U.S. patent application number 09/759514 was filed with the patent office on 2003-05-01 for polypeptide sequences of human edg-1c.
Invention is credited to Bergsma, Derk J., Chambers, Jonathan K., Chan, Winnie, Jensen, Pamela Joy, Johnson, Randall K., Khandoudi, Nassirah, Livi, George P., Robert, Phillipe, Stadel, Jeffrey M., Wilson, Shelagh.
Application Number | 20030082743 09/759514 |
Document ID | / |
Family ID | 26759190 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082743 |
Kind Code |
A1 |
Bergsma, Derk J. ; et
al. |
May 1, 2003 |
Polypeptide sequences of human EDG-1c
Abstract
Human EDG-1c polypeptidees and polynucleotides and methods for
producing such polypeptides by recombinant techniques are
disclosed. Human EDG-1c is identified as a selective receptor for
sphingosine-1-phosphate ("S-1-P") and for di-hydro S-1-P. Also
disclosed are methods for discovering agonists and antagonists of
the interaction between S-1-P and di-hydro S-1-P and their cellular
receptor, human EDG-1c, which may have utility in the treatment of
several human diseases and disorders, including, but not limited to
the treatment of infections such as bacterial, fungal, protozoan
and viral infections, particularly infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity;
anorexia; bulimia; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
congestive heart failure; left ventricular hypertrophy; arrythmias;
restenosis after coronary artery angioplasty; vascular sclerosis;
deleterious fibrosis; atherosclerosis; inflammation; angiogenesis;
wound healing; ulcers; asthma; allergies; benign prostatic
hypertrophy; migraine; vomiting; psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
depression, delirium, dementia, and severe mental retardation;
degenerative diseases, such as neurodegenerative diseases and
ischemic stroke; and dyskinesias, such as Huntington's disease or
Gilles dela Tourett's syndrome.
Inventors: |
Bergsma, Derk J.; (Berwyn,
PA) ; Chan, Winnie; (West Chester, PA) ;
Chambers, Jonathan K.; (Cambridge, GB) ; Johnson,
Randall K.; (Ardmore, PA) ; Khandoudi, Nassirah;
(Saint Gregoire, FR) ; Livi, George P.;
(Havertown, PA) ; Robert, Phillipe; (Saint
Gregoire, FR) ; Stadel, Jeffrey M.; (Wayne, PA)
; Jensen, Pamela Joy; (Wayne, PA) ; Wilson,
Shelagh; (Hertford, GB) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Family ID: |
26759190 |
Appl. No.: |
09/759514 |
Filed: |
January 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09759514 |
Jan 12, 2001 |
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09262477 |
Mar 4, 1999 |
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6423508 |
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60077369 |
Mar 9, 1998 |
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60087102 |
May 28, 1998 |
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Current U.S.
Class: |
435/69.4 ;
435/320.1; 435/325; 530/388.25; 530/399; 536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
C40B 40/00 20130101; C07K 14/723 20130101; A61P 9/06 20180101; A61P
9/04 20180101 |
Class at
Publication: |
435/69.4 ;
435/320.1; 435/325; 530/399; 530/388.25; 536/23.5 |
International
Class: |
A61K 038/18; C07K
014/475; C12P 021/02; C12N 005/06; C07H 021/04; C07K 016/22 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a nucleotide sequence
encoding the polypeptide of SEQ ID NO:2; or a nucleotide sequence
complementary to said nucleotide sequence.
2. The polynucleotide as claimed in claim 1, wherein said
polynucleotide is DNA or RNA.
3. The polynucleotide as claimed in claim 1, wherein said
nucleotide sequence comprises SEQ ID NO:1.
4. An isolated polypeptide comprising the polypeptide sequence set
forth in SEQ ID NO:2.
5. An expression system comprising a polynucleotide capable of
producing a polypeptide as claimed in claim 4 when said expression
system is in a compatible host cell.
6. A process for producing a recombinant host cell comprising the
step of introducing the expression system as claimed in claim 5
into a cell, such that the host cell, under appropriate culture
conditions, produces said polypeptide.
7. A recombinant host cell produced by the process as claimed in
claim 6.
8. A membrane of a recombinant host cell as claimed in claim 7
expressing said polypeptide.
9. A process for producing a polypeptide comprising culturing a
host cell as claimed in claim 6 under conditions sufficient for the
production of said polypeptide and recovering the polypeptide from
the culture.
10. An antibody immunospecific for the polypeptide as claimed in
claim 4.
11. A method for identifying agonist or antagonist of the of
polypeptide as claimed in claim 4 comprising: (a) contacting a cell
expressing on the surface thereof the polypeptide, said polypeptide
being associated with a second component capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide, with a compound to be screened under conditions to
permit binding to the polypeptide; and (b) determining whether the
compound binds to and activates or inhibits the polypeptide by
measuring the level of a signal generated from the interaction of
the compound with the polypeptide.
12. The method as claimed in claim 11, wherein said method further
comprises conducting the identification of an agonist or antagonist
in the presence of labeled or unlabeled sphingosine 1-phosphate or
di-hydo sphingosine 1-phosphate.
13. A method for identifying an agonist or antagonist of the
polypeptide as claimed in claim 4 comprising: determining the
inhibition of binding of a ligand to cells expressing the
polypeptide on the surface thereof, or to cell membranes containing
the polypeptide, in the presence of a candidate compound under
conditions to permit binding to the polypeptide, and determining
the amount of ligand bound to the polypeptide, such that a compound
capable of causing reduction of binding of a ligand is an agonist
or antagonist.
14. The method as claimed in claim 13, wherein the ligand is
labeled or unlabeled sphingosine-1-phosphate or di-hydro
sphingosine 1-phosphate.
15. A method for screening to identify compounds that stimulate or
that inhibit a function or level of the polypeptide as claimed in
claim 4, comprising a method selected from the group consisting of:
(a) measuring or, quantitatively or qualitatively, detecting the
binding of a candidate compound to the polypeptide (or to the cells
or membranes bearing the polypeptide) or a fusion protein thereof
by means of a label directly or indirectly associated with the
candidate compound; (b) measuring the competition of the binding of
a candidate compound to the polypeptide (or to the cells or
membranes bearing the polypeptide) or a fusion protein thereof in
the presence of a labeled competitor, preferably
sphingosine-1-phosphate or di-hydro sphingosine 1-phosphate; (c)
testing whether the candidate compound results in a signal
generated by activation or inhibition of the polypeptide, using
detection systems appropriate to the cells or cell membranes
bearing the polypeptide; (d) mixing a candidate compound with a
solution comprising said polypeptide to form a mixture, measuring
activity of the polypeptide in the mixture, and comparing the
activity of the mixture to a to a control mixture which contains no
candidate compound; or (e) detecting the effect of a candidate
compound on the production of mRNA encoding said polypeptide and
said polypeptide in cells.
16. An antagonist identified by the method as claimed in claim
15.
17. An agonist identified by the method as claimed in claim 15.
18. A method for the treatment of a subject having need to inhibit
activity or expression of human EDG-1c polypeptide comprising: (a)
administering to the subject a therapeutically effective amount of
an antagonist as claimed in claim 16.
