Identification of compounds affecting specific interaction of peptide binding pairs

This invention relates to novel modified host cells which express heterologous fused proteins and methods of screening for test samples having peptide-binding activity; wherein the modified host cell comprises: (a) a gene sequence encoding a heterologous fusion protein; said fusion protein comprising a first peptide of a peptide binding pair, or segment of said first peptide, which is joined to either a DNA binding domain or its corresponding transcriptional activation domain of a transcriptional activation protein; (b) a gene sequence encoding a heterologous fusion protein, said fusion protein comprising a second peptide of the peptide binding pair in (a), or a segment thereof, fused to either a DNA binding domain or its corresponding transcriptional activation domain, whichever one is not employed in (a); (c) a reporter gene operatively associated with the transcriptional activation protein, or a portion thereof; (d) optionally, a deletion or mutation in the chromosomal DNA of the host cell for the transcriptional activation protein if present in the selected host cell.

FIELD OF THE INVENTION 
This invention relates to novel cells which express heterologous fused 
proteins and methods of screening for compounds having peptide-binding 
activity; wherein the methods employ the novel cells of this invention. 
BACKGROUND OF THE INVENTION 
The specific binding of a pair of peptides to each other triggers a vast 
number of functions in a living cell. For example, the specific binding of 
a ligand to a surface receptor serves as the trigger for cellular 
responses to many external signals. In mammals, cells respond to a wide 
variety of circulating peptide hormones, often through single 
transmembrane domain receptors. It is certainly recognized that the 
cytokine receptor superfamily illustrates the diverse aspects of cellular 
function, and physiological responses. Recent examinations of cytokine 
receptor function have revealed differing ligand-receptor protein 
stoichiometries including both 2-protein (ligand/receptor) (Cunningham et 
al., 1991; Staten et al., 1993) and 3-protein (ligand/receptor/receptor or 
ligand/receptor/transducer) interactions (Young, 1992; Taga and Kishimoto, 
1992; Mui and Miyajima, 1994). The intricacies of such protein 
associations have been investigated using in vitro, often laborious, 
methods (Fuh et al., 1992; 1993; Davis et al., 1993) since genetically 
malleable expression systems have been unavailable. The present invention 
is directed to novel modified host systems which can be used for such 
protein investigations, yet the novel systems are significantly less 
laborious. 
Recently reported systems in the art refer to a "2-hybrid" system as 
discussed by Fields and Song, 1989 and also Chein et al., 1991. The 
"2-hybrid" system involves differential interactions between the separable 
DNA binding and activation domains of the yeast transcriptional activator, 
Gal4. Heterologous proteins are expressed as hybrid proteins fused to 
either half of Gal4 (see FIG. 1; Fields and Song, 1989; Chein et al., 1991 
for discussion of the 2-hybrid system). The productive interaction of the 
heterologous proteins brings the two halves of the Gal4 protein in close 
proximity, activating expression of a scorable reporter gene. To this 
date, such 2-hybrid systems have been disclosed as useful for determining 
whether a first given test peptide sequence has binding activity for the 
known sequence of second peptide; wherein the affinity of the test peptide 
for the known peptide is unknown. Studies using such a system have been 
directed to analyzing intracellular proteins such as transcription factors 
and kinase-target protein interactions (Yang et al., 1992; Durfee et al., 
1993; Li et al., 1994). 
The novel modified cells of this invention and novel methods incorporating 
these cells provide significant advancement for the study and discovery of 
peptide mimics, including ligand mimics and receptor mimics. At this time 
no one has developed an efficient and specific screening system to 
investigate these areas. By employing in the cell a peptide binding pair 
for which the binding affinity is known, the present invention permits the 
investigation of peptide binding pairs, such as a ligand and receptor, 
wherein the peptides bind via extracellular interactions. The present 
invention creates exponential advantages for the discovery of compounds 
which can interact as ligands for specific receptors or transducers. 
Potential ligands include, but are not limited to, mammalian hormones with 
the receptors being a cognate extracellular ligand-binding peptide. 
Furthermore, the present invention describes the use of cell systems which 
express multiple heterologous proteins, including the two heterologous 
fused proteins to establish the specific and reversible binding of the 
ligand and receptor. The specific interaction of the above-described 
binding is readily detected by a measurable change in cellular phenotype, 
e.g. growth on selective medium. 
SUMMARY OF THE INVENTION 
One aspect of the present invention relates to novel modified host cells 
for the expression of heterologous fusion proteins. The novel modified 
host cells comprise: 
a) a gene sequence encoding a heterologous fusion protein; said fusion 
protein comprising a first peptide of a peptide binding pair, or segment 
of said first peptide, which is joined to either a DNA binding domain or 
its corresponding transcriptional activation domain of a transcriptional 
activation protein; 
b) a gene sequence encoding a heterologous fusion protein, said fusion 
protein comprising a second peptide of the peptide binding pair in (a), or 
a segment of said second peptide, fused to either a DNA binding domain or 
its corresponding transcriptional activation domain, whichever one is not 
employed in (a); 
c) a reporter gene operatively associated with the transcriptional 
activation protein, or a portion thereof. 
d) optionally, a deletion or mutation in the chromosomal DNA of the yeast 
host cell for the transcriptional activation protein if present in the 
host cell. 
These novel modified host cells of the present invention can be used to 
determine the interaction of a test sample with a selected peptide of a 
peptide binding pair; e.g. the cell can be used to determine the 
interaction of a test sample with selected ligand or receptor. 
A second aspect of the present invention relates to novel modified cells 
and screening methods which indicate the interaction of a test sample with 
a selected peptide and receptor by a recognizable change in phenotype. The 
cell exhibits the change in phenotype only in the presence of test 
compound having binding affinity for a peptide of the peptide binding 
pair, e.g. binding affinity for a ligand or its receptor. 
A third aspect of the present invention relates to novel cells and 
screening methods which permit determining to which peptide of a peptide 
binding pair a test sample binds. 
A fourth aspect of the present invention relates to novel cells which 
express three or more heterologous components for the study of higher 
order multi-protein associations between three or more peptides (e.g. such 
as the study of ligand dependent dimerization). 
Defined Terms: 
The term peptide binding pair refers to any pair of peptides having a known 
binding affinity for which the DNA sequence is known or can be deduced. 
The peptides of the peptide binding pair must exhibit preferential binding 
for each other over any other components of the modified cell. 
The term peptide as used in the above summary and herein means any peptide, 
polypeptide or protein, unless stated otherwise. As noted above the 
peptides of a peptide binding pair can be a ligand and its corresponding 
receptor, or a ligand and any peptide having a known binding affinity for 
the ligand. 
