Methods for identifying modulators of insulin receptor phosphorylation

The present invention relates to cell lines useful for the screening and identification of compounds that by modulating phosphotyrosine phosphatase activity, modulate insulin receptor type tyrosine kinase mediated signal transduction. Genetically engineered cells expressing IR in culture overcome the effect of insulin on morphology and adhesion when they are also coexpressing RPTP.alpha. or RPTP.epsilon.. Such engineered cell lines may be used to screen and identify non-toxic compounds that could elicit or modulate insulin signal transduction even in the absence of insulin.

INTRODUCTION 
The present invention relates to genetically engineered cells useful for 
the screening and identifying of compounds that affect insulin 
receptor-type tyrosine kinase mediated signal transduction. 
The present invention further relates to methods for screening and 
identifying of specific compounds, that by modulating the activity of the 
controlling protein phosphotyrosine phosphatases, have uses in the 
treatment of diabetes and other diseases. 
BACKGROUND OF THE INVENTION 
SIGNAL TRANSDUCTION 
Cellular signal transduction is a fundamental mechanism whereby external 
stimuli regulate diverse cellular processes are relayed to the interior of 
cells. The process is generally initiated by the binding of extracellular 
factors (such as hormones and growth factors) to membrane receptors on the 
cell surface. The biochemical pathways through which signals are 
transmitted within cells comprise a circuitry of directly or functionally 
connected interactive proteins. 
One of the key biochemical mechanisms of signal transduction involves the 
reversible phosphorylation of tyrosine residues on proteins. The 
phosphorylation state of a protein may affect its conformation and/or 
enzymic activity as well as its cellular location. The phosphorylation 
state of a protein is modified through the reciprocal actions of protein 
tyrosine kinases (PTKs) and protein phosphotyrosine phosphatases (PTPs). 
Generally, the level of tyrosine phosphorylation increases after the cell 
has been stimulated by an extracellular factor. Research has largely 
focussed on the protein kinases (Sefton et al., 1980, Cell 20:807-16; 
Heldin and Westermark, 1984, Cell 37:9-20; Yarden and Ullrich, 1988, Ann. 
Rev. Biochem. 57:443-78; Ullrich and Schlessinger, 1990, Cell, 61:203-12). 
Protein tyrosine kinases comprise a large family of transmembrane as well 
as cytoplasmic enzymes with multiple functional domains (Taylor et al., 
1992, Ann. Rev. Cell Biol. 8:429-62). The binding of an extracellular 
factor or ligand allosterically transduces the signal to the inner face of 
the cell membrane where the cytoplasmic portion of the receptor protein 
tyrosine kinase (RPTKs) initiates a cascade of molecular interactions that 
disseminate the signal throughout the cell and into the nucleus. 
Ligand-induced activation of the kinase domain and its signalling potential 
are mediated by receptor dimerization. Once activated, the receptor 
self-phosphorylates (autophosphorylation or transphosphorylation) on 
specific tyrosine residues of the cytoplasmic domain. (Schlessinger, 1988, 
Trends Biochem. Sci. 13:443-7, Schlessinger and Ullrich, 1992, Neuron, 
9:383-91, and references therein). 
Like the PTKs, the protein phosphotyrosine phosphatases (PTP) comprise a 
family of transmembrane and cytoplasmic enzymes. (Hunter, 1989, Cell, 
58:1013-16; Fischer et al., 1991, Science, 253:401-6; Saito and Streuli, 
1991, Cell growth and differentiation, 2:59-65; Pot and Dixon, 1992, 
Biochim. Biophys. Acta, 1136:35-43). As presently understood by those in 
the art, in general PTKs play a triggering role in signal transduction, 
while PTPs guarantee that the trigger is reset thereby serving to 
deactivate the pathway. However, the specific functions of PTPs have not 
yet been defined (Walton et al., 1993, Ann. Rev. Biochem., 66:101-20). 
In addition to a homologous core catalytic domain, mammalian PTPs share 
diverse noncatalytic sequences. While some receptor protein tyrosine 
phosphatases (RPTPS) contain in their extracellular portions Ig-like 
and/or fibronectin type III repeats (e.g., LAR, Streuli et al., 1988, J. 
Exp. Med. 168:1523); others have small extracellular glycosylated segments 
(e.g., RPTP.alpha., Sap et al., 1990, Proc. Natl. Acad. Sci. USA, 87:6112; 
and RPTP.epsilon., Krueger et al., 1990, EMBO J, 9:3241). In all cases, 
the putative ligands have yet to be identified. Other phosphotyrosine 
phosphatases such as PTP1B, PTP.mu., PTP1C, TC-PTP, PTPH1, RPTP.kappa. and 
CD45 have been cloned and their cDNAs are described in Chernoff et al., 
1990, Proc. Natl. Acad. Sci. USA, 87:2735-9; Gebbink et al., 1991, FEBS 
Lett. 290:123-30; Shen et al., 1991, Nature, 352:736-9; Jiang et al., 
1993, Mol. Cell Biol., 13:2942-51 and; Charbonneau et al., 1988, Proc. 
Natl. Acad. Sci. USA, 85:7182-6 respectively. 
Abnormal PTK/PTP signal transduction has been associated with a variety of 
diseases including psoriasis, cancer and diabetes. 
THE INSULIN RECEPTOR AND DIABETES MELLITUS 
The insulin receptor (IR)(Ullrich et al., Nature, 313:756-61, 1985) is the 
prototype for a family of RPTKs structurally defined as a heterotetrameric 
species of two .alpha. and two .beta. subunits. Other members of the 
insulin receptor-type protein tyrosine kinase (IR-PTK) family include, for 
example, the receptor for insulin-like growth factor 1 (IGF-1 R, Ullrich 
et al., 1986, EMBO J. 5:2503-12) and insulin related receptor (IRR, Zhang 
et al., 1992, J. Biol. Chem. 267:18320-8) the ligand(s) for which is at 
present unknown. 