19. The method as claimed in claim 18, wherein the subject is
afflicted with a disease selected from the group consisting of:
congestive heart failure, left ventricular hypertrophy, and
arrythmias.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit to the earlier provisional
U.S. application Ser. Nos. 60/077,369, filed on Mar. 9, 1998, and
60/087,102, filed on May 28, 1998, the contents of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to newly identified polypeptides and
polynucleotides encoded by them and to the use of such
polynucleotides and polypeptides, and to their production. More
particularly, the polynucleotides and polypeptides of the present
invention relate to the G-protein coupled receptors, hereinafter
referred to as human EDG-1c receptor. This invention also relates
to methods for discovering agonists and antagonists of the
interaction between sphingosine 1-phosphate (hereinafter referred
to as "S-1-P") and di-hydro sphingosine 1-phosphate (also known as
sphingoanine 1-phosphate and hereinafter referred to as "di-hydro
S-1-P") and their cellular receptor, human EDG-1c receptor. The
invention also relates to the use of human EDG-1c polynucleotides
and polypeptides in therapy and in identifying compounds which may
be agonists, antagonists and/or inhibitors which are potentially
useful in therapy, and to production of such polypeptides and
polynucleotides.
BACKGROUND OF THE INVENTION
[0003] The drug discovery process is currently undergoing a
fundamental revolution as it embraces `functional genomics`, that
is, high throughput genome- or gene-based biology. This approach is
rapidly superseding earlier approaches based on `positional
cloning`. A phenotype, that is a biological function or genetic
disease, would be identified and this would then be tracked back to
the responsible gene, based on its genetic map position.
[0004] Functional genomics relies heavily on the various tools of
bioinformatics to identify gene sequences of potential interest
from the many molecular biology databases now available. There is a
continuing need to identify and characterize further genes and
their related polypeptides/proteins, as targets for drug
discovery.
[0005] It is well established that many medically significant
biological processes are mediated by proteins participating in
signal transduction pathways that involve G-proteins and/or second
messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354).
Herein, these proteins are referred to as proteins participating in
pathways with G-proteins. Some examples of these proteins include
the G-protein coupled receptors, such as those for adrenergic
agents and dopamine (Kobilka, B. K., et al., Proc. Natl Acad. Sci.,
USA, 1987, 84:46-50; Kobilka, B. K., et al., Science, 1987,
238:650-656; Bunzow, J. R., et al., Nature, 1988, 336:783-787),
G-proteins themselves, effector proteins, e.g., phospholipase C,
adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g.,
protein kinase A and protein kinase C (Simon, M. I., et al.,
Science, 1991, 252:802-8).
[0006] For example, in one form of signal transduction, the effect
of hormone binding is activation of the enzyme, adenylate cyclase,
inside the cell. Enzyme activation by hormones is dependent on the
presence of the nucleotide GTP. GTP also influences hormone
binding. A G-protein connects the hormone receptor to adenylate
cyclase. G-protein was shown to exchange GTP for bound GDP when
activated by a hormone receptor. The GTP-carrying form then binds
to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed
by the G-protein itself, returns the G-protein to its basal,
inactive form. Thus, the G-protein serves a dual role, as an
intermediate that relays the signal from receptor to effector, and
as a clock that controls the duration of the signal.
[0007] The membrane protein gene superfamily of G-protein coupled
receptors has been characterized as having seven putative
transmembrane domains. The domains are believed to represent
transmembrane a-helices connected by extracellular or cytoplasmic
loops. G-protein coupled receptors include a wide range of
biologically active receptors, such as hormone, viral, growth
factor and neuroreceptors.
[0008] G-protein coupled receptors (otherwise known as 7TM
receptors) have been characterized as including these seven
conserved hydrophobic stretches of about 20 to 30 amino acids,
connecting at least eight divergent hydrophilic loops. The
G-protein family of coupled receptors includes dopamine receptors
which bind to neuroleptic drugs used for treating psychotic and
neurological disorders. Other examples of members of this family
include, but are not limited to, calcitonin, adrenergic,
endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin,
histamine, thrombin, kinin, follicle stimulating hormone, opsins,
endothelial differentiation gene-1, rhodopsins, odorant, and
cytomegalovirus receptors.
[0009] Most G-protein coupled receptors have single conserved
cysteine residues in each of the first two extracellular loops
which form disulfide bonds that are believed to stabilize
functional protein structure. The 7 transmembrane regions are
designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been
implicated in signal transduction.
[0010] Phosphorylation and lipidation (palmitylation or
farnesylation) of cysteine residues can influence signal
transduction of some G-protein coupled receptors. Most G-protein
coupled receptors contain potential phosphorylation sites within
the third cytoplasmic loop and/or the carboxy terminus. For several
G-protein coupled receptors, such as the b-adrenoreceptor,
phosphorylation by protein kinase A and/or specific receptor
kinases mediates receptor desensitization.
[0011] For some receptors, the ligand binding sites of G-protein
coupled receptors are believed to comprise hydrophilic sockets
formed by several G-protein coupled receptor transmembrane domains,
said socket being surrounded by hydrophobic residues of the
G-protein coupled receptors. The hydrophilic side of each G-protein
coupled receptor transmembrane helix is postulated to face inward
and form polar ligand binding site. TM3 has been implicated in
several G-protein coupled receptors as having a ligand binding
site, such as the TM3 aspartate residue. TM5 serines, a TM6
asparagine and TM6 or TM7 phenylalanines or tyrosines are also
implicated in ligand binding.
[0012] G-protein coupled receptors can be intracellularly coupled
by heterotrimeric G-proteins to various intracellular enzymes, ion
channels and transporters (see, Johnson, et al., Endoc. Rev., 1989,
10:317-331) Different G-protein a-subunits preferentially stimulate
particular effectors to modulate various biological functions in a
cell. Phosphorylation of cytoplasmic residues of G-protein coupled
receptors have been identified as an important mechanism for the
regulation of G-protein coupling of some G-protein coupled
receptors. G-protein coupled receptors are found in numerous sites
within a mammalian host.
[0013] Over the past 15 years, nearly 350 therapeutic agents
targeting 7 transmembrane (7TM) receptors have been successfully
introduced onto the market.
SUMMARY OF THE INVENTION
[0014] In one aspect, the invention relates human EDG-1c
polypeptides and recombinant materials and methods for their
production. Another aspect of the invention relates to methods for
using such human EDG-1c polypeptides and polynucleotides. Such uses
include the treatment of infections such as bacterial, fungal,
protozoan and viral infections, particularly infections such as
bacterial, fungal, protozoan and viral infections, particularly
infections caused by HIV-1 or HIV-2; pain; cancers; diabetes,
obesity; anorexia; bulimia; asthma; Parkinson's disease; acute
heart failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
congestive heart failure; left ventricular hypertrophy; arrythmias;
restenosis after coronary artery angioplasty; vascular sclerosis;
deleterious fibrosis; atherosclerosis; inflammation; angiogenesis;
wound healing; ulcers; asthma; allergies; benign prostatic
hypertrophy; migraine; vomiting; psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
depression, delirium, dementia, and severe mental retardation;
degenerative diseases, such as neurodegenerative diseases and
ischemic stroke; and dyskinesias, such as Huntington's disease or
Gilles dela Tourett's syndrome, among others.
[0015] In accordance with another aspect of the present invention
there are provided methods of screening for compounds which bind to
and activate (agonist) or inhibit activation (antagonist) of human
EDG-1c polypeptides (receptors), and for their ligands.
[0016] In particular, the preferred method for identifying agonist
or antagonist of a human EDG-1c polypeptide comprises:
[0017] (a) contacting a cell expressing on the surface thereof the
polypeptide, said polypeptide being associated with a second
component capable of providing a detectable signal in response to
the binding of a compound to said polypeptide, with a compound to
be screened under conditions to permit binding to the polypeptide;
and
[0018] (b) determining whether the compound binds to and activates
or inhibits the polypeptide by measuring the level of a signal
generated from the interaction of the compound with the
polypeptide.
[0019] In a further preferred embodiment, the method further
comprises conducting the identification of agonist or antagonist in
the presence of labeled or unlabeled S-1-P or di-hydro S-1-P.