Heterologous as used in the above summary and herein means peptides which 
(1) are not expressed by the naturally-occurring host cell or (2) are 
expressed by the modified host cell by an expression method other than the 
expression method by which the host cell would normally express the 
peptide. 
Unless specified otherwise, the term receptor as used herein encompasses 
the terms receptor.sub.r, soluble receptor, transducer and binding 
protein. In preferred embodiments of the invention, the receptor employed 
is a receptor.sub.r or soluble receptor, with receptor.sub.r being more 
preferred. 
Receptor.sub.r as used herein means plasma membrane proteins that bind 
specific molecules, such as growth factors, hormones or neurotransmitters, 
and then transmits a signal to the cells' interior that causes a cell to 
respond in a specific manner. This includes single transmembrane proteins. 
Soluble receptor means a non-transmembrane form of a receptor which is able 
to bind ligand. These are receptors released from a cell either by 
proteolysis or by alternatively spliced mRNA. 
Binding protein means proteins that demonstrate binding affinity for a 
specific ligand. Binding proteins may be produced from separate and 
distinct genes. For a given ligand, the binding proteins that are produced 
from specific genes are distinct from the ligand binding domain of the 
receptor.sub.r or its soluble receptor. 
Transducer means a molecule that allows the conversion of one kind of 
signal into another, and the molecule is readily known as a transducer for 
one or more the peptides of a peptide binding pair, e.g. a transducer for 
a ligand/receptor group.

DETAILED DESCRIPTION OF THE INVENTION 
The modified cell of this invention employs a host cell. An effective host 
cell for use in the present invention simply requires that it is defined 
genetically in order to engineer the appropriate expression of 
heterologous fused proteins, reporter(s) and any other desired genetic 
manipulations. The host cell can be any eukaryotic cell, vertebrate or 
non-vertebrate. The host cell can be mammalian as well as amphibian, e.g. 
a Xenopus egg cell. Preferably, the host cell is a fungal cell, e.g. 
Aspergilla or Neuropora. In more preferred embodiments the host cell is a 
yeast cell. In alternatively preferred embodiments the yeast host cell is 
Saccharomyces cerevisiae, Schizosaccharomyces pombe or Pichia pastoris. 
The modified host cell employs at least two genes for expressing separately 
the two heterologous fusion proteins. One of these fusion proteins 
comprises a first peptide of a peptide binding pair, or segment of said 
first peptide, which is joined to either a DNA binding domain, or its 
corresponding transcriptional activation domain, of a transcriptional 
activation protein. A second fusion protein comprising the second peptide 
of the peptide binding pair, or a segment thereof. The second peptide is 
fused to either a DNA binding domain or its corresponding transcriptional 
activation domain, whichever one is not employed in the first heterologous 
fused protein. The activity of the binding between the peptides of the 
peptide binding pairs is monitored by the use of a reporter gene, which is 
operatively associated with the transcriptional activation protein 
employed in the two fusion proteins. 
The transcriptional activation protein can vary widely as long as the DNA 
binding domains and the activation domains are known or can be deduced by 
available scientific methods. The transcriptional activation protein can 
be any protein having two components, a DNA binding component and an 
activation component, wherein the transcriptional activation protein 
contains an acidic alpha helix for the activation of transcription. 
Preferably, the transcriptional activation protein is selected from Gal4, 
Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, 
VP16, LexA, non-mammalian nuclear receptors (e.g.ecdysone) or mammalian 
nuclear receptors (e.g. estrogen, androgens, glucocorticoids, 
mineralocorticoids, retinoic acid and progesterone; see also Picard et 
al., 1990). Preferably, the transcriptional activation protein is a yeast 
protein, and more preferably, the transcriptional yeast protein is 
selected from Gal4, Gcn4 or Adr1. It is noted that any DNA binding protein 
can be used which functions with an activation domain. A DNA binding 
protein can be substituted for the DNA binding domain of a transcriptional 
activation protein if the recognition sequences operatively associated 
with the reporter gene are correspondingly engineered. Illustrative of 
non-yeast DNA binding proteins are mammalian steroid receptors and 
bacterial LexA (see Wilson et al., 1990) 
The reporter gene is generally selected in order that the binding of the 
domains of the transcriptional activation protein can be monitored by 
well-known and straightforward techniques. Preferably, the reporter gene 
is selected based on its cost, ease of measuring its activity and low 
background (i.e. the activity can be determined at relatively low levels 
of expression of the reporter gene because of a high signal to background 
ratio and/or a relatively low or no un-induced activity). The reporter can 
be any reporter for which its activity can be detected by any means 
available. Illustrative of reporters which can be used in the present 
invention are reporter genes selected from the group of: 
a) lacZ, Luciferase gene, green fluorescent protein gene, CAT 
b) genes complementing auxotrophies, such as HIS, URA, LEU, ARG, MET, ADE, 
LYS, TRP. 
c) gene conferring antibiotic resistance, such as neo.sup.r, KAN, 
d) genes conferring sensitivity to a chemical such as CYH2 (cycloheximide 
resistance), CAN1 (canavanine resistance). In many embodiments it may be 
convenient for the reporter gene to prevent growth (CYH2). Preferably, the 
activity of the reporter gene is indicated by colorimetric or fluorsecent 
methods and/or by measuring growth of the yeast cell. 
As noted previously, the peptide employed in the modified cell is a peptide 
of a peptide binding pair for which the DNA sequence is known as well as 
the sequence of the second peptide of the binding pair. The peptides can 
also be peptides of a peptide binding complex which contains two or more 
peptides which bind each other to form the binding complex. The peptides 
of the peptide binding pair can be a specific ligand and a corresponding 
receptor or any other peptides which bind to each other preferentially, 
such subunits of an enzyme. 
One of the significant advantages of this invention is the discovery that 
the modified cell employing the DNA binding and activation domains of a 
transcriptional protein can be used to monitor the binding of peptides of 
a peptide binding pair which bind through extracellular interaction. 
Certainly, if desired peptides which bind through intracellular 
interaction can also be employed in any of the novel modified cells and 
methods of this invention. The peptide can be from a mammalian cell or 
non-mammalian cell. One of the most important embodiments of the present 
invention relates to the application of the novel modified cells and 
corresponding screening methods of this invention for studying numerous 
mammalian peptide interactions. The mammalian peptides include mammalian 
ligand/receptor interactions, such as hormone/receptor interactions. 