The binding of insulin to the insulin receptor triggers a variety of 
metabolic and growth promoting effects. Metabolic effects include glucose 
transport, biosynthesis of glycogen and fats, inhibition of triglyceride 
breakdown, and growth promoting effects include DNA synthesis, cell 
division and differentiation. It is known that some of these biological 
effects of insulin can be mimicked by vanadium salts such as vanadates and 
pervanadates. However, this class of compounds appears to inhibit 
phosphotyrosine phosphatases generally, and are potentially toxic because 
they contain heavy metal (U.S. Pat. No. 5,155,031; Fantus et al., 1989, 
Biochem., 28:8864-71; Swarup et al., 1982, Biochem. Biophys. Res. Commun. 
107:1104-9). 
Diabetes mellitus is a heterogeneous primary disorder of carbohydrate 
metabolism with multiple etiologic factors that generally involve insulin 
deficiency or insulin resistance or both. Type I, or juvenile onset, or 
insulin-dependent diabetes mellitus, is present in patients with little or 
no endogenous insulin secretory capacity. These patients develop extreme 
hyperglycemia and are entirely dependent on exogenous insulin therapy for 
immediate survival. Type II, or adult onset, or non-insulin-dependent 
diabetes mellitus, occurs in patients who retain some endogenous insulin 
secretory capacity but the great majority of them are both insulin 
deficient and insulin resistant. Insulin resistence can be due to 
insufficient insulin receptor expression, reduced insulin-binding 
affinity, or any abnormality at any step along the insulin signaling 
pathway (Olefsky, 1988, in "Cecil Textbook of Medicine," 18th Ed., 
2:1360-81) 
Overall, in the United States the prevalence of diabetes is probably 
between 2 and 4 per cent, with Type I comprising 7 to 10 per cent of all 
cases. Secondary complications of diabetes have serious clinical 
implications, such as amputations (primarily of toes, feet, and legs) and 
blindness. 
Insulin is the primary mode of therapy in all patients with Type I diabetes 
and in many with Type II diabetes. Oral hypoglycemic agents such as 
sulfonylureas are effective in Type II diabetic patients but approximately 
10 to 20 per cent of patients do not respond or cease to respond 12-24 
months after treatment began. 
Effective control of glucose level is difficult to achieve for prolonged 
periods even with the most meticulous mode of insulin therapy in the most 
motivated patients. Transplantation of the pancreas or islet cells, which 
normally produce insulin, continues to receive extensive study as a 
potential treatment. In addition, efforts towards developing newer and 
better external or implantable insulin-delivery devices integrated with a 
glucose sensor continues. 
SUMMARY OF THE INVENTION 
The present invention relates to cell lines useful for the screening and 
identification of compounds that modulate insulin receptor-type tyrosine 
kinase (IR-PTK) mediated signal transduction. 
The invention is based, in part, on the discovery that genetically 
engineered cells coexpressing IR and RPTP.alpha. or RPTP.epsilon. in 
culture are not sensitive to the effects of insulin on cell morphology and 
adhesion. The phenotype of the cells may be used as an indicator of 
insulin mediated signal transduction. The claimed cell lines of the 
invention are, therefore, useful in screening assays for non-toxic 
compounds, that by modulating phosphatase activity, modulate or prolong 
IR-PTK signal transduction. 
In specific embodiments of the present invention detailed in the example 
section infra, the stable coexpression of IR and RPTP.alpha. or 
RPTP.epsilon. in baby hamster kidney (BHK) cells, and the development of 
cell-based assay system for IR signal transduction are described.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to cell lines useful in screening assays 
for compounds that modulate insulin receptor-type tyrosine kinase (IR-PTK) 
mediated signal transduction. The IR-PTKs include insulin receptor, 
insulin-like growth factor 1 (IGF-1 R) and insulin receptor related 
receptor (IRR). As used herein, the term signal transduction is not 
limited to transmembrane signalling, and includes the multiple pathways 
that branch off throughout the cell and into the nucleus. The term ligand 
is synonymous with extracellular signalling molecules, and includes 
insulin, IGF-1, IGF-2 and other hormones, growth factors or cytokines that 
may interact with IR-PTKs. 
Genetically engineered cells expressing IR are sensitive to the presence of 
insulin in culture and this sensitivity is easily detected. More 
specifically, the cells respond to insulin by losing their normaly flat 
and adherent phenotype, and instead, round up and become detached from the 
culture dish. However, when these IR-expressing cells are transfected with 
DNA encoding RPTP.alpha. or RPTP.epsilon., the cells coexpressing IR and 
the phosphatase are able to grow normally in the presence of insulin. 
Although, the inventors do not want to be bound by any specific 
theoretical mechanism, it is possible that the presence of the phosphatase 
restores balance to the signal transduction pathways activated by the 
insulin receptor in the presence of its ligand. 
In a preferred embodiment of the invention, genetically engineered cell 
lines coexpressing IR and RPTP.alpha. or RPTP.epsilon. may be used to 
screen and identify compounds which, by modulating the activity of 
RPTP.alpha. or RPTP.epsilon., elicit, modulate or prolong insulin receptor 
signal transduction. 
COEXPRESSION OF RPTPs AND IR-PTK AND GENERATION OF ENGINEERED CELL LINES 
In accordance with the invention, RPTP.alpha., RPTP.epsilon. and IR 
nucleotide sequences or functional equivalents thereof may be used to 
generate recombinant DNA molecules that direct the coexpression of 
RPTP.alpha. or RPTP.epsilon. and IR proteins or a functionaly equivalent 
thereof, in appropriate host cells. The nucleotide sequences of 
RPTP.alpha., RPTP.epsilon. and IR are reported in Sap et al., 1990, Proc. 