[0020] In another embodiment, the method for identifying agonist or
antagonist of a human EDG-1c polypeptide comprises:
[0021] determining the inhibition of binding of a ligand to cells
which have the polypeptide on the surface thereof, or to cell
membranes containing the polypeptide, in the presence of a
candidate compound under conditions to permit binding to the
polypeptide, and determining the amount of ligand bound to the
polypeptide, such that a compound capable of causing reduction of
binding of a ligand is an agonist or antagonist. Preferably, the
ligand is S-1-P or di-hydro S-1-P. Yet more preferably, S-1-P or
di-hydro S-1-P is labeled.
[0022] Furthermore, the present invention relates to treating
conditions associated with human EDG-1c receptor imbalance with the
identified compounds. Yet another aspect of the invention relates
to diagnostic assays for detecting diseases associated with
inappropriate EDG-1 activity or levels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows the nucleotide sequence of the human EDG-1c
receptor (SEQ ID NO: 1).
[0024] FIG. 2 shows the deduced amino acid sequence of the human
EDG-1c receptor (SEQ ID NO:2).
[0025] FIG. 3 shows concentration response curves for S-1-P against
endogeneous HEK293 cells.
[0026] FIG. 4 shows an agar plate assay of the dose response for
S-1-P against yeast cells containing the pathway-inducible
fus1-lacZ reporter and expressing the human EDG-1c receptor in
combination with either the endogenous yeast G? protein (GPA1) or a
chimeric yeast G?/human G?i2.
[0027] FIG. 5 shows concentration response curves for S-1-P against
yeast cells containing the pathway-inducible fus1-lacZ reporter and
expressing the human EDG-1c receptor in combination with GPA1 in a
liquid lacZ assay format.
[0028] FIG. 6 shows dose dependent cellular hypertrophy in rat
neonatal myocytes in culture induced by S-1-P.
[0029] FIG. 7 shows concentration-response curves for S-1-P in RBL
2H3 cells stably transfected with the EDG-1c receptor.
[0030] FIG. 8 shows agonist activity for a number of ligands in RBL
2H3 EDG-1c cells.
DESCRIPTION OF THE INVENTION
Definitions
[0031] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0032] "Human EDG-1c" refers generally to polypeptides having the
amino acid sequence set forth in SEQ ID NO:2 or an allelic variant
thereof.
[0033] "S-1-P (sphingosine-1-phosphate)" refers to the sphingolipid
metabolite having the structure: 1
[0034] "Di-hydro S-1-P" (di-hydro sphingosine-1-phosphate)"
(hereinafter referred to as "di-hydro S-1-P") refers to the
sphingolipid metabolite having the structure: 2
[0035] "Receptor Activity" or "Biological Activity of the Receptor"
refers to the metabolic or physiologic function of said human
EDG-1c including similar activities or improved activities or these
activities with decreased undesirable side-effects. Also included
are antigenic and immunogenic activities of said human EDG-1c.
[0036] "Human EDG-1c polypeptides" refers to polypeptides with
amino acid sequences sufficiently similar to human EDG-1c
preferably exhibiting at least one biological activity of the
receptor.
[0037] "Human EDG-1c gene" refers to a polynucleotide having the
nucleotide sequence set forth in SEQ ID NO:1 or allelic variants
thereof and/or their complements.
[0038] "Human EDG-1c polynucleotides" and refers to polynucleotides
containing a nucleotide sequence which encodes a human EDG-1c
polypeptide of SEQ ID NO:2, or a nucleotide sequence which has
sufficient identity to a nucleotide sequence contained in SEQ ID
NO:1 to hybridize under conditions useable for amplification or for
use as a probe or marker.
[0039] "Antibodies," as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
[0040] "Isolated" means altered "by the hand of man" from its
natural state, i.e., if it occurs in nature, it has been changed or
removed from its original environment, or both. For example, a
polynucleotide or a polypeptide naturally present in a living
organism is not "isolated," but the same polynucleotide or
polypeptide separated from the coexisting materials of its natural
state is "isolated", as the term is employed herein. Moreover, a
polynucleotide or polypeptide that is introduced into an organism
by transformation, genetic manipulation or by any other recombinant
method is "isolated" even if it is still present in said organism,
which organism may be living or non-living.
[0041] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications has been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0042] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the 20 gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination. See,
for instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and
Wold, F., Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter et al., "Analysis for protein modifications and nonprotein
cofactors", Meth Enzymol (1990) 182:626-646 and Rattan, et al.,
"Protein Synthesis: Posttranslational Modifications and Aging", Ann
NY Acad Sci (1992) 663:48-62.
[0043] "Variant," as the term is used herein, is a polynucleotide
or polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence, as discussed below. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical. A
variant and reference polypeptide may differ in amino acid sequence
by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurring such as
an allelic variant, or it may be a variant that is not known to
occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis
techniques or by direct synthesis.
[0044] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as determined by comparing the sequences. In the art,
"identity" also means the degree of sequence relatedness between
polypeptide or polynucleotide sequences, as the case may be, as
determined by the match between strings of such sequences.
"Identity" and "similarity" can be readily calculated by known
methods, including but not limited to those described in
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991; and Carillo, H., and
Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred
methods to determine identity are designed to give the largest
match between the sequences tested. Methods to determine identity
and similarity are codified in publicly available computer
programs. Preferred computer program methods to determine identity
and similarity between two sequences include, but are not limited
to, the GCG program package (Devereux, J., et al., Nucleic Acids
Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul,
et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is
publicly available from NCBI and other sources (BLAST Manual,
Altschul, et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S.,
et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith
Waterman algorithm may also be used to determine identity.
[0045] Preferred parameters for polypeptide sequence comparison
include the following:
[0046] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0047] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
[0048] Gap Penalty: 12
[0049] Gap Length Penalty: 4
[0050] A program useful with these parameters is publicly available
as the "gap" program from Genetics Computer Group, Madison Wis. The
aforementioned parameters are the default parameters for peptide
comparisons (along with no penalty for end gaps).
[0051] Preferred parameters for polynucleotide comparison include
the following:
[0052] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0053] Comparison matrix: matches=+10, mismatch=0
[0054] Gap Penalty: 50
[0055] Gap Length Penalty: 3
[0056] Available as: The "gap" program from Genetics Computer
Group, Madison Wis. These are the default parameters for nucleic
acid comparisons.
[0057] By way of example, a polynucleotide sequence of the present
invention may be identical to the reference sequence of SEQ ID
NO:1, that is be 100% identical, or it may include up to a certain
integer number of nucleotide alterations as compared to the
reference sequence. Such alterations are selected from the group
consisting of at least one nucleotide deletion, substitution,
including transition and transversion, or insertion, and wherein
said alterations may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among the
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence. The number of nucleotide
alterations is determined by multiplying the total number of
nucleotides in SEQ ID NO:1 by the numerical percent of the
respective percent identity(divided by 100) and subtracting that
product from said total number of nucleotides in SEQ ID NO:1,
or:
n.sub.n.ltoreq.x.sub.n-(x.sub.n.multidot.y),
[0058] wherein n.sub.n is the number of nucleotide alterations,
x.sub.n is the total number of nucleotides in SEQ ID NO:1, and y
is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90
for 90%, 0.95 for 95%, etc., and wherein any non-integer product of
x.sub.n and y is rounded down to the nearest integer prior to
subtracting it from x.sub.n. Alterations of a polynucleotide
sequence encoding the polypeptide of SEQ ID NO:2 may create
nonsense, missense or frameshift mutations in this coding sequence
and thereby alter the polypeptide encoded by the polynucleotide
following such alterations.
