 
TyrThrCysPheAlaGluAsnAsnArgGlyLysAlaAsnSerThrGly 
485490495 
ThrLeuValIleThrAsnProThrArgIleIleLeuAlaProIleAsn 
500505510 
AlaAspIleThrValGlyGluAsnAlaThrMetGlnCysAlaAlaSer 
515520525 
PheAspProSerLeuAspLeuThrPheValTrpSerPheAsnGlyTyr 
530535540 
ValIleAspPheAsnLysGluIleThrHisIleHisTyrGlnArgAsn 
545550555560 
PheMetLeuAspAlaAsnGlyGluLeuLeuIleArgAsnAlaGlnLeu 
565570575 
LysHisAlaGlyArgTyrThrCysThrAlaGlnThrIleValAspAsn 
580585590 
SerSerAlaSerAlaAspLeuValValArgGlyProProGlyProPro 
595600605 
GlyGlyLeuArgIleGluAspIleArgAlaThrSerValAlaLeuThr 
610615620 
TrpSerArgGlySerAspAsnHisSerProIleSerLysTyrThrIle 
625630635640 
GlnThrLysThrIleLeuSerAspAspTrpLysAspAlaLysThrAsp 
645650655 
ProProIleIleGluGlyAsnMetGluSerAlaLysAlaValAspLeu 
660665670 
IleProTrpMetGluTyrGluPheArgValValAlaThrAsnThrLeu 
675680685 
GlyThrGlyGluProSerIleProSerAsnArgIleLysThrAspGly 
690695700 
AlaAlaProAsnValAlaProSerAspValGlyGlyGlyGlyGlyThr 
705710715720 
AsnArgGluLeuThrIleThrTrpAlaProLeuSerArgGluTyrHis 
725730735 
TyrGlyAsnAsnPheGlyTyrIleValAlaPheLysProPheAspGly 
740745750 
GluGluTrpLysLysValThrValThrAsnProAspThrGlyArgTyr 
755760765 
ValHisLysGluThrMetThrProSerThrAlaPheGlnValLysVal 
770775780 
LysAlaPheAsnAsnLysGlyAspGlyProTyrSerLeuIleAlaVal 
785790795800 
IleAsnSerAlaGlnAspAlaProSerGluAlaProThrGluValGly 
805810815 
ValLysValLeuSerSerSerGluIleSerValHisTrpLysHisVal 
820825830 
LeuGluLysIleValGluSerTyrGlnIleArgTyrAlaGlyHisAsp 
835840845 
LysGluAlaAlaAlaHisArgValGlnValThrSerGlnGluTyrSer 
850855860 
AlaArgLeuGluAsnLeuLeuProAspThrGlnTyrPheIleGluVal 
865870875880 
GlyAlaCysAsnSerAlaGlyCysGlyProSerSerAspValIleGlu 
885890895 
ThrPheThrArgLysAlaProProSerGlnProProArgIleIleSer 
900905910 
SerValArgSerGlySerArgTyrIleIleThrTrpAspHisValVal 
915920925 
AlaLeuSerAsnGluSerThrValThrGlyTyrLysIleLeuTyrArg 
930935940 
ProAspGlyGlnHisAspGlyLysLeuPheSerThrHisLysHisSer 
945950955960 
IleGluValProIleProArgAspGlyGluTyrValValGluValArg 
965970975 
AlaHisSerAspGlyGlyAspGlyValValSerGlnValLysIleSer 
980985990 
GlyValSerThrLeuSerSerGlyLeuLeuSerLeuLeuLeuProSer 
99510001005 
LeuGlyPheLeuValPheTyrSerGluPhe 
10101015 
__________________________________________________________________________