Natl. Acad. Sci. USA, 87:6112-6 and Kaplan et al., 1990, Proc. Natl. Acad. 
Sci. USA, 87:7000-4; Krueger et al., 1990, EMBO J, 9:3241-52; and Ullrich 
et al., 1985, Nature 313:756-61 respectively and are incorporated by 
reference herein in their entirety. The specific interaction between 
RPTP.alpha., RPTP.epsilon. and IR may involve the formation of a transient 
or stable multimolecular complex, hereinafter, referred to as 
RPTP.alpha.-IR, RPTP.epsilon.-IR complex or generally RPTP-IR-PTK complex. 
As used herein, a functionaly equivalent RPTP.alpha., RPTP.epsilon. or IR 
refers to an enzyme with essentially the same catalytic function, but not 
necessarily the same catalytic activity as its native counterpart. A 
functionally equivalent receptor refers to a receptor which binds to its 
cognate ligand, but not necessarily with the same binding affinity of its 
counterpart native receptor. 
Due to the inherent degeneracy of the genetic code, other DNA sequences 
which encode substantially the same or a functionally equivalent amino 
acid sequence, may be used in the practice of the invention for the 
coexpression of the RPTP.alpha. or RPTP.epsilon. and IR proteins. Altered 
DNA sequences which may be used in accordance with the invention include 
deletions, additions or substitutions. For example, mutations may be 
introduced using techniques which are well known in the art, e.g. site 
directed mutagenesis, to insert new restriction sites, to alter 
glycosylation patterns, phosphorylation, etc. Amino acid substitutions may 
be made on the basis of similarity in polarity, charge, solubility, 
hydrophobicity, hydrophilicity, and/or the amphipatic nature of the 
residues involved. 
The RPTP.alpha., RPTP.epsilon. or IR or a modified RPTP.alpha., 
RPTP.epsilon. or IR sequence may be ligated to a heterologous sequence to 
encode a fusion protein. For example, for screening of peptide libraries 
it may be useful to encode a chimeric RPTP.alpha., RPTP.epsilon. or IR 
protein expressing a heterologous epitope that is recognized by an 
antibody. A fusion protein may also be engineered to contain the 
ligand-binding, regulatory or catalytic domain of another PTP or PTK. 
The coding sequence of RPTP.alpha., RPTP.epsilon. or IR could be 
synthesized in whole or in part, using chemical methods well known in the 
art. See, for example, Caruthers, et al., 1980, Nuc. Acids Res. Symp. Ser. 
7:215-233; Crea and Horn, 180, Nucleic Acids Res. 9(10):2331; Matteucci 
and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981, 
Nucleic Acids Res. 9(12):2807-2817. 
In order to coexpress a biologically active RPTP.alpha., RPTP.epsilon. or 
IR, the nucleotide sequence coding for RPTP.alpha., RPTP.epsilon. or IR, 
or their functional equivalent(s) as described supra, is inserted into one 
or more appropriate expression vector(s), i.e., a vector which contains 
the necessary elements for the transcription and translation of the 
inserted coding sequence(s). The RPTP.alpha. and/or RPTP.epsilon. gene(s) 
may be placed in tandem with the IR sequence under the control of the same 
or different promoter used to control the expression of the other coding 
sequence. The two phosphatases, RPTP.alpha. and RPTP.epsilon. may also be 
coexpressed together with IR. 
Methods which are well known to those skilled in the art can be used to 
construct expression vectors containing the RPTP.alpha., RPTP.epsilon. 
and/or IR coding sequence(s) and appropriate transcriptional/translational 
control signals. These methods include in vitro recombinant DNA 
techniques, synthetic techniques and in vivo recombination/genetic 
recombination. See, for example, the techniques described in Maniatis et 
al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor 
Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular 
Biology, Greene Publishing Associates and Wiley Interscience, N.Y. 
A variety of host-expression vector systems may be utilized to coexpress 
the RPTP.alpha., RPTP.epsilon., or IR coding sequences. These include but 
are not limited to microorganisms such as bacteria transformed with 
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression 
vectors containing the RPTP.alpha., RPTP.epsilon., or IR coding 
sequence(s) (see, Current Protocols in Molecular Biology, Vol. 2, 1988, 
Ed. Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Section 
16.1); yeast transformed with recombinant yeast expression vectors 
containing the RPTP.alpha., RPTP.epsilon., or IR coding sequence(s) 
(Bitner, 1987, Heterologous Gene Expression in Yeast, Methods Enzymol, 
Eds. Berger & Mimmel, Acad. Press, N.Y. 152:673-84); insect cell systems 
infected with recombinant virus expression vectors (e.g., baculovirus, see 
Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No. 4,215,051) 
containing the RPTP.alpha., RPTP.epsilon.and/or IR coding sequence(s); 
plant cell systems infected with recombinant virus expression vectors 
(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or 
transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) 
containing the RPTP.alpha., RPTP.epsilon.and/or IR coding sequence(s) (see 
Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic 
Press, NY); or animal cell systems. 
In mammalian host cells, a number of viral based expression systems may be 
utilized. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 
81:3655-3659, Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 
79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864). 