Illustrative of peptide hormones which can be used in the present 
invention are peptides selected from, but not limited to, one of the 
following groups: (a) the group consisting of cytokines, interleukins, 
hematopoietic growth factors, insulin, insulin-like growth factors, growth 
hormone, prolactin, interferons, and growth factors; (b) ligands for 
G-protein coupled receptors; (c) ligands for nonvertebrate receptors; (d) 
ligands for guanylyl cyclase receptors; and (e) ligands for tyrosine 
phosphatase receptors. 
In alternative embodiments, the peptide is a growth factor selected from 
epidermal GF, nerve GF, leukemia inhibitory factor, fibroblast GF, 
platelet-derived GF, vascular endothelial GF, tumor necrosis factor, 
oncostatin M, ciliary neurotrophic factor, erythropoietin, steel factor, 
placental lactogen and transforming GF.beta.. 
In various preferred embodiments the peptide hormone is a ligand for a 
G-protein coupled receptor, such as growth hormone releasing factor, 
secretin, vasoactive inhibitory peptide, glucagon, thyrotropin, 
interleukin-8, luteinizing hormone (LH) and follicle stimulating hormone 
(FSH). 
In additional alternative embodiments the peptide employed is a 
nonvertebrate peptide, such as those selected from the group consisting of 
plant systemin and insect differentiation peptides. However, in preferred 
embodiments the peptide is selected from the group consisting of mammalian 
peptides, and more preferably, mammalian peptide hormones. 
It is also noted that specific types of receptors may also be a peptide of 
a peptide binding pair or peptide binding complex. Illustrative of various 
receptors are those selected from one of the following groups: (a) a cell 
adhesion molecule; (b) an immunomodulatory, antigen recognition or 
presentation molecule or other related peptides. Illustrative of cell 
adhesion molecules are ICAM, VCAM, ECAM, fibronectin, integrin, selectin 
and fibrinogen. Illustrative of an immunomodulatory, antigen recognition 
or presentation molecule are T cell receptor complex, B cell receptor 
complex, Fc receptors, major histocompatibility complex I, major 
histocompatibility complex II, CD4, CD8, CD27, CD30, MAC complex. 
It is also noted that specific types of transducers may also be used as a 
peptide of a peptide binding pair or peptide binding complex. The 
transducer proteins employed can be any transducer protein which binds at 
least one of the peptides of the peptide binding pair or peptide binding 
complex. Transducer proteins include gp130, kh97, AIC2A, AIC2B. 
Preferably, the heterologous fused proteins are expressed by transformation 
of the yeast cell with an autonomously-replicating plasmid capable of 
expressing the fusion protein although they can be expressed by 
chromosomal modification. 
As noted, the screening methods of this invention are designed in order to 
detect the ability of a test sample to affect the binding of a peptide 
binding pair, e.g. ligand-receptor interaction. Basically, the method 
comprises determining the activity of the reporter gene upon adding a test 
sample to a modified host cell of the present invention under conditions 
suitable to detect the activity in the presence of a sample or under a 
condition for which the modified host cell exhibits such activity only in 
the presence of a sample having binding interaction with the peptide 
binding pair. Preferably, the activity of the reporter gene is determined 
by measuring a change in selected phenotype which is directly correlated 
to activity of the reporter. 
The novel modified cells of this invention are readily applied in various 
screening methods for determining the binding ability of a test sample. 
The test sample may be a peptide, which is preferably about two amino 
acids in length, or a non-peptide chemical compound. The non-peptide test 
sample includes compounds, complexes and salts as well as natural product 
samples, such as plant extracts and materials obtained from fermentation 
broths. The modified host cells are cultured under suitable conditions for 
growth to study the interaction of a test sample on the binding 
interaction of the peptide binding pair. The modified host cells are 
placed in a growth medium, which preferably contains agar, with the test 
sample applied to the surface of the growth medium. The growth medium is 
preferably a conventional liquid medium of growth reagents and water, such 
as yeast synthetic medium (YSM available from BIO101 (also see Rose et 
al., Methods in Yeast Genetics, 1990). 
One of the embodiments of the present invention is directed to a novel 
modified host cell and screening method which indicate the interaction of 
a test compound with a selected peptide binding pair by a recognizable 
change in phenotype. This modified host cell exhibits the change in 
phenotype only in the presence of test compound having binding affinity 
for one of the peptides of the peptide binding pair. This host cell is 
referred to herein as a "rescue" system. Normally, a cell response is 
exhibited when the two domains of the transcriptional activation protein 
interact. However, in a rescue system a positive indication of change in 
the phenotype does not occur when the two domains of the transcriptional 
activation protein interact. A positive indication of change in the 
phenotype occurs only when a test sample interrupts the interaction of the 
two domains of the transcriptional activation protein. In a rescue system, 
a modified host cell is capable of expressing at least two heterologous 
fusion proteins. Further, the host cell comprises: a reporter gene 
operatively associated with the transcriptional activation protein; 
wherein said reporter gene prevents the exhibition of a specific phenotype 
on a selective medium due to the expression of the transcriptional 
activation protein, or a portion thereof. A mutation in the chromosomal 
DNA of the host cell allows for reversal of the detectable phenotype, on 
the selective medium, in the absence of expression of the reporter gene. 
If needed, there is a deletion or mutation in the chromosomal DNA of the 
host cell for a transcriptional activation protein in order that 
transcriptional activation only occurs upon productive interaction of the 
selected binding pair. Only when a test sample interrupts the interaction 
of the two domains of the transcriptional activation protein will the 
modified cell grow or survive, or exhibit another selected phenotype. 
Preferably, the phenotype corresponds to the growth of the cell. 
Once a screening method, as discussed above, is used to determine whether a 
test sample interacts with, or rather disrupts, the peptide binding 
observed in the absence of a test sample, a secondary screen is employed 
to determine the specific binding affinity of the test sample, i.e. to 
which peptide of the peptide binding pair the test sample binds. The 
secondary screens employ the novel cells of this invention wherein cells 
are adapted to exhibit a phenotype, or phenotypic change only in the 
presence of a test sample which binds one peptide of the peptide binding 
pair. One of the preferred methods for determining the specific binding 
characteristics of the test sample involves employing cells which contain 
an effective (relatively high) copy number of either fusion protein 
containing one of the peptides. An effective copy number is any copy 
number sufficient to enable determination of the specific binding of the 
test sample. Preferably, the gene copy number is at least about 5, and 
preferably ranges from about 5 to about 50, with the higher copy numbers 
being the most preferred. The other fusion protein is maintained at a 
relatively low (1 to about 2 copies per cell) by either integration into a 
cell chromosome or by utilizing chromosomal centromeric sequences on the 
expression plasmid. If a high copy number of a first peptide is used in a 
cell of this invention, the cell will be more sensitive to the presence of 
the test sample which binds the second peptide of the peptide binding pair 
since the limiting amount of second peptide determines the level of the 
activity of the reporter gene (i.e. the change in phenotype observed). 