[0059] Similarly, a polypeptide sequence of the present invention
may be identical to the reference sequence of SEQ ID NO:2, that is
be 100% identical, or it may include up to a certain integer number
of amino acid alterations as compared to the reference sequence
such that the % identity is less than 100%. Such alterations are
selected from the group consisting of at least one amino acid
deletion, substitution, including conservative and non-conservative
substitution, or insertion, and wherein said alterations may occur
at the amino- or carboxy-terminal positions of the reference
polypeptide sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids in the
reference sequence or in one or more contiguous groups within the
reference sequence. The number of amino acid alterations for a
given % identity is determined by multiplying the total number of
amino acids in SEQ ID NO:2 by the numerical percent of the
respective percent identity(divided by 100) and then subtracting
that product from said total number of amino acids in SEQ ID NO:2,
or:
n.sub.a.ltoreq.x.sub.a-(x.sub.a.multidot.y)
[0060] wherein n.sub.a is the number of amino acid alterations,
x.sub.a is the total number of amino acids in SEQ ID NO:2, and y
is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and
wherein any non-integer product of x.sub.a and y is rounded down to
the nearest integer prior to subtracting it from x.sub.a.
[0061] Polypeptides of the Invention
[0062] The human EDG-1c polypeptides of the present invention
include the polypeptide of SEQ ID NO:2 (in particular the mature
polypeptide).
[0063] The human EDG-1c polypeptides may be in the form of the
"mature" protein or may be a part of a larger protein such as a
fusion protein. It is often advantageous to include an additional
amino acid sequence which contains secretory or leader sequences,
pro-sequences, sequences which aid in purification such as multiple
histidine residues, or an additional sequence for stability during
recombinant production.
[0064] Biologically active fragments of the human EDG-1c
polypeptides are also included in the invention. A fragment is a
polypeptide having an amino acid sequence that entirely is the same
as part, but not all, of the amino acid sequence of the
aforementioned human EDG-1c polypeptides. As with human EDG-1c
polypeptides, fragments may be "free-standing," or comprised within
a larger polypeptide of which they form a part or region, most
preferably as a single continuous region. Representative examples
of polypeptide fragments of the invention, include, for example,
fragments from about amino acid number 1-20, 21-40, 41-60, 61-80,
81-100, and 101 to the end of human EDG-1c polypeptides. In this
context "about" includes the particularly recited ranges larger or
smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or
at both extremes.
[0065] Preferred fragments include, for example, truncation
polypeptides having the amino acid sequence of human EDG-1c
polypeptides, except for deletion of a continuous series of
residues that includes the amino terminus, or a continuous series
of residues that includes the carboxyl terminus or deletion of two
continuous series of residues, one including the amino terminus and
one including the carboxyl terminus. Also preferred are fragments
characterized by structural or functional attributes such as
fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions.
Biologically active fragments are those that mediate receptor
activity, including those with a similar activity or an improved
activity, or with a decreased undesirable activity. Also included
are those that are antigenic or immunogenic in an animal,
especially in a human.
[0066] Thus, the polypeptides of the invention include polypeptides
having the amino acid sequence set forth in SEQ ID NO:2.
Preferably, all of these polypeptides retain the biological
activity of the receptor, including antigenic activity. Included in
this group are variants of the defined sequence and fragments.
Preferred variants are those that vary from the referents by
conservative amino acid substitutions--i.e., those that substitute
a residue with another of like characteristics. Typical such
substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;
among the acidic residues Asp and Glu; among Asn and Gln; and among
the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
Particularly preferred are variants in which several, 5-10, 1-5, or
1-2 amino acids are substituted, deleted, or added in any
combination.
[0067] The human EDG-1c polypeptides of the invention can be
prepared in any suitable manner. Such polypeptides include isolated
naturally occurring polypeptides, recombinantly produced
polypeptides, synthetically produced polypeptides, or polypeptides
produced by a combination of these methods. Means for preparing
such polypeptides are well understood in the art.
[0068] Polynucleotides of the Invention
[0069] Another aspect of the invention relates to isolated
polynucleotides which encode the EDG-1 polypeptides and
polynucleotides closely related thereto.
[0070] The nucleotide sequence of SEQ ID NO:1 shows homology with
human EDG-1 receptor (Hla, T., and T. Maciag; 1990; J. Biol. Chem.
265: 9309-9313). The nucleotide sequence of SEQ ID NO:1 is a cDNA
sequence and comprises a polypeptide encoding sequence (nucleotide
1 to 1149) encoding a polypeptide of 382 amino acids, the
polypeptide of SEQ ID NO:2. The nucleotide sequence encoding the
polypeptide of SEQ ID NO:2 may be identical to the polypeptide
encoding sequence contained in SEQ ID NO:1 or it may be a sequence
other than the one contained in SEQ ID NO:1, which, as a result of
the redundancy (degeneracy) of the genetic code, also encodes the
polypeptide of SEQ ID NO:2. The polypeptide of the SEQ ID NO:2 is
structurally related to other proteins of the G-coupled Protein
Receptors family, having homology and/or structural similarity with
human EDG-1 receptor (Hla, T., and T. Maciag; 1990; J. Biol. Chem.
265: 9309-9313).
[0071] One polynucleotide of the present invention encoding human
EDG-1c may be obtained using standard cloning and screening, from a
cDNA library derived from mRNA in cells of human placenta using the
expressed sequence tag (EST) analysis (Adams, M. D., et al. Science
(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992)
355:632-634; Adams, M. D., et al., Nature (1995) 377 Supp:3-174).
Polynucleotides of the invention can also be obtained from natural
sources such as genomic DNA libraries or can be synthesized using
well known and commercially available techniques.
[0072] Thus, the nucleotide sequence encoding human EDG-1c
polypeptides may be identical over its entire length to the coding
sequence in FIG. 1 (SEQ ID NO:1).
[0073] When the polynucleotides of the invention are used for the
recombinant production of human EDG-1c polypeptide, the
polynucleotide may include the coding sequence for the mature
polypeptide or a fragment thereof, by itself; the coding sequence
for the mature polypeptide or fragment in reading frame with other
coding sequences, such as those encoding a leader or secretory
sequence, a pre-, or pro- or prepro- protein sequence, or other
fusion peptide portions. For example, a marker sequence which
facilitates purification of the fused polypeptide can be encoded.
In certain preferred embodiments of this aspect of the invention,
the marker sequence is a hexa-histidine peptide, as provided in the
pQE vector (Qiagen, Inc.) and described in Gentz, et al., Proc Natl
Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide
may also contain non-coding 5' and 3' sequences, such as
transcribed, non-translated sequences, splicing and polyadenylation
signals, ribosome binding sites and sequences that stabilize
mRNA.
[0074] Among particularly preferred embodiments of the invention
are polynucleotides encoding human EDG-1c polypeptides having the
amino acid sequence of set out in FIG. 1 (SEQ ID NO:2) and variants
thereof.
[0075] Further preferred embodiments are polynucleotides encoding
human EDG-1c variants that have the amino acid sequence of the
human EDG-1c of FIG. 1 (SEQ ID NO:2) in which several, 5-10, 1-5,
1-3, 1-2 or 1 amino acid residues are substituted, deleted or
added, in any combination.
[0076] The present invention further relates to polynucleotides
that hybridize to the herein above-described sequences. In this
regard, the present invention especially relates to polynucleotides
which hybridize under stringent conditions to the herein
above-described polynucleotides. As herein used, the term
"stringent conditions" means hybridization will occur only if there
is at least 95% and preferably at least 97% identity between the
sequences.
[0077] Polynucleotides of the invention, which are sufficiently
identical to a nucleotide sequence contained in SEQ ID NO:1, may be
used as hybridization probes for cDNA and genomic DNA, to isolate
full-length cDNAs and genomic clones encoding human EDG-1c and to
isolate cDNA and genomic clones of other genes that have a high
sequence similarity to the human EDG-1c gene. Such hybridization
techniques are known to those of skill in the art. Typically these
nucleotide sequences are 70% identical, preferably 80% identical,
more preferably 90% identical to that of the reference sequence.