A host cell of a particular cell type may also be chosen for the cell 
type-specific cofactors which may be required for the specific signalling 
pathway. A host cell strain may also be chosen which modulates the 
expression of the inserted sequences, or modifies and processes the gene 
product in the specific fashion desired. Such modifications (e.g., 
glycosylation) and processing (e.g., cleavage) of protein products may be 
important for the function of the protein. Different host cells have 
characteristic and specific mechanisms for the post-translational 
processing and modification of proteins. Appropriate cells lines or host 
systems can be chosen to ensure the correct modification and processing of 
the foreign protein expressed. To this end, eukaryotic host cells which 
possess the cellular machinery for proper processing of the primary 
transcript, glycosylation, and phosphorylation of the gene product may be 
used. Such mammalian host cells include but are not limited to CHO, VERO, 
BHK, HeLa, COS, MDCK, 293, WI38, PC12 etc. 
Stable expression is preferred for long-term, high-yield production of 
recombinant proteins in animal cells. Rather than using expression vectors 
which contain viral origins of replication, host cells can be transformed 
with RPTP.alpha., RPTP.epsilon., or IR DNA controlled by appropriate 
expression control elements (e.g., promoter, enhancer, sequences, 
transcription terminators, polyadenylation sites, etc.), and a selectable 
marker. Following the introduction of foreign DNA, engineered cells may be 
allowed to grow for 1-2 days in an enriched media, and then are switched 
to a selective media. The selectable marker in the recombinant plasmid 
confers resistance to the selection and allows cells to stably integrate 
the plasmid into their chromosomes and grow to form foci which in turn can 
be cloned and expanded into cell lines. This method, which is demonstrated 
in the examples below, may advantageously be used to engineer cell lines 
which stably coexpress both the RTP and IR-PTK, and which respond to 
ligand mediated signal transduction. Such engineered cell lines are 
particularly useful in screening PTP inhibitors, stimulators and analogs. 
A number of selection systems may be used, including but not limited to the 
herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), 
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 
1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine 
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can be 
employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells, respectively. Also, 
antimetabolite resistance can be used as the basis of selection for dhfr, 
which confers resistance to methotrexate (Wigler, et al., 1980, Natl. 
Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 
78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & 
Berg, 1981), Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers 
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. 
Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin 
(Santerre, et al., 1984, Gene 30:147) genes. Recently, additional 
selectable genes have been described, namely trpB, which allows cells to 
utilize indole in place of tryptophan; hisD, which allows cells to utilize 
histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. 
Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase) which confers 
resistance to the ornithine decarboxylase inhibitor, 
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current 
Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.). 
As the IR-PTK and RPTP may be coexpressed from different expression 
plasmids in the same cell, a different amplifiable selection system (for 
example, dhfr and adenosine deaminase) may be used for each individual 
plasmid. By applying different concentrations of the selecting drugs, the 
expression level of individual protein may be controlled separately as 
required (Wood et al., 1990, J. Immunol. 145:3011-16). 
The host cells which contain the coding sequences and which express the 
biologically active gene products may be identified by at least three 
general approaches; (a) DNA--DNA or DNA-RNA hybridization; (b) the 
presence or absence of "marker" gene functions; and (c) detection of the 
gene products as measured by immunoassay or by their biological activity. 
In the first approach, the presence of the RPTP.alpha., RPTP.epsilon. or IR 
coding sequence(s) inserted in the expression vector(s) can be detected by 
DNA--DNA or DNA-RNA hybridization using probes comprising nucleotide 
sequences that are homologous to the RPTP.alpha., RPTP.epsilon. or IR 
coding sequence(s), respectively, or portions or derivatives thereof. 
In the second approach, the recombinant expression vector/host system can 
be identified and selected based upon the presence or absence of certain 
"marker" gene functions (e.g., thymidine kinase activity, resistance to 
antibiotics, resistance to methotrexate, transformation phenotype, 
occlusion body formation in baculovirus, etc.). For example, if the 
RPTP.alpha., RPTP.epsilon. or IR coding sequence(s) is inserted within a 
marker gene sequence of the vector, recombinant cells containing the 
RPTP.alpha., RPTP.epsilon. or IR coding sequence(s) can be identified by 
the absence of the marker gene function. Alternatively, a marker gene can 
be placed in tandem with the RPTP.alpha., RPTP.epsilon. or IR sequence 
under the control of the same or different promoter used to control the 
expression of the RPTP.alpha., RPTP.epsilon. or IR coding sequence(s). 
Expression of the marker in response to induction or selection indicates 
expression of the RPTP.alpha., RPTP.epsilon. or IR coding sequence(s). 
In the third approach, the expression of the RPTP.alpha., RPTP.epsilon. or 
IR protein product can be assessed immunologically, for example by Western 
blots, immunoassays such as immunoprecipitation, enzyme-linked 
immunoassays and the like. The ultimate test of the success of the 
expression system, however, involves the detection of the biologically 
active RPTP.alpha., RPTP.epsilon. or IR proteins. A number of assays can 
be used to detect activity including but not limited to ligand binding 
assays, phosphorylation assays, dephosphorylation assays, and biological 
assays using engineered cell lines as the test substrate. 
The RPTP.alpha., RPTP.epsilon. or IR gene products as well as host cells or 
cell lines transfected or transformed with recombinant RPTP.alpha., 
RPTP.epsilon. and IR expression vector(s) can be used for a variety of 
purposes. These include but are not limited to the screening and selection 
of RPTP.alpha. or RPTP.epsilon. analogs, or drugs that act by interacting 
with RTP-IR-PTK complex, or generating antibodies (i.e., monoclonal or 
polyclonal) that bind to the RTP-IR-PTK complex, including those that 
competitively inhibit the formation of such complexes. These gene products 
or host cells or cell lines may also be used for identifying other 
signalling molecules or their genes that are engaged in the insulin 
signalling pathway. 