Conversely, if a high copy number of a gene encoding the second peptide of 
a peptide binding pair is used, the cell will be more sensitive to the 
presence of a test sample which binds the first peptide since the limiting 
amount of the second peptide determines the level of the activity of the 
reporter gene (i.e. the change in phenotype observed). A direct comparison 
of effects of a test compound on the phenotypes of the two strains 
(receptor&gt;&gt;&gt;ligand versus ligand&gt;&gt;&gt;receptor) demonstrates the specific 
protein interaction of the compound. As discussed supra, the genes 
expressing the peptides as well as the reporter gene are preferably 
expressed by transformation of the yeast cell with an 
autonomously-replicating plasmid. 
An additional modified host cell of this invention is directed to cells 
which can be used to study peptides ligands which employ dual receptors or 
a receptor and a transducer for activation or transmission of a signal 
from the binding of multiple peptide binding components, i.e. three or 
more peptide binding components. 
Receptor dimerization is a critical first step for signal transduction for 
certain classes of receptors. Dimer receptor structures can be composed of 
identical receptor units (examples: insulin receptor, IGF-I receptor, PDGF 
receptor, kinase inert domain receptor (KDR), or colony stimulating factor 
(CSF)-I receptor) or non-identical receptor units (example: IL-6R+gp130; 
insulin-IGF-I hybrid receptor; LIF+gp130; CNTF+gp130; various interferon 
receptors). 
The components of a modified host cell for monitoring the binding activity 
of a peptide having "dual receptor" system are as follows: the gene 
sequence (a) is a gene sequence encoding a heterologous fusion protein; 
said fusion protein comprising one peptide of a multiple peptide binding 
complex, or segment of said peptide, which is joined to either a DNA 
binding domain or its corresponding transcriptional activation domain of a 
transcriptional activation protein; and the gene sequence (b) is a gene 
sequence encoding a heterologous fusion protein; said fusion protein 
comprising a second peptide of said multiple peptide binding complex, or a 
segment of said receptor, fused to either a DNA binding domain or its 
corresponding transcriptional activation domain, whichever one is not 
employed in (a). The modified host cell for studying a multiple peptide 
binding complex, such as a dual receptor system, also comprises an 
appropriate reporter gene and chromosomal mutations for specific analysis 
of the peptide (ligand/receptor) interaction as discussed infra. One can 
express a third peptide (e.g. a ligand) to establish a control for 
comparative or competitive testing. 
As noted above, for the study of multiple binding peptide complexes, i.e. 
higher-order proteins which contain three or more peptides, one can 
actually use the modified host cell of the present invention to express 
three or more peptides. In the case of a tripeptide binding complex, any 
two of the peptides can be fused to the two components of the 
transcriptional activation protein. For example, to study the interaction 
of a ligand which interacts via receptor dimerization, one can express the 
receptors as fused proteins with the ligand being expressed as a nonfusion 
protein. This host cell sytem can be also be applied in studying 
multiprotein enzyme complexes. For any multipeptide binding complex, one 
can identify novel peptides which interact with the complex by expressing 
novel proteins from random complementary DNA sequences (e.g. a cDNA 
library) fused to one of the domains of a transcriptional activation 
protein. In such a sytem, one of the known peptides of the peptide binding 
complex is fused to the other domain of the transcriptional activation 
protein while other units of the peptide binding complex are expressed as 
nonfusion peptides. It is further noted that the number of peptides 
expressed by the modified host cell should only be limited by the 
available detection means and the capacity of the host cell. 
The novel screening methods can be utilized to identify compounds 
interacting with any peptide binding pair, e.g. any receptor and/or 
ligand. Also, this modified cell system with a reporter gene to create a 
screen can be applied to any protein-protein interaction to discover novel 
compounds that disrupt that interaction. As specific examples: a) protein 
kinases implicated in cancers can be inserted into the system to rapidly 
screen for novel compounds that block the kinase-target interaction and 
thus may serve as unique cancer therapeutics; b) viral coat proteins, such 
as human immunodeficiency virus glycoproteins, and corresponding cell 
surface receptor proteins, such as CD4, can be inserted into the system to 
rapidly screen for compounds that disrupt this interaction, and may serve 
as anti-viral agents: c) the two subunits for the Plasmodium 
ribonucleotide reductase enzyme can be expressed in the system to screen 
for compounds which prevent this specific protein association and thus may 
serve as novel anti-malarial agents. 
The following Examples are provided to further illustrate various aspects 
of the present invention. They are not to be construed as limiting the 
invention. 
EXAMPLE 1 
Specific and Reversible Ligand-Receptor Interaction 
Genes encoding fusion proteins are generated by cloning growth hormone (GH) 
and growth hormone receptor (GHR) cDNA sequences into plasmids containing 
the coding region for the domains of Gal4. DNA binding domain (Gal4) 
fusions are constructed in pAS2, which is described in Wade Harper et al. 
Gene activation domain (Gal4) fusions are constructed in pACT-II, which is 
identical to pACT (described in Durfee et al., 1993) except with a 
modification of the polylinker region. Into the Bg1 II site is added the 
following sequence: Bgl II-Hemagglutinin 
epitope-NdeI-NcoI-SmaI-BamHI-EcoRI-XhoI-Bgl II, as adapted from the 
polylinker sequence of pAS2 (Wade Harper et al., 1993). The cDNA encoding 
the mature peptide for porcine GH is generated using standard polymerase 
chain reaction (PCR) techniques (see Finney, 1993). 