The probes generally will comprise at least 15 nucleotides.
Preferably, such probes will have at least 30 nucleotides and may
have at least 50 nucleotides. Particularly preferred probes will
range between 30 and 50 nucleotides.
[0078] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to animal and human
disease.
[0079] Vectors, Host Cells, Expression
[0080] The present invention also relates to vectors which comprise
a polynucleotide or polynucleotides of the present invention, and
host cells which are genetically engineered with vectors of the
invention and to the production of polypeptides of the invention by
recombinant techniques. Cell-free translation systems can also be
employed to produce such proteins using RNAs derived from the DNA
constructs of the present invention.
[0081] For recombinant production, host cells can be genetically
engineered to incorporate expression systems or portions thereof
for polynucleotides of the present invention. Introduction of
polynucleotides into host cells can be effected by methods
described in many standard laboratory manuals, such as Davis, et
al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook, et
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as
calcium phosphate transfection, DEAE-dextran mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection.
[0082] Representative examples of appropriate hosts include
bacterial cells, such as streptococci, staphylococci, E. coli,
Streptomyces and Bacillus subtilis cells; fungal cells, such as
yeast cells and Aspergillus cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa,
C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
[0083] A great variety of expression systems can be used. Such
systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate nucleotide
sequence may be inserted into an expression system by any of a
variety of well-known and routine techniques, such as, for example,
those set forth in Sambrook, et al., MOLECULAR CLONING, A
LABORATORY MANUAL (supra).
[0084] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the desired polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0085] If the human EDG-1c polypeptide is to be expressed for use
in screening assays, generally, it is preferred that the
polypeptide be produced at the surface of the cell. In this event,
the cells may be harvested prior to use in the screening assay. If
human EDG-1c polypeptide is secreted into the medium, the medium
can be recovered in order to recover and purify the polypeptide; if
produced intracellularly, the cells must first be lysed before the
polypeptide is recovered.
[0086] Human EDG-1c polypeptides can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography is employed for
purification. Well known techniques for refolding proteins may be
employed to regenerate active conformation when the polypeptide is
denatured during isolation and or purification.
[0087] Diagnostic Assays
[0088] This invention also relates to the use of human EDG-1c
polynucleotides for use as diagnostic reagents. Detection of a
mutated form of human EDG-1c gene associated with a dysfunction
will provide a diagnostic tool that can add to or define a
diagnosis of a disease or susceptibility to a disease which results
from under-expression, over-expression or altered expression of
human EDG-1c. Individuals carrying mutations in the human EDG-1c
gene may be detected at the DNA level by a variety of
techniques.
[0089] Nucleic acids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biopsy or autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to labeled human EDG-1c nucleotide sequences.
Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase digestion or by differences in melting
temperatures. DNA sequence differences may also be detected by
alterations in electrophoretic mobility of DNA fragments in gels,
with or without denaturing agents, or by direct DNA sequencing.
See, e.g., Myers, et al., Science (1985) 230:1242. Sequence changes
at specific locations may also be revealed by nuclease protection
assays, such as RNase and S1 protection or the chemical cleavage
method. See Cotton, et al., Proc Natl Acad Sci USA (1985) 85:
4397-4401.
[0090] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to infections such as bacterial,
fungal, protozoan and viral infections, particularly through
detection of mutation in the EDG-1 gene by the methods
described.
[0091] In addition, infections such as bacterial, fungal, protozoan
and viral infections, particularly infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity;
anorexia; bulimia; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
congestive heart failure; left ventricular hypertrophy; arrythmias;
restenosis after coronary artery angioplasty; vascular sclerosis;
deleterious fibrosis; atherosclerosis; inflammation; angiogenesis;
wound healing; ulcers; asthma; allergies; benign prostatic
hypertrophy; migraine; vomiting; psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
depression, delirium, dementia, and severe mental retardation;
degenerative diseases, such as neurodegenerative diseases and
ischemic stroke; and dyskinesias, such as Huntington's disease or
Gilles dela Tourett's syndrome, can be diagnosed by methods
comprising determining from a sample derived from a subject an
abnormally decreased or increased level of human EDG-1c polypeptide
or human EDG-1c mRNA. Decreased or increased expression can be
measured at the RNA level using any of the methods well known in
the art for the quantitation of polynucleotides, such as, for
example, PCR, RT-PCR, RNase protection, Northern blotting and other
hybridization methods. Assay techniques that can be used to
determine levels of a protein, such as human EDG-1c, in a sample
derived from a host are well-known to those of skill in the art.
Such assay methods include radioimmunoassays, competitive-binding
assays, Western Blot analysis and ELISA assays.
[0092] Chromosome Assays
[0093] The nucleotide sequences of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. The mapping of relevant
sequences to chromosomes according to the present invention is an
important first step in correlating those sequences with gene
associated disease. Once a sequence has been mapped to a precise
chromosomal location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. Such data are
found, for example, in V. McKusick, Mendelian Inheritance in Man
(available on line through Johns Hopkins University Welch Medical
Library). The relationship between genes and diseases that have
been mapped to the same chromosomal region are then identified
through linkage analysis (coinheritance of physically adjacent
genes).
[0094] The differences in the cDNA or genomic sequence between
affected and unaffected individuals can also be determined. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
[0095] Antibodies
[0096] The polypeptides of the invention or their fragments or
analogs thereof, or cells expressing them can also be used as
immunogens to produce antibodies immunospecific for the human
EDG-1c polypeptides. The term "immunospecific" means that the
antibodies have substantially greater affinity for the polypeptides
of the invention than their affinity for other related polypeptides
in the prior art.
[0097] Antibodies generated against the human EDG-1c polypeptides
can be obtained by administering the polypeptides or
epitope-bearing fragments, analogs or cells to an animal,
preferably a nonhuman, using routine protocols. For preparation of
monoclonal antibodies, any technique which provides antibodies
produced by continuous cell line cultures can be used. Examples
include the hybridoma technique (Kohler, G. and Milstein, C.,
Nature (1975) 256:495-497), the trioma technique, the human B-cell
hybridoma technique (Kozbor, et al., Immunology Today (1983) 4:72)
and the EBV-hybridoma technique (Cole, et al., MONOCLONAL
ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc.,
1985).
[0098] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can also be adapted to produce single
chain antibodies to polypeptides of this invention. Also,
transgenic mice, or other organisms including other mammals, may be
used to express humanized antibodies.
[0099] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or to purify the
polypeptides by affinity chromatography.
[0100] Antibodies against human EDG-1c polypeptides may also be
employed to treat infections such as bacterial, fungal, protozoan
and viral infections, particularly infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity;
anorexia; bulimia; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
congestive heart failure; left ventricular hypertrophy; arrythmias;
restenosis after coronary artery angioplasty; vascular sclerosis;
deleterious fibrosis; atherosclerosis; inflammation; angiogenesis;
wound healing; ulcers; asthma; allergies; benign prostatic
hypertrophy; migraine; vomiting; psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
depression, delirium, dementia, and severe mental retardation;
degenerative diseases, such as neurodegenerative diseases and
ischemic stroke; and dyskinesias, such as Huntington's disease or
Gilles dela Tourett's syndrome, among others.