ASSAY SYSTEMS FOR DRUG SCREENING 
In one embodiment of the invention, the RPTPs, the RPTP-IR-PTK complex, or 
cell lines that express the RPTPs or RPTP-IR-PTK complex, may be used to 
screen for molecules that modulate RPTP activity. Such molecules may 
include small organic or inorganic compounds, antibodies, peptides, or 
other molecules that modulate RPTP.alpha.'s or RPTP.epsilon.'s 
dephosphorylation activity toward IR, or that promote or prevent the 
formation of RPTP.alpha.-IR or RPTP.epsilon.-IR complex. Synthetic 
compounds, natural products, and other sources of potentially biologically 
active materials can be screened in a number of ways. 
The ability of a test molecule to modulate the activity of RPTP.alpha. or 
RPTP.epsilon. toward IR, hence signal transduction, may be measured using 
standard biochemical techniques, such as those described in section 6.1. 
Other responses such as activation or suppression of catalytic activity, 
phosphorylation or dephosphorylation of other proteins, activation or 
modulation of second messenger production, changes in cellular ion levels, 
association, dissociation or translocation of signalling molecules, or 
transcription or translation of specific genes may also be monitored. 
These assays may be performed using conventional techniques developed for 
these purposes in the course of screening. 
Ligand binding to its cellular receptor may, via signal transduction 
pathways, affect a variety of cellular processes. Cellular processes under 
the control of insulin signalling pathway may include, but are not limited 
to, normal cellular functions such as carbohydrate metabolism, 
proliferation, differentiation, maintenance of cell shape, and adhesion, 
in addition to abnormal or potentially deleterious processes such as 
apoptosis, loss of contact inhibition, blocking of differentiation or cell 
death. The qualitative or quantitative observation and measurement of any 
of the described cellular processes by techniques known in the art may be 
advantageously used as a means of scoring for signal transduction in the 
course of screening. 
Described in this section are methods of the invention for screening, 
identification and evaluation of compounds that interact with RPTP.alpha., 
RPTP.epsilon. and IR and may affect various cellular processes under the 
control of the insulin signalling pathway. 
The present invention includes a method for identifying a compound which is 
capable of, by modulating phosphotyrosine phosphatase activity of 
RPTP.alpha. and/or RPTP.epsilon., modulating insulin receptor type protein 
kinase IR-PTK signal transduction, comprising: 
(a) contacting the compound with RPTP.alpha. and/or RPTP.epsilon. and IR 
or, a functional derivatives thereof, in pure form, in a membrane 
preparation, or in a whole live or fixed cell; 
(b) incubating the mixture of step (a) for an interval sufficient for the 
compound to stimulate or inhibit the phosphotyrosine phosphatase enzymatic 
activity or the signal transduction; 
(c) measuring the phosphotyrosine phosphatase enzymatic activity or the 
signal transduction; 
(d) comparing the phosphotyrosine phosphatase enzymatic activity or the 
signal transduction activity to that of RPTP.alpha., and/or RPTP.epsilon. 
and IR, incubated without the compound, thereby determining whether the 
compound stimulates or inhibits signal transduction. 
RPTP.alpha. and/or RPTP.epsilon. and IR, or functional derivatives thereof, 
for example, having amino acid deletions and/or insertions and/or 
substitutions while maintaining signal transduction, can also be used for 
the testing of compounds. A functional derivative may be prepared from a 
naturally occurring or recombinantly expressed RPTP.alpha., RPTP.epsilon. 
and IR by proteolytic cleavage followed by conventional purification 
procedures known to those skilled in the art. Alternatively, the 
functional derivative may be produced by recombinant DNA technology by 
expressing only these parts of RPTP.alpha., RPTP.epsilon. or IR in 
suitable cells. Cells expressing RPTP.alpha. and/or RPTP.epsilon. and IR 
may be used as a source of RPTP.alpha., RPTP.epsilon. and/or IR, crude or 
purified, or in a membrane preparation, for testing in these assays. 
Alternatively, whole live or fixed cells may be used directly in those 
assays. The cells may be genetically engineered to coexpress RPTP.alpha., 
RPTP.epsilon. and IR. The cells may also be used as host cells for the 
expression of other recombinant molecules with the purpose of bringing 
these molecules into contact with RPTP.alpha., RPTP.epsilon. and/or IR 
within the cell. 
IR-PTK signal transduction activity may be measured by standard biochemical 
techniques or by monitoring the cellular processes controlled by the 
signal. To assess modulation of phosphatase activity, the test molecule is 
added to a reaction mixture containing the phosphorylated substrate and 
the phosphatase. To assess modulation of kinase activity of the IR-PTK, 
the test molecule is added to a reaction mixture containing the IR-PTK and 
its substrate (in the case of autophosphorylation, the IR-PTK is also the 
substrate). Where the test molecule is intended to mimic ligand 
stimulation the assay is conducted in the absence of insulin. Where the 
test molecule is intended to reduce or inhibit insulin activity the 
presence of insulin. The kinase reaction is then initiated with the 
addition of ATP. An immunoassay is performed on the kinase or phosphatase 
reaction to detect the presence of absence of the phosphorylated tyrosine 
residues on the substrate, and results are compared to those obtained for 
controls i.e., reaction mixtures not exposed to the test molecule. The 
immunoassay used to detect the phosphorylated substrate in the cell lysate 
or the in vitro reaction mixture may be carried out with an 
anti-phosphotyrosine antibody. Signal transduction is mimicked if the 
cellular processes under the control of the signalling pathway are 
affected in a way similar to that caused by ligand binding. Such compounds 
may be naturally occurring or synthetically produced molecules that could 
replace the administration of insulin in the treatment of diabetes. 