Oligonucleotides prepared on an ABI oligosynthesizer are designed according 
to the published cDNA sequence for pig GH (see Su and El-Gewely, 1988). A 
30 base 5' oligonucleotide contains a NcoI site 
(5'-CATGCCATGGAGGCCTTCCCAGCCATGCCC 3' SEQ. ID. NO.1) and a 27 base 3' 
oligonucleotide contains a BamHI site (5'-CGGGATCCGCAACTAGAAGGCACAGCT-3' 
SEQ. ID. NO.2). The GH cDNA is generated using a pig pituitary lambda gt11 
library as template source. A 540 bp fragment is obtained, ligated into 
pCR II vector (Invitrogen Corp.), recombinants are confirmed by 
restriction enzyme digest, and the DNA produced as described in Maniatus 
et al., 1982. The cDNA sequence is confirmed by dye-deoxy terminator 
reaction using reagents and protocols from Perkin-Elmer Cetus Corp. and an 
ABI 373A automated sequencer. The GH cDNA is directionally cloned into 
pACT-II via NcoI and BamHI sites. The cDNA encoding the extracellular 
domain of the GHR is generated using standard PCR methods. A 33 base 5' 
oligonucleotide containing a NcoI site 
(5'-CATGCCATGGAGATGTTTCCTGGAAGTGGGGCT-3' SEQ. ID. NO.3) and a 39 base 3' 
oligonucleotide containing a termination codon, followed by a NcoI site 
5-CATGCCATGGCCTACCGGAAATCTTCTTCACATGCTGCC-3' SEQ. ID. NO.4) are used to 
generate a 742 bp fragment encoding amino acids 1-247 of the rat GHR 
(Baumbach et al., 1989). This GHR cDNA is cloned into pCRII vector as 
previously described above, and then subcloned into the NcoI site of the 
pAS2 vector. DNA of final recombinant vectors is transformed into yeast 
strain(s) by the lithium acetate method (Rose et al., 1990). 
A yeast host (Y190) containing a UAS.sub.GAL -HIS reporter gene is prepared 
according to the procedure described in Wade Harper et al., 1993. The 
genotype of strain Y190 is MATa leu2-3,112 ura3-52 trp1-901 his3d200 
ade2-101 gal4 gal80 URA3::GAL-lacZ LYS2::GAL-HIS3 cyh.sup.r. Strain Y190 
is transformed with both fusion constructs or with a single fusion 
construct plus the opposing vector containing no heterologous sequences. 
All strains are found to exhibit equal growth on nonselective medium (FIG. 
2A). These strains are then tested for growth on selective medium (i.e. a 
growth medium lacking an amino acid which is synthesized by activation of 
the reporter gene). Only the strain containing both hybrid proteins 
(CY722) is able to grow while the strains containing either the ligand or 
receptor fusion alone do not grow (CY724 and CY723, respectively; FIG. 
2B). Two independent samples of each strain are streaked on synthetic 
medium containing 2% glucose, yeast nitrogen base, ammonium sulfate, 0.1 
mM adenine and 60 mM 3-amino-triazole (plate B) or on the same medium 
supplemented with histidine (plate A). Plate A is incubated at 30 C. for 
three days; plate B for five days. These results demonstrate that GH and 
GHR can mediate the Gal4-dependent activation of the reporter gene in an 
interaction suggestive of ligand-receptor binding. 
EXAMPLE 1A 
Competing Expressed Free Ligand (GH) in the Presence of GH and GHR Fusion 
Proteins 
To substantiate the apparent binding of GH to its receptor in the foreign 
environment of a yeast nucleus, the system is modified to add a third 
plasmid mediating expression of "free" ligand to show that the GH peptide 
competes with the GH-Gal4 fusion protein, reversing the 2-hybrid 
interaction shown in Example 1. The parental strain Y190 (Wade Harper et 
al., 1993) is grown on a medium containing 5-fluoro-orotic acid to select 
for derivatives that spontaneously lose the URA3 gene (see Rose et al., 
1990). The resultant strain, designated CY770, is utilized for all 
experiments examining effects of protein expressed concurrently from the 
third component (i.e. third plasmid). The cDNA encoding GH is generated by 
PCR methods using a 38 base 5' oligonucleotide containing an EcoRI site 
(5'-CCGAATTCAAAATGGCCTTCCCAGCCATGCCCTTGTCC-3' SEQ. ID. NO.5) and a 26 base 
3' oligonucleotide containing a HindIII site 
(5'CCAAGCTTCAACTAGAAGGCACAGCT-3' SEQ. ID. NO.6) for subsequent subcloning 
into the vector pCUP. pCUP is an inducible yeast expression vector derived 
from pRS316 (Hill et al., 1986). Briefly, this vector is constructed by 
inserting the 3' end of the yeast PGK gene (from pPGK; Kang et al., 1990) 
into the pRS316 cloning region as a BamHI-SalI fragment to serve as a 
transcriptional terminator. To this plasmid, the CUP1 promoter region 
(Butt et al., 1984) is amplified by PCR as a SacI-EcoRI fragment and 
inserted into corresponding sites of the plasmid to create pCUP. The GH 
expression plasmid (GH-pCUP) is then co-transformed with the GH and GHR 
fusion constructs into strain CY770 to generate CY781. Concurrent 
expression of free GH with the GH and GHR fusion proteins (CY781) is shown 
to block GH-GHR-dependent cell growth on selective medium (FIG. 2B). This 
experiment typifies an in vivo competition assay and demonstrates the 
reversibility of the observed ligand-receptor interaction. 
EXAMPLE 1B 
Binding of Peptide Hormone Prolactin (PRL) and its Receptor 
To expand and validate this technology, a similar system was developed 
using the peptide hormone prolactin (PRL) and its receptor. Prolactin is 
structurally related to GH and the prolactin receptor (PRLR) is also a 
member of the cytokine receptor superfamily. Unlike human GH, sub-primate 
GH does not readily bind the PRLR (Young and Bazer, 1989); nor does PRL 
readily bind the GHR (Leung et al., 1987). Mature porcine PRL is generated 
as a fusion to the GAL4 activation domain. oligonucleotides are designed 
to pig PRL (obtained from Genbank X14068), and used to generate the mature 
pig PRL protein hormone from a pig pituitary lambda gt11 library using 
standard PCR methods. A 31 base 5' oligonucleotide includes an EcoRI site 
(5'-CGGAATTCTGCCCATCTGCCCCAGCGGGCCT-3' SEQ. ID. NO.7) and corresponds to 
sequences encoding amino acids 1-7. A 30 base 3' oligonucleotide contains 
an EcoRI site (5'-GAATTCACGTGGGCTTAGCAGTTGCTGTCG-3' SEQ. ID. NO.8) and 
corresponds to a region of cDNA 3' to the endogenous termination codon. A 
600 bp fragment is obtained, ligated into pCR II vector, and confirmed by 
restriction enzyme digest and sequence analysis. The PRL cDNA is cloned 
into pACT-II via the EcoRI site. 