[0101] Vaccines
[0102] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with human EDG-1c polypeptide, or a fragment
thereof, adequate to produce antibody and/or T cell immune response
to protect said animal from infections such as bacterial, fungal,
protozoan and viral infections, particularly infections such as
bacterial, fungal, protozoan and viral infections, particularly
infections caused by HIV-1 or HIV-2; pain; cancers; diabetes,
obesity; anorexia; bulimia; asthma; Parkinson's disease; acute
heart failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
congestive heart failure; left ventricular hypertrophy; arrythmias;
restenosis after coronary artery angioplasty; vascular sclerosis;
deleterious fibrosis; atherosclerosis; inflammation; angiogenesis;
wound healing; ulcers; asthma; allergies; benign prostatic
hypertrophy; migraine; vomiting; psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
depression, delirium, dementia, and severe mental retardation;
degenerative diseases, such as neurodegenerative diseases and
ischemic stroke; and dyskinesias, such as Huntington's disease or
Gilles dela Tourett's syndrome, among others. Yet another aspect of
the invention relates to a method of inducing immunological
response in a mammal which comprises, delivering human EDG-1c gene
via a vector directing expression of human EDG-1c polypeptide in
vivo in order to induce such an immunological response to produce
antibody to protect said animal from diseases.
[0103] A further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to a human EDG-1c polypeptide wherein the
composition comprises a human EDG-1c polypeptide or human EDG-1c
gene. The vaccine formulation may further comprise a suitable
carrier. Since human EDG-1c polypeptide may be broken down in the
stomach, it is preferably administered parenterally (including
subcutaneous, intramuscular, intravenous, intradermal etc.
injection). Formulations suitable for parenteral administration
include aqueous and non-aqueous sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the recipient;
and aqueous and non-aqueous sterile suspensions which may include
suspending agents or thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampoules and vials and may be stored in a freeze-dried
condition requiring only the addition of the sterile liquid carrier
immediately prior to use. The vaccine formulation may also include
adjuvant systems for enhancing the immunogenicity of the
formulation, such as oil-in water systems and other systems known
in the art. The dosage will depend on the specific activity of the
vaccine and can be readily determined by routine
experimentation.
[0104] Screening Assays
[0105] The EDG1-c polypeptide of the present invention may be
employed in a process for screening for compounds that bind to and
activate the EDG1-c polypeptides of the present invention (called
agonists), or inhibit the interaction of the EDG1-c polypeptides
with receptor ligands (called antagonists). Thus, polypeptides of
the invention may also be used to assess the binding of small
molecule substrates and ligands in, for example, cells, cell-free
preparations, chemical libraries, and natural product mixtures.
These substrates and ligands may be natural substrates and ligands
or may be structural or functional mimetics. See Coligan, et al.,
Current Protocols in Immunology 1(2):Chapter 5 (1991).
[0106] EDG1-c proteins are responsible for many biological
functions, including many pathologies. Provided by the invention
are screening methods to identify compounds and drugs that
stimulate EDG1-c or that inhibit the function or level of the
polypeptide. In general, agonists are employed for therapeutic and
prophylactic purposes for such conditions as infections such as
bacterial, fungal, protozoan and viral infections, particularly
infections caused by HIV-1 or HIV-2; pain; cancers; diabetes,
obesity; anorexia; bulimia; asthma; Parkinson's disease; acute
heart failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; stroke;
congestive heart failure; left ventricular hypertrophy; arrythmias;
restenosis after coronary artery angioplasty; vascular sclerosis;
deleterious fibrosis; atherosclerosis; inflammation; angiogenesis;
wound healing; ulcers; asthma; allergies; benign prostatic
hypertrophy; migraine; vomiting; psychotic and neurological
disorders, including anxiety, schizophrenia, manic depression,
depression, delirium, dementia, and severe mental retardation;
degenerative diseases, such as neurodegenerative diseases and
iscbemic stroke; and dyskinesias, such as Huntington's disease or
Gilles dela Tourett's syndrome, among others.
[0107] In general, such screening procedures involve providing
appropriate cells that express the receptor polypeptide of the
present invention on the surface thereof. Such cells include cells
from mammals, yeast, Drosophila or E. coli. In particular, a
polynucleotide encoding the receptor of the present invention is
employed to transfect cells to thereby express the EDG1-c
polypeptide. The expressed receptor is then contacted with a test
compound to observe binding, stimulation or inhibition of a
functional response.
[0108] One such screening procedure involves the use of
melanophores that are transfected to express the EDG1-c polypeptide
of the present invention. Such a screening technique is described
in PCT WO 92/01810, published Feb. 6, 1992. Such an assay may be
employed to screen for a compound which inhibits activation of the
receptor polypeptide of the present invention by contacting the
melanophore cells that encode the receptor with both a receptor
ligand, such as S-1-P or di-hydro S-1-P, and a compound to be
screened. Inhibition of the signal generated by the ligand
indicates that a compound is a potential antagonist for the
receptor, i.e., inhibits activation of the receptor.
[0109] The technique may also be employed for screening of
compounds that activate the receptor by contacting such cells with
compounds to be screened and determining whether such compound
generates a signal, i.e., activates the receptor.
[0110] Other screening techniques include the use of cells which
express the EDG1-c polypeptide (for example, transfected CHO cells)
in a system that measures extracellular pH changes caused by
receptor activation. In this technique, compounds may be contacted
with cells expressing the receptor polypeptide of the present
invention. A second messenger response, e.g., signal transduction
or pH changes, is then measured to determine whether the potential
compound activates or inhibits the receptor.
[0111] Another screening technique involves expressing the EDG1-c
polypeptide in which the receptor is linked to phospholipase C or
D. Representative examples of such cells include, but are not
limited to: endothelial cells, smooth muscle cells, and embryonic
kidney cells. The screening may be accomplished as hereinabove
described by detecting activation of the receptor or inhibition of
activation of the receptor from the phospholipase second
signal.
[0112] Another method involves screening for compounds that are
antagonists, and thus inhibit activation of the receptor
polypeptide of the present invention by determining inhibition of
binding of labeled ligand, such as S-1-P or di-hydro S-1-P, to
cells expressing the receptor on their surface, or cell membranes
containing the receptor. Such a method involves transfecting a
eukaryotic cell with DNA encoding the EDG1-c polypeptide, such that
the cell expresses the receptor on its surface. The cell is then
contacted with a potential antagonist in the presence of a labeled
form of a ligand, such as S-1-P or di-hydro S-1-P. The ligand can
be labeled, e.g., by radioactivity. The amount of labeled ligand
bound to the receptors is measured, e.g., by measuring
radioactivity associated with transfected cells or membrane from
these cells. If the compound binds to the receptor, the binding of
labeled ligand to the receptor is inhibited as determined by a
reduction of labeled ligand which binds to the receptors. This
method is called binding assay. Naturally, this same technique can
be used to look for an agonist.
[0113] The screening method may simply measure the binding of a
candidate compound to the polypeptide, or to cells or membranes
bearing the polypeptide, or a fusion protein thereof by means of a
label directly or indirectly associated with the candidate
compound. Alternatively, the screening method may involve measuring
or, qualitatively or quantitatively, detecting the competition of
binding of a candidate compound to the polypeptide with a labeled
competitor (e.g. agonist or antagonist). Further, these screening
methods may test whether the candidate compound results in a signal
generated by activation or inhibition of the polypeptide, using
detection systems appropriate to the cells bearing the polypeptide.
Inhibitors of activation are generally assayed in the presence of a
known agonist and the effect on activation by the agonist by the
presence of the candidate compound is observed. Further, the
screening methods may simply comprise the steps of mixing a
candidate compound with a solution containing a polypeptide of the
present invention, to form a mixture, measuring EDG-1c activity in
the mixture, and comparing the EDG-1c activity of the mixture to a
control mixture which contains no candidate compound.