The invention also includes a method whereby a molecule capable of binding 
to RPTP.alpha. and/or RPTP.epsilon. and IR in a chemical or biological 
preparation may be identified comprising: 
(a) immobilizing RPTP.alpha. and/or RPTP.epsilon. and IR, or fragments 
thereof, to a solid phase matrix; 
(b) contacting the chemical or biological preparation with the solid phase 
matrix produced in step (a), for an interval sufficient to allow the 
compound to bind; 
(c) washing away any unbound material from the solid phase matrix; 
(d) detecting the presence of the compound bound to the solid phase, 
thereby identifying the compound. 
The above method may further include the step of: 
(e) eluting the bound compound from the solid phase matrix, thereby 
isolating the compound. 
The term "compound capable of binding to RPTP.alpha. and/or RPTP.epsilon. 
and IR" refers to a naturally occurring or synthetically produced molecule 
which interacts with RPTP.alpha. and/or RPTP.epsilon. and IR. Such a 
compound may directly or indirectly modulate IR-PTK signal transduction 
and may include molecules that are natively associated with RPTP.alpha., 
RPTP.epsilon. and/or IR inside a cell. Examples of such compounds are (i) 
a natural substrate, of RPTP.alpha. and/or RPTP.epsilon.; (ii) a naturally 
occurring molecule which is part of the signalling complex; iii) a natural 
substrate of IR-PTK, iv) a naturally occuring signalling molecule produced 
by other cell types. 
The present invention also includes methods for identifying the specific 
site(s) of RPTP.alpha., or RPTP.epsilon. interaction with IR. Using the 
methods described herein, and biochemical and molecular biological methods 
well-known in the art, it is possible to identify the corresponding 
portions of RPTP.alpha., RPTP.epsilon. and IR involved in this 
interaction. For example, site-directed mutagenesis of DNA encoding either 
RPTP.alpha., RPTP.epsilon. or IR may be used to destroy or inhibit he 
interaction between the two molecules. Biophysical methods such as X-ray 
crystallography and nuclear magnetic resonance may also be used to map and 
study these sites of interaction. Once these sites have been identified, 
the present invention provides means for promoting or inhibiting this 
interaction, depending upon the desired biological outcome. Based on the 
foregoing, given the physical information on the sites of interaction is 
known, compounds that modulate catalytic activity and signal transduction 
may be elaborated by standard methods well known in the field of rational 
drug design. 
The present invention further provides an assay for identifying a compound, 
which can block the interaction of RPTP.alpha. or RPTP.epsilon. and IR. 
For example, a cell transfected to coexpress RPTP.alpha. or RPTP.epsilon. 
and IR, in which the two proteins interact to form a RPTP.alpha.-IR or 
RPTP.epsilon.-IR complex, can be incubated with an agent suspected of 
being able to inhibit this interaction, and the effect on the interaction 
measured. Any of a number of means for measuring the interaction and its 
disruption, such as coimmunoprecipitation, are available. The present 
invention also provides an assay method to identify and test a compound 
which stabilizes and promotes the interaction, using the same approach 
described above for a potential inhibitor. 
Random peptide libraries consisting of all possible combinations of amino 
acids may be used to identify peptides that are able to bind to the 
substrate binding site of RPTP.alpha. or RPTP.epsilon., or other 
functional domains of RPTP.alpha. or RPTP.epsilon.. Similarily, such 
libraries may also be used to identify peptides that are able to bind to 
the IR's site of interaction with RPTP.alpha. or RPTP.epsilon.. 
Identification of molecules that are able to bind to RPTP.alpha., 
RPTP.epsilon. and IR may be accomplished by screening a peptide library 
with recombinant RPTP.alpha., RPTP.epsilon. or IR proteins or recombinant 
soluble forms of RPTP.alpha. or RPTP.epsilon. or IR protein. 
Alternatively, the phosphatase and extracellular ligand binding domains of 
RPTP.alpha. or RPTP.epsilon. may be separately expressed and used to 
screen peptide libraries. 
One way to identify and isolate the peptide that interacts and forms a 
complex with RPTP.alpha. or RPTP.epsilon. and IR, may involve labeling or 
"tagging" RPTP.alpha. or RPTP.epsilon. and IR proteins. The RPTP.alpha. or 
RPTP.epsilon. and IR proteins may be conjugated to enzymes such as 
alkaline phosphatase or horseradish peroxidase or to other reagents such 
as fluorescent labels which may include fluorescein isothyiocynate (FITC), 
phycoerythrin (PE) or rhodamine. Conjugation of any given label, to 
RPTP.alpha. or RPTP.epsilon. and IR, may be performed using techniques 
that are routine in the art. Alternatively, RPTP.alpha., RPTP.epsilon. or 
IR expression vectors may be engineered to express a chimeric RPTP.alpha., 
RPTP.epsilon. or IR protein containing an epitope for which a commercially 
available antibody exists. The epitope specific antibody may be tagged 
using methods well known in the art including labeling with enzymes, 
fluorescent dyes or colored or magnetic beads. 
The present invention also includes a method for identifying and isolating 
a nucleic acid molecule encoding a gene product which is capable of, by 
modulating phosphotyrosine phosphatase activity RPTP.alpha. and/or 
RPTP.epsilon., modulating IR-PTK signal transduction, comprising: 
(a) introducing the nucleic acid molecule into host cells coexpressing 
RPTP.alpha. and/or RPTP.epsilon. and IR or fragments thereof; 
(b) culturing the cells so that the gene product encoded by the nucleic 
acid molecule is expressed in the host cells and interacts with 
RPTP.alpha. and/or RPTP.epsilon. and IR or fragments thereof; 
(c) measuring the phosphotyrosine phosphatase enzymatic activity of 
RPTP.alpha. and/or RPTP.epsilon. or IR-PTK signal transduction activity; 
(d) comparing the phosphotyrosine phosphatase enzymatic activity or signal 
transduction to that of RPTP.alpha. and/or RPTP.epsilon. and IR, or 
fragments thereof in cells without the nucleic acid molecule, thereby 
determining whether the gene product encoded by the nucleic acid molecule 
modulates IR-PTK signal transduction. 