The extracellular domain of the porcine PRL receptor (PRLR) is generated as 
a fusion to GAL4 DNA binding domain. Oligonucleotides are designed based 
on sequence of the mouse PRLR (Davis and Linzer, 1989) A 31 base 5' 
oligonucleotide contains a SmaI site 
(5'-TCCCCCGGGGATGTCATCTGCACTTGCTTAC-3' SEQ. ID. NO.9) while the 31 base 3' 
oligonucleotide contains a termination codon followed by a SalI site 
(5'TCCGTCGACGGTCTTTCAAGGTGAAGTCATT-3' SEQ. ID. NO.10). These 
oligonucleotides flank the extracellular domain of the PRLR, encoding 
amino acids 1-229. A pig pituitary lambda gt11 library is used as template 
source. Using standard PCR methods, a 687 bp fragment is generated, 
ligated into pCRII, and the nucleotide sequence is confirmed. The PRLR 
cDNA is cloned into the pAS2 vector via the SmaI and SalI restriction 
sites. 
Strain Y190 was transformed with the PRL or PRLR fusion expression plasmids 
either alone (CY727 or CY728, respectively) or together (CY726). Cells 
expressing both the PRL and PRLR fusions are able to grow on selective 
medium while the strains containing either the ligand or receptor fusion 
alone can not. These results mirror those observed in the GH-GHR system in 
the examples above and establish the general utility of the 2-hybrid 
system for examination of ligand binding to members of this receptor 
superfamily. 
EXAMPLE 1C 
Additional Confirmation of Ligand-Receptor Specificity for the Novel Yeast 
Host Cell System 
Additional strains are developed to assess ligand-receptor specificity. URA 
minus strains expressing GH and GHR fusion proteins are transformed with 
pCUP or PRL-pCUP; while strains expressing PRL and PRLR fusion proteins 
are transformed with pCUP, or PRL-pCUP. Briefly, PRL-pCUP is constructed 
in a fashion similar to that described for GH-pCUP. The PRL cDNA is 
generated by PCR using a 33 base 5' oligonucleotide with an EcoRI site 
(5'-GAATTCAAAATGCTGCCCATCTGCCCCAGCGGG-3' SEQ. ID. NO.11) and the 3' 
oligonucleotide in example 1B. The resulting fragment is introduced into 
pCUP via the EcoRI site. As demonstrated in the above Examples, a strain 
expressing the GH and GHR fusions with no competitor grows on selective 
medium and this growth is abolished with coexpression of free GH. The 
prolactin experiment produces similar results which confirm the 
specificity of the ligand-receptor binding in the yeast cell. A strain 
carrying PRL and PRLR fusions (CY787) can grow on selective medium and 
this growth is abrogated by expression of free PRL (CY786; Table 1). 
To test selectivity of the GHR, a strain containing the GH and GHR fusions 
is transformed with PRL-pCUP. This strain grows on selective medium 
(CY785; Table 1). These data indicate that GH binding to its receptor in 
this system can be efficiently competed by excess GH (CY751) binding but 
not by the related PRL peptide (CY755). The results from the above 
experiments, expressing three heterologous proteins, illustrates the 
specificity of ligand-receptor interaction(s) in the system of this 
invention. 
TABLE 1 
______________________________________ 
Strain list and bioassay results.sup.a 
Designation 
AD fusion.sup.b 
BD fusion.sup.c 
pCUP.sup.d 
Growth.sup.e 
______________________________________ 
CY700 -- -- -- 0 
CY722 GH GHR -- + 
CY723 vector GHR -- 0 
CY724 GH vector -- 0 
CY726 PRL PRLR -- + 
CY770 -- -- -- 0 
CY781 GH GHR GH 0 
CY784 GH GHR vector + 
CY785 GH GHR PRL + 
CY786 PRL PRLR PRL 0 
CY787 PRL PRLR vector + 
______________________________________ 
.sup.a All yeast strains are derived from strain Y190 (Wade Harper et al. 
1993). The genotype is MATa gal4 gal80 his3 trpl901 ade2101 ura352 
leu23,112 URA3::GALlacZ LYS2::GALHIS3 cyh.sup.r. Strains with number 
designations equal to or greater than 770 do not have the URA3::GALlacZ 
gene. A dash indicates that a strain does not contain the denoted plasmid 
.sup.b AD fusions are pACT derivatives; GH or PRL fused to the Gal4 
activation domain. 
.sup.c BD fusions are pAS2 derivatives; extracellular domains of GH or PR 
receptors fused to the DNA binding domain of Gal4. 
.sup.d pCUP denotes peptides expressed from the pCUP plasmid. 
.sup.e Summary of bioassay results. Each strain is grown on selective 
medium for 3 to 5 days at 30 C. then scored for cell growth, indicated by 
a plus. 
EXAMPLE 2 
Screen for compounds disrupting ligand-receptor interaction 
Low-copy-number plasmids expressing GHR- or GH-Gal4 fusion proteins (pOZ153 
and pOZ152, respectively) are constructed to reduce expression of these 
proteins. In addition, a novel reporter gene is constructed that prevents 
cell proliferation on selective medium unless expression is abrogated. To 
construct the GHR fusion expression plasmid, a SacI-BamHI restriction 
fragment containing a yeast constitutive promoter and GAL4 sequences is 
isolated from pAS1 (Durfee et al., 1993) and cloned into pUN30 (Elledge 
and Davis, 1988). The extracellular domain of GHR is then fused to GAL4 by 
ligation as an NcoI fragment as described in Example 1 to create pOZ153. 
To construct the GH fusion expression construct, the entire GH-Gal4 region 
with promoter and terminator sequences is isolated from the plasmid 
described in Example 1 as a PvuI-SalI fragment. This DNA segment is cloned 
into pUN100 (Elledge and Davis, 1988) generating pOZ152. A reporter gene 
is constructed by isolating the yeast CYH2 coding region and operatively 
linking it to a GAL promoter in a yeast expression plasmid. Briefly, the 
GAL1 promoter region is inserted into YEp352 (Hill et al., 1986) as a 685 
bp EcoRI-BamHI fragment. CYH2 sequences are amplified by PCR using 
oligonucleotides primers (5'-GGATCCAATCAAGAATGCCTTCCAGAT-3' SEQ. ID. NO.12 
and 5'-GCATGCGTCATAGAAATAATACAG-3' SEQ. ID. NO.13) and pAS2 as the 
template. The PCR product is digested with BamHI plus SphI and cloned into 
the corresponding sites in the YEp352-GAL vector. These plasmids are 
transformed into yeast strain CY770 which carries a mutation at the 
chromosomal cyh2 gene rendering the strain resistant to the protein 
synthesis inhibitor cycloheximide. The presence of all three plasmids is 
necessary to confer cycloheximide sensitivity (cyh). 