[0114] Another screening procedure involves the use of mammalian
cells (CHO, HEK 293, Xenopus Oocytes, RBL-2H3, etc.) that are
transfected to express the receptor of interest. The cells are
loaded with an indicator dye that produces a fluorescent signal
when bound to calcium, and the cells are contacted with a test
substance and a receptor agonist, such as
[0115] S-1-P or di-hydro S-1-P. Any change in fluorescent signal is
measured over a defined period of time using, for example, a
fluorescence spectrophotometer or a fluorescence imaging plate
reader. A change in the fluorescence signal pattern generated by
the ligand indicates that a compound is a potential antagonist or
agonist for the receptor.
[0116] Another screening procedure involves use of mammalian cells
(CHO, HEK293, Xenopus Oocytes, RBL-2H3, etc.) that are transfected
to express the receptor of interest, and that are also transfected
with a reporter gene construct that is coupled to activation of the
receptor (for example, luciferase or beta-galactosidase behind an
appropriate promoter). The cells are contacted with a test
substance and the receptor agonist (ligand), such as S-1-P or
di-hydro S-1-P, and the signal produced by the reporter gene is
measured after a defined period of time. The signal can be measured
using a luminometer, spectrophotometer, fluorimeter, or other such
instrument appropriate for the specific reporter construct used.
Inhibition of the signal generated by the ligand indicates that a
compound is a potential antagonist for the receptor.
[0117] Another screening technique for antagonists or agonists
involves introducing RNA encoding the EDG1-c polypeptide into
Xenopus oocytes (or CHO, HEK 293, RBL-2H3, etc.) to transiently or
stably express the receptor. The receptor oocytes are then
contacted with the receptor ligand, such as S-1-P or di-hydro
S-1-P, and a compound to be screened. Inhibition or activation of
the receptor is then determined by detection of a signal, such as,
cAMP, calcium, proton, or other ions.
[0118] Another method involves screening for EDG1-c polypeptide
inhibitors by determining inhibition or stimulation of EDG1-c
polypeptide-mediated cAMP and/or adenylate cyclase accumulation or
dimunition. Such a method involves transiently or stably
transfecting a eukaryotic cell with EDG1-c polypeptide receptor to
express the receptor on the cell surface. The cell is then exposed
to potential antagonists in the presence of EDG1-c polypeptide
ligand, such as S-1-P or di-hydro S-1-P. The changes in levels of
cAMP is then measured over a defined period of time, for example,
by radioimmuno or protein binding assays (for example using
Flashplates or a scintillation proximity assay). Changes in cAMP
levels can also be determined by directly measuring the activity of
the enzyme, adenylyl cyclase, in broken cell preparations. If the
potential antagonist binds the receptor, and thus inhibits EDG1-c
polypeptide-ligand binding, the levels of EDG1-c
polypeptide-mediated cAMP, or adenylate cyclase activity, will be
reduced or increased.
[0119] Another screening method for agonists and antagonists relies
on the endogenous pheromone response pathway in the yeast,
Saccharomyces cerevisiae. Heterothallic strains of yeast can exist
in two mitotically stable haploid mating types, MATa and MATa. Each
cell type secretes a small peptide hormone that binds to a
G-protein coupled receptor on opposite mating-type cells which
triggers a MAP kinase cascade leading to G1 arrest as a prelude to
cell fusion. Genetic alteration of certain genes in the pheromone
response pathway can alter the normal response to pheromone, and
heterologous expression and coupling of human G-protein coupled
receptors and humanized G-protein subunits in yeast cells devoid of
endogenous pheromone receptors can be linked to downstream
signaling pathways and reporter genes (e.g., U.S. Pat. Nos.
5,063,154; 5,482,835; 5,691,188). Such genetic alterations include,
but are not limited to: (i) deletion of the STE2 or STE3 gene
encoding the endogenous G-protein coupled pheromone receptors; (ii)
deletion of the FAR1 gene encoding a protein that normally
associates with cyclin-dependent kinases leading to cell cycle
arrest; and (iii) construction of reporter genes fused to the FUS1
gene promoter (where FUS1 encodes a membrane-anchored glycoprotein
required for cell fusion). Downstream reporter genes can permit
either a positive growth selection (e.g., histidine prototrophy
using the FUS1-HIS3 reporter), or a colorimetric, fluorimetric or
spectrophotometric readout, depending on the specific reporter
construct used (e.g., b-galactosidase induction using a FUS1-LacZ
reporter).
[0120] The yeast cells can be further engineered to express and
secrete small peptides from random peptide libraries, some of which
can permit autocrine activation of heterologously expressed human
(or mammalian) G-protein coupled receptors (Broach, et al., Nature
384: 14-16, 1996; Manfredi, et al., Mol. Cell. Biol. 16: 4700-4709,
1996). This provides a rapid direct growth selection (e.g., using
the FUS1-HIS3 reporter) for surrogate peptide agonists that
activate characterized or orphan receptors. Alternatively, yeast
cells that functionally express human (or mammalian) G-protein
coupled receptors linked to a reporter gene readout (e.g.,
FUS1-LacZ) can be used as a platform for high-throughput screening
of known ligands, fractions of biological extracts and libraries of
chemical compounds for either natural or surrogate ligands.
Functional agonists of sufficient potency (whether natural or
surrogate) can be used as screening tools in yeast cell-based
assays for identifying G-protein coupled receptor antagonists. For
example, agonists will promote growth of a cell with FUS-HIS3
reporter or give positive readout for a cell with FUS1-LacZ.
However, a candidate compound that inhibits growth or negates the
positive readout induced by an agonist is an antagonist. For this
purpose, the yeast system offers advantages over mammalian
expression systems due to its ease of utility and null receptor
background (lack of endogenous G-protein coupled receptors), which
often interferes with the ability to identify agonists or
antagonists.
[0121] The present invention also provides a method for identifying
new ligands not known to be capable of binding to an EDG1-c
polypeptide. The screening assays described above for identifying
agonists may be used to identify new ligands.
[0122] The present invention also contemplates agonists and
antagonists obtained from the above described screening
methods.
[0123] Examples of potential EDG1-c polypeptide receptor
antagonists include peptidomimetics, synthetic organic molecules,
natural products, antibodies, etc., that bind to the receptor but
do not elicit a second messenger response, such that the activity
of the receptor is prevented.
[0124] Potential antagonists also include proteins which are
closely related to the ligand of the EDG1-c polypeptide receptor,
i.e., a fragment of the ligand, which have lost biological
function, and when they bind to the EDG1-c polypeptide receptor,
elicit no response.
[0125] Thus in another aspect, the present invention relates to a
screening kit for identifying agonists, antagonists, and ligands
for EDG1-c polypeptides, comprising:
[0126] (a) a EDG1-c polypeptide, preferably that of SEQ ID NO:2;
and further preferably comprises labeled or unlabeled S-1-P or
di-hydro S-1-P;
[0127] (b) a recombinant cell expressing a EDG1-c polypeptide,
preferably that of SEQ ID NO:2; and further preferably comprises
labeled or unlabeled S-1-P or di-hydro S-1-P; or
[0128] (c) a cell membrane expressing EDG1-c polypeptide;
preferably that of SEQ ID NO:2; and further preferably comprises
labeled or unlabled S-1-P or di-hydro S-1-P.
[0129] It will be appreciated that in any such kit, (a), (b), or
(c) may comprise a substantial component.
[0130] As noted above, a potential antagonist is a small molecule
which binds to the EDG1-c polypeptide receptor, making it
inaccessible to ligands such that normal biological activity is
prevented. Examples of small molecules include, but are not limited
to, small peptides or peptide-like molecules.
[0131] Potential antagonists also include soluble forms of EDG1-c
polypeptide receptor, e.g., fragments of the receptor, which bind
to the ligand and prevent the ligand from interacting with membrane
bound EDG1-c polypeptide receptors.