The above method may further include the step of: 
(e) selecting and culturing the cells identified in step (d), recovering 
the nucleic acid molecule, thereby isolating the nucleic acid molecule. 
By the term "nucleic acid molecule" is meant a naturally occurring or 
recombinantly generated nucleic acid molecule containing a nucleotide 
sequence operatively associated with an element that controls expression 
of the nucleotide sequence. An expression library may be created by 
introducing into host cells a pool of different nucleic acid molecules 
encoding different gene products. The host cells may be genetically 
engineered to coexpress RPTP.alpha., RPTP.epsilon. and IR. Such a gene 
library may be screened by standard biochemical techniques or by 
monitoring the cellular processes controlled by the signal. This approach 
is especially useful in identifying other native signalling molecules that 
are also involved in the signalling pathway. 
ANTIBODY PRODUCTION AND SCREENING 
Various procedures known in the art may be used for the production of 
antibodies to epitopes of the recombinantly produced RPTP.alpha., 
RPTP.epsilon., IR, RPTP.alpha.-IR and RPTP.epsilon.-IR complex. Such 
antibodies include but are not limited to polyclonal, monoclonal, 
chimeric, single chain, Fab fragments and fragments produced by an Fab 
expression library. Neutralizing antibodies i.e., those which compete for 
the substrate binding site of RPTP.alpha. or RPTP.epsilon., or the IR's 
site of interaction with RPTP.alpha. or RPTP.epsilon. are especially 
preferred for therapeutics. 
For the production of antibodies, various host animals may be immunized by 
injection with RPTP.alpha., RPTP.epsilon., IR, RPTP.alpha.-IR or 
RPTP.epsilon.-IR complex, or genetically engineered cells expressing 
RPTP.alpha., RPTP.epsilon. and IR, including but not limited to rabbits, 
mice, rats, etc. Various adjuvants may be used to increase the 
immunological response, depending on the host species, including but not 
limited to Freund's (complete and incomplete), mineral gels such as 
aluminum hydroxide, surface active substances such as lysolecithin, 
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet 
hemocyanin, dinitrophenol, and potentially useful human adjuvants such as 
BCG (bacille Calmette-Guerin) and Corynebacterium parvum. 
Monoclonal antibodies to RPTP.alpha., RPTP.epsilon., IR, RPTP.alpha.-IR and 
RPTP.epsilon.-IR complex may be prepared by using any technique which 
provides for the production of antibody molecules by continuous cell lines 
in culture. These include but are not limited to the hybridoma technique 
originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), 
the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology 
Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and 
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and 
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques 
developed for the production of "chimeric antibodies" (Morrison et al., 
1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, 
Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing 
the genes from a mouse antibody molecule of appropriate antigen 
specificity together with genes from a human antibody molecule of 
appropriate biological activity can be used. Alternatively, techniques 
described for the production of single chain antibodies (U.S. Pat. No. 
4,946,778) can be adapted to produce RPTP.alpha., RPTP.epsilon., IR, 
RPTP.alpha.-IR or RPTP.epsilon.-IR complex-specific single chain 
antibodies. 
Antibody fragments which contain specific binding sites of RPTP.alpha., 
RPTP.epsilon., IR, RPTP.alpha.-IR or RPTP.epsilon.-IR complex may be 
generated by known techniques. For example, such fragments include but are 
not limited to: the F(ab').sub.2 fragments which can be produced by pepsin 
digestion of the antibody molecule and the Fab fragments which can be 
generated by reducing the disulfide bridges of the F(ab').sub.2 fragments. 
Alternatively, Fab expression libraries may be constructed (Huse et al., 
1989, Science, 246:1275-1281) to allow rapid and easy identification of 
monoclonal Fab fragments with the desired specificity to RPTP.alpha., 
RPTP.epsilon., IR, RPTP.alpha.-IR or RPTP.epsilon.-IR complex. 
EXAMPLE: DEMONSTRATION OF AN IN VIVO SELECTION SYSTEM FOR INSULIN RECEPTOR 
ACTIVATION 
In the example described below, host cells were engineered to express both 
the IR and a series of PTPs. The cells expressing IR alone or IR plus an 
ineffective PTP display an altered phenotype when exposed to insulin. The 
results show that coexpression of RPTP.alpha. or RPTP.epsilon. inhibits 
phosphorylation of the IR and restores normal cell phenotype. The results 
demonstrate that RPTP-.alpha. and RPTP-.epsilon. modulate with IR signal 
transduction. 
MATERIAL AND METHODS 
IR/BHK cells were maintained in DMEM/high glucose, 10% fetal calf serum, 10 
mM glutamine, 1 .mu.M methotrexate plus antibiotics. The cDNAs for 
RPTP.alpha. or RPTP.epsilon. were cloned into a cytomegalovirus early 
promoter-based expression plasmid pCMV (Eaton et al., Biochemistry, 
25:8343-47, 1986). Plasmid DNA were transfected into 10.sup.7 BHK cells/10 
cm.sup.2 plate according to the protocol of Chen and Okayama (Mol. Cell 
Biol., 7:2745-52, 1987). Eighteen hours after the addition of DNA 
precipitate, cells were washed once and supplied with fresh medium 
containing 0.5% serum. Forty-eight hours after transfection, the cells 
were split at least 1:10. Medium containing 1 .mu.M insulin was added 12 
hours later. Medium containing insulin was changed 3 times a day. Cells in 
culture were washed thoroughly with PBS each time the media was changed in 
order to remove detached cells. 