The strain (CY857) containing the ligand and receptor fusion plasmids plus 
the reporter plasmid forms the basis of a simple primary screen for 
compounds that disrupt the binding of GH to its receptor. Strain CY857 is 
embedded in standard yeast growth medium containing 10.0 .mu.g/ml 
cycloheximide. Due to the ligand/receptor interaction driving expression 
of the CYH2 reporter gene, the strain is cyh.sup.s and thus unable to 
grow. Chemical compounds are placed on this test medium. Compounds which 
impair GH-GHR binding are identified by the growth of cells surrounding 
the compound because in the absence of CYH2 expression the cells become 
resistant to cycloheximide present in the medium. 
Secondary Screen to Determine Target of Sample 
Disruption of ligand-receptor binding in this assay can result from 
reaction of the compound with either the receptor or ligand fusion 
component. The specific target of the novel compound is determined by a 
simple secondary assay utilizing strains overexpressing one of the fusion 
proteins. Strain CY858 expresses the GHR-GAL4 fusion in large excess due 
to the construct being maintained within the cells at high copy number 
(pOZ149), while the GH-fusion (pOZ152) is maintained at levels similar to 
the base strain (CY857). Conversely, strain CY859 expresses the GH-GAL4 
fusion in large excess due to this construct being maintained within the 
cells at high copy number (pKY14), while the GHR fusion (pOZ153) is 
maintained at levels similar to base strain (CY857). Compounds rescuing 
growth in the primary screen using CY857 (GH and GHR fusions expressed on 
low copy numbers plasmids) are then assayed in the same manner using CY858 
(GHR&gt;&gt;GH) or CY859 (GH&gt;&gt;GHR) as the test strain. For example, when 
ligand-receptor binding is inhibited by a compound reacting with the GHR, 
the secondary screen will demonstrate a detectable change for the 
phenotype measured. Secondary testing of the rescuing compound on strain 
CY858 which overexpresses the GHR fusion produces a smaller growth in the 
presence of the compound than that observed for CY859. This detectable 
change in the measured phenotype occurs because the overabundance of GHR 
titrates the compound thereby increasing CYH2 expression and inhibiting 
cell growth. CY859 produces a detectable change similar to CY857 because 
the GHR fusion protein is limiting. A compound interacting with the ligand 
fusion demonstrates the inverse change in measured phenotype in this 
secondary assay. 
EXAMPLE 3 
Demonstration of Ligand Dependent Receptor Dimerization 
Multiple protein interactions (for example; ligand-receptor-receptor) are 
investigated with the expanded system which expresses a third protein 
using the following scheme. 
One unit of the receptor dimer is generated as a fusion protein with either 
the Gal 4 DNA binding or activation domain. The other unit of the receptor 
dimer is generated as a fusion protein with corresponding Gal DNA binding 
or activation domain, whichever is not used for the first fusion. The gene 
encoding the ligand is expressed from the third plasmid and is produced as 
a free (non-fusion) ligand. Interaction of the fusion proteins occurs only 
in the presence of ligand (see FIG. 3). 
The interaction of vascular endothelial cell growth factor (VEGF) with the 
ligand binding domain of its cognate receptor (KDR, kinase insert domain 
containing receptor) is described as an example for this system. KDR is a 
tyrosine kinase receptor, and dimer formation (1 ligand-2 receptors) is 
suggested to be important for hormone-induced receptor function. The cDNA 
encoding the ligand domain of KDR (Terman et al., 1991) is isolated as an 
Nco I-BamHI fragment and cloned into both the pACT-II and pAS2 vectors. 
The cDNA encoding the mature protein for VEGF is generated using standard 
PCR techniques. oligonucleotides are designed from published sequence (see 
Tischer et al., 1991). A 34 base 5' oligonucleotide containing an EcoRI 
site (5'-CGGAATTCGAAGTATGGCACCCATGGCAGAAGGA-3' SEQ. ID. NO.14) and a 28 
base 3' oligonucleotide containing an EcoRI site 
(5'-CGGAATTCGGATCCTCATTCATTCATCA-3' SEQ. ID. NO.15) are used to generate a 
450 bp fragment encoding the mature protein and cloned into the EcoRI site 
of pCUP. DNA of final recombinant vectors is transformed into yeast by the 
lithium acetate method to generate appropriate strains. 
The yeast host strain (CY770) is transformed with KDR-pACT-II, KDR-pAS2 and 
VEGF-pCUP to generate strain CY846; or transformed with both receptor 
fusions and pCUP to generate strain CY847. Additionally, both KDR-T-II 
and KDR-pAS2 are transformed together (CY845) or separately (CY843 or 
CY844) or VEGF-pCUP alone (CY841) as control strains. Strains are tested 
for growth on selective medium. The strain (CY846) that expresses the VEGF 
ligand plus the two receptor fusion proteins exhibits substantial growth 
on selective media in comparison to the strain CY847, which does not 
express the VEGF ligand (see FIG. 4). These results demonstrate that the 
effective cells of this invention can be used to study ligand-dependent 
dimerization of the receptor. 
EXAMPLE 4 
Screen for Compounds that Act as Ligands in a Dimer Receptor System 
Dimerization (oligomerization) of receptor units is often an important 
first step in activation of receptors such as those for the growth 
factors, cytokines and those describe supra. The novel cell system 
described in example 3 can be applied to the discovery of novel compounds 
which promote (or block) receptor dimerization. Such novel interacting 
compounds may serve as effective therapeutic agents for pathologies 
associated with these receptors. 
Plasmids expressing the dimer receptor unit(s) as fusion proteins are 
generated as discussed in example 3. The strain (CY845) containing the 
KDR-pACT-II and KDR-pAS2 fusions serves as an example of a simple primary 
screen for receptors which exhibit a dimer structure. Strain CY845 is 
embedded in synthetic agar medium deficient in histidine (Rose et al., 
1990). Test compounds are applied to the top of this test medium. Chemical 
compounds which induce interaction of the two receptor fusions (in the 
absence of ligand) results in the reconstitution of the endogenous 
transcriptional activator, which is linked to a reporter gene, such as 
HIS3. The reconstitution is identified by growth of cells surrounding the 
compound. 
PUBLICATIONS REFERENCED ABOVE 
Baumbach W R, Horner D. and J S Logan. (1989) The growth hormone-binding 
protein in rat serum is an alternatively spliced form of the rat growth 
hormone receptor. Genes & Devel. 3:1199-1205. 
Butt T R, Sternberg E J, Gorman J A, Clark P, Hamer D, Rosenberg M and S T 
Crooke. (1984) Copper metallothionein of yeast, structure of the gene and 
regulation of expression. Proc. Natn. Acad. Sci. USA 81:3332-3336. 