[0132] The screening method may simply measure the binding of a
candidate compound to the polypeptide, or to cells or membranes
bearing the polypeptide, or a fusion protein thereof by means of a
label directly or indirectly associated with the candidate
compound. Alternatively, the screening method may involve
competition with a labeled competitor. Further, these screening
methods may test whether the candidate compound results in a signal
generated by activation or inhibition of the polypeptide, using
detection systems appropriate to the cells bearing the polypeptide.
Inhibitors of activation are generally assayed in the presence of a
known agonist and the effect on activation by the agonist by the
presence of the candidate compound is observed. Constitutively
active polypeptides may be employed in screening methods for
inverse agonists or inhibitors, in the absence of an agonist or
inhibitor, by testing whether the candidate compound results in
inhibition of activation of the polypeptide. Further, the screening
methods may simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide of the present
invention, to form a mixture, measuring EDG-1c activity in the
mixture, and comparing the EDG-1c activity of the mixture to a
standard. Fusion proteins, such as those made from Fc portion and
EDG-1c polypeptide, as hereinbefore described, can also be used for
high-throughput screening assays to identify antagonists for the
polypeptide of the present invention (see D. Bennett et al., J.
Mol. Recognition, 8:52-58 (1995); and K. Johanson et al., J. Biol.
Chem., 270(16):9459-9471 (1995).
[0133] Polypeptides of the present invention may be employed in
conventional low capacity screening methods and also in
high-throughput screening (HTS) formats. Such HTS formats include
not only the well-established use of 96- and, more recently,
384-well microtiter plates but also emerging methods such as the
nanowell method described by Schullek, et al., Anal Biochem., 246:
20-29 (1997).
[0134] Prophylactic and Therapeutic Methods
[0135] This invention provides methods of treating an abnormal
conditions related to both an excess of and insufficient amounts of
human EDG-1c receptor activity.
[0136] If the activity of human EDG-1c receptor is in excess,
several approaches are available. One approach comprises
administering to a subject an inhibitor compound (antagonist) as
hereinabove described along with a pharmaceutically acceptable
carrier in an amount effective to inhibit activation by blocking
binding of ligands to the human EDG-1c receptor, or by inhibiting a
second signal, and thereby alleviating the abnormal condition.
[0137] In another approach, soluble forms of human EDG-1c
polypeptides still capable of binding the ligand in competition
with endogenous human EDG-1c may be administered. Typical
embodiments of such competitors comprise fragments of the human
EDG-1c polypeptide.
[0138] In still another approach, expression of the gene encoding
endogenous human EDG-1c can be inhibited using expression blocking
techniques. Known such techniques involve the use of antisense
sequences, either internally generated or separately administered.
See, for example, O'Connor, J Neurochem (1991) 56:560 in
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Alternatively, oligonucleotides
which form triple helices with the gene can be supplied. See, for
example, Lee, et al., Nucleic Acids Res (1979) 6:3073; Cooney et
al., Science (1988) 241:456; Dervan, et al., Science (1991)
251:1360. These oligomers can be administered per se or the
relevant oligomers can be expressed in vivo.
[0139] For treating abnormal conditions related to an
under-expression of human EDG-1c receptor and its activity, several
approaches are also available. One approach comprises administering
to a subject a therapeutically effective amount of a compound which
activates human EDG-1c receptor, i.e., an agonist as described
above, in combination with a pharmaceutically acceptable carrier,
to thereby alleviate the abnormal condition. Alternatively, gene
therapy may be employed to effect the endogenous production of
human EDG-1c receptor by the relevant cells in the subject. For
example, a polynucleotide of the invention may be engineered for
expression in a replication defective retroviral vector, as
discussed above. The retroviral expression construct may then be
isolated and introduced into a packaging cell transduced with a
retroviral plasmid vector containing RNA encoding a polypeptide of
the present invention such that the packaging cell now produces
infectious viral particles containing the gene of interest. These
producer cells may be administered to a subject for engineering
cells in vivo and expression of the polypeptide in vivo. For
overview of gene therapy, see Chapter 20, Gene Therapy and other
Molecular Genetic-based Therapeutic Approaches, (and references
cited therein) in Human Molecular Genetics, T Strachan and A P
Read, BIOS Scientific Publishers Ltd. (1996).
[0140] Formulation and Administration
[0141] Peptides, such as the soluble form of human EDG-1c
polypeptides, and agonists and antagonist peptides or small
molecules, may be formulated in combination with a suitable
pharmaceutical carrier. Such formulations comprise a
therapeutically effective amount of the polypeptide or compound,
and a pharmaceutically acceptable carrier or excipient. Such
carriers include but are not limited to, saline, buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof.
Formulation should suit the mode of administration, and is well
within the skill of the art. The invention further relates to
pharmaceutical packs and kits comprising one or more containers
filled with one or more of the ingredients of the aforementioned
compositions of the invention.
[0142] Polypeptides and other compounds of the present invention
may be employed alone or in conjunction with other compounds, such
as therapeutic compounds.
[0143] Preferred forms of systemic administration of the
pharmaceutical compositions include injection, typically by
intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if properly
formulated in enteric or encapsulated formulations, oral
administration may also be possible. Administration of these
compounds may also be topical and/or localized, in the form of
salves, pastes, gels and the like.
[0144] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the subject's condition, and the judgment of the
attending practitioner. Suitable dosages, however, are in the range
of 0.1-100 .mu.g/kg of subject. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
compounds available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization, as is
well understood in the art.
[0145] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
EXAMPLES
Example 1
Yeast Cell Expression
[0146] The receptors of the present invention was constitutively
expressed in Saccharomyces cerevisiae using the PGK1 promoter
carried on a standard 2-micron-based S. cerevisiae-E.coli shuttle
plasmid containing the gene for ampillicin resistance, the ColE1
origin of replication and the S. cerevisiae LEU2 gene. The human
EDG-1c cDNA was modified by trimming away the 5' and 3' UTRs and
subcloned into the yeast expression vector. Following introduction
into yeast cells using standard yeast genetic techniques, human
EDG-1c polypeptide expression was detected by western blotting
using a C-terminally tagged human EDG-1c construct and antibodies
to the epitope tag. Functional expression of human EDG-1c
polypeptide (untagged) was determined as described in Example
9.
Example 2
Ligand Bank for Binding and Functional Assays
[0147] A bank of over 600 putative receptor ligands has been
assembled for screening. The bank comprises: transmitters, hormones
and chemokines known to act via a human seven transmembrane (7TM)
receptor; naturally occurring compounds which may be putative
agonists for a human 7TM receptor, non-mammalian, biologically
active peptides for which a mammalian counterpart has not yet been
identified; and compounds not found in nature, but which activate
7TM receptors with unknown natural ligands. This bank is used to
initially screen the receptor for known ligands, using both
functional (i.e., calcium, cAMP, microphysiometer, oocyte
electrophysiology, etc., see below) as well as binding assays.
Example 3
Ligand Binding Assays
[0148] Ligand binding assays provide a direct method for
ascertaining receptor pharmacology and are adaptable to a high
throughput format. The purified ligand for a receptor is
radiolabeled to high specific activity (50-2000 Ci/mmol) for
binding studies. A determination is then made that the process of
radiolabeling does not diminish the activity of the ligand towards
its receptor. Assay conditions for buffers, ions, pH and other
modulators such as nucleotides are optimized to establish a
workable signal to noise ratio for both membrane and whole cell
receptor sources. For these assays, specific receptor binding is
defined as total associated radioactivity minus the radioactivity
measured in the presence of an excess of unlabeled competing
ligand. Where possible, more than one competing ligand is used to
define residual nonspecific binding.
Example 4
Functional Assay in Xenopus Oocytes
[0149] Capped RNA transcripts from linearized plasmid templates
encoding the receptor cDNAs of the invention are synthesized in