The presence of insulin does not cause cell death but detachment, so it is 
necessary to maintain the selective pressure of insulin presence until 
stable co-transfected clones have grown to sufficient numbers to be 
isolated and characterized. This process took approximately four weeks. 
Antibodies used in the analysis of protein expression and phosphorylation 
were the mouse monoclonal antiphosphotyrosine antibody 5E2 (Fendly et al., 
1990, Cancer Res., 50:1550-8), mouse anti-IR monoclonal antibody 18-34 and 
rabbit antisera against the phosphatases. The rabbit antisera to 
RPTP.alpha. and RPTP.epsilon. were prepared by standard techniques using 
peptide fragments derived from the C-terminus of RPTP.alpha. and 
RPTP.epsilon. as immunogen. For detection of phosphotyrosine and protein 
antigens on immunoblots, the ECL system (Amersham) was used in conjunction 
with goat anti-mouse and anti-rabbit antibodies (Biorad). For reprobing, 
blots were stripped in 67 mM Tris-HCl (pH 6.8), 2% SDS, and 0.1% 
.beta.-mercaptoethanol at 50.degree. C. for 30 minutes. 
SELECTION AND ANALYSIS OF CELLS BY TRANSFECTION WITH cDNAS ENCODING PTPS 
The specificity of each PTP for the insulin receptor was determined by 
assaying insulin-induced phenotypic changes in the cells and 
phosphorylation of insulin receptor .beta.-subunit by Western blot as 
described below. 
INSULIN-INDUCED CHANGE IN PHENOTYPE 
In the presence of 1 .mu.M insulin IR/BHK cells display an abnormal 
phenotype, i.e., rounding up and becoming detached from the plastic 
surface (FIG. 1A). The change in the morphology and the loss of adhesion 
to the substratum induced by insulin was most pronounced at low cell 
density and in the presence of 10% fetal calf serum. IR/BHK cells were 
transfected with cDNAs coding for PTP1B, PTP1B.DELTA.299, PTP1C, PTP.mu., 
CD45, RPTP.kappa., RPTP.alpha., RPTP.epsilon., LAR, and LAR (domain 1). To 
determine which of these PTPs were capable of modulating IR activity 
thereby preventing these phenotypic changes of the cells. Only RPTP.alpha. 
and RPTP.epsilon., were able to restore the normal phenotype. After 24 
hours of selection, small clones consisting of 4-8 cells could be seen. 
These transfected cells exhibited the normal phenotype and did not respond 
in the same manner to high doses of insulin as the cells transfected with 
IR alone (FIG. 1B). 
AUTOPHOSPHORYLATION ASSAY BY WESTERN BLOT 
Two stably cotransfected clones for each transfection (IR+RPTP.alpha. and 
IR+RPTP.epsilon.) were starved overnight in DMEM/high glucose containing 
0% fetal calf serum then stimulated with 1 .mu.M insulin for 10 minutes. 
The cells were lysed and the phosphotyrosine content of insulin receptor 
.beta.-subunit was detected by Western blotting (FIGS. 2 and 3) using 
anti-phosphotyrosine antibodies. 
FIG. 2A shows the phosphorylation status of IR in stable BHK cell clones 
coexpressing IR and RPTP.alpha.. In control cells a strong tyrosine 
phosphorylation of insulin receptors .beta.-subunit could be detected. 
This phosphorylation level was lower with the clones obtained after 
transfection with cDNA encoding RPTP.alpha.. FIG. 2B shows the level of 
RPTP.alpha. expression in the cotransfected clones. An additional band 
immunoreactive with anti-RPTP.alpha. antibodies, could be detected in 
these cotransfected clones. FIG. 2C shows the level of IR expression in 
control and cotransfected clones, which was similar. Stable BHK cell clone 
5 coexpressing IR and RPTP.alpha. was deposited with the American Type 
Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 on Jan. 20, 
1994, and assigned accession number ATCC CRL 11528. 
As shown in FIG. 3A, 3B and 3C, the pattern of IR phosphorylation and 
protein expression levels in stable cell clones coexpressing IR and 
RPTP.epsilon. are similar to that of IR and RPTP.alpha.. The data suggests 
that the restoration of normal phenotype of the cotransfected cells was 
associated with the dephosphorylation of the insulin receptor or 
downstream key signalling event. Stable BHK cell clone 6 coexpressing IR 
and RPTP.epsilon. was deposited with the American Type Culture Collection, 
12301 Parklawn Drive, Rockville, Md. 20852 on Jan. 20, 1994, and assigned 
accession number ATCC CRL 11529. 
The results described clearly indicate that RPTP.alpha. and RPTP.epsilon. 
interact specifically with IR. In the presence of insulin, RPTP.alpha. and 
RPTP.epsilon. modulate IR signal transduction and downstream cellular 
processes, which prevent changes in cell morphology and adhesion 
properties. These cell lines could be used in a drug screen whereby any 
biological effect of the test compound in vivo on insulin signal 
transduction may be monitored by changes in the cell morphology and 
adhesion properties or by phosphorylation state of the insulin receptor. 
Drugs that interfere with RPTP.alpha. or RPTP.epsilon. activity would make 
the cells respond to insulin and re-exhibit the insulin-sensitive 
phenotype. 
The present invention is not to be limited in scope by the specific 
embodiments described which are intended as single illustrations of 
individual aspects of the invention, and functionally equivalent methods 
and components are within the scope of the invention. Indeed, various 
modifications of the invention, in addition to those shown and 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.