Chein, C-T., Bartel, P L., Sternglanz R. and S. Fields (1991) The 
two-hybrid system: A method to identify and clone genes for proteins that 
interact with a protein of interest. Proc. Natl. Acad. Sci., USA 88: 
9578-9582. 
Cunningham B C, Ultsch M, De Vos A M, Mulkerrin M G, Clauser K R and J A 
Wells. (1991) Dimerization of the extracellular domain of the human growth 
hormone receptor by a single hormone molecule. Science 254:821-825. 
Davis J and D I H Linzer. (1989) Expression of multiple forms of the 
prolactin receptor in the mouse liver. Mol. Endocrinol. 3:674-680. 
Davis S, Aldrich T H, Stahl N, Pan L, Taga T, Kishimoto T, Ip N Y and G 
Yancopoulos. (1993) LIFR and gp 130 as heterodimerizing signal transducers 
of the tripartate CNTF receptor. Science 260:1805-1808. 
Durfee T., Becherer K., Chen P-L., Yeh, S-H., Yang Y., Kilburn A E., Lee 
W-H. and S J Elledge. (1993) The retinoblastoma protein associated with 
the protein phosphatase type 1 catalytic subunit. Genes and Devel. 
7:555-569. 
Elledge S J and R W Davis. (1988) A family of versitile centromeric vectors 
designed for use in the sectering-shuffle mutagenesis assay in 
Saccharomyces cerevisiae. Gene 70:303-312. 
Fields S and O. Song. (1989) A novel genetic system to detect 
protein-protein interactions. Nature 340:245-246. 
Finny M. (1992) The polymerase chain reaction. In: Current Protocols in 
Molecular Biology. Chapter 15 Eds (F M Ausubel, R Brent, R E Kingston, D D 
Moore, J G Seidman, J Smith and K Struhl) John Wiley & Sons, NY. 
Fuh G., Cunningham B C, Fukunaga R, Nagata S, Goeddel D V and J A Wells. 
(1992) Rational design of potent antagonists to the human growth hormone 
receptor. Science 256:1677-1680. 
Fuh G, Colosi P, Wood W I and J A Wells. (1993) Mechanism--based design of 
prolactin receptor antagonists. J. Biol. Chem. 8:5376-5381. 
Hill J E, Myers A M, Koerner T J and A Tzagoloff. (1986) Yeast/E.Coli 
shuttle vectors with multiple unique restriction sites. Yeast 2:163-167. 
Kang Y-S, Kane J, Kurjan J, Stadel J M and D J Tipper. (1990) Effects of 
expression of mammalian G and hybrid mammalian-yeast G proteins on the 
yeast pheromone response signal transduction pathway. Mol. Cell. Biol. 
10:2582-2590. 
Kondo M, Takeshita T, Ishii N, Nakamura M, Watenabe S, Arai K-i and K 
Sugamura. (1993) Sharing of the interleukin-2 (IL-2) receptor chain 
between receptors for IL-2 and IL-4. Science 262: 1874-1877. 
Leung D W, Spencer S A, Cachianes G, Hammonds G, Collins C, Henzel W J, 
Barnard R, Waters M J and W I Wood. (1987) Growth hormone receptor and 
serum binding protein: purification, cloning and expression. Nature 
330:537-543. 
Li, J J and I. Herskowitz (1993) Isolation of ORC6, a component of the 
yeast origin recognition complex by a one-hybrid system. Science 
262:1870-1874. 
Maniatus T, Fritsch E F and J Sambrook. (1982) Molecular Cloning. Cold 
Spring Harbor Laboratory Press. 
Mui A and A Miyajima (1994) Cytokine receptors and signal transduction. In: 
Progress in Growth Factor Research. pp 15-35. Pergamon Press. NY. 
Noguchi M, Nakamura Y, Russell S M, Zeigler S F, Tsang M, Cao X and W J 
Leonard. (1993) Interleukin-2 receptor chain: A functional component of 
the interleukin-7 receptor. Science 262:1877-1880. 
Picard D, Schena M and K R Yamamoto. (1990) An inducible expression vector 
for both fission and budding yeast. Gene 86:257-261. 
Rose M D, Winston F, and P Hieter. (1990) Methods in yeast genetics. Cold 
Spring Harbor Laboratory Press. 
Staten N R, Byatt J C and G G Krivi. (1993) Ligand-specific dimerization of 
the extracellular domain of the bovine growth hormone receptor. J. Biol. 
Chem. 268:18467-18473. 
Su T-Z and M R El-Geweley. (1988) A multisite--directed mutagenesis using 
T7 DNA polymerase: application for reconstructing a mammalian gene. Gene 
69:81-89. 
Taga T and T Kishimoto (1993) Cytokine receptors and signal transduction. 
FASEB J. 7:3387-3396. 
Taga T, Hibi M, Matsuda T, Hirano T and T. Kishimoto. (1993) IL-6-induced 
homodimerization of gp130 and associated activation of a tyrosine kinase. 
Science 260:1808-1810. 
Terman B I, Dougher-Vermanzen M, Carrion M E, Dimitrov D, Armellina D C, 
Gospodarowicz D and P. Bohlen (1992) Identification of the KDR tyrosine 
kinase as a receptor for vascular endothelial cell growth factor. Biochem. 
Biophys. Res. Comm. 187:1579-1586. 
Terman B I, Carrion M E, Kovach E, Rasmussen B A, Eddy R L and Shaws B. 
(1991). Identification of a new endothelial cell growth factor receptor 
tyrosine kinase. Oncogene 6: 1677-1683. 
Tischer E, Mitchell R, Hartman T, Silva T. Gospodarowicz D, Fiddes J C, and 
J A Abraham. (1991) The human gene for vascular endothelial growth factor. 
Multiple protein forms are encoded through alternative exon splicing. J. 
Biol. Chem. 266:11947-11954. 
Yang X, Hubbard J A, and M Carlson (1992) A protein kinase substrate 
identified by the two-hybrid system. Science 257:31-33. 
Young K H and F W Bazer (1989) Porcine endometrial prolactin receptors 
detected by homologous radioreceptor assay. Mol. and Cell. Endocrinol. 
64:145-154. 
Young P R (1992) Protein hormones and their receptors. Curr. Opin. Biotech. 
3:408-421. 
Wade Harper J, Adami G R, Wei N, Keyomarsk K and S J Elledge. (1993) The 
p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 
Cyclin-dependent kinases. Cell 75:805-816. 
Wilson T E, Fahrner T J, Johnston M and J Milbrandt. (1991) Identification 
of the DNA binding site for NGFI-B by genetic selection in yeast. Science 
252:1296-1300. 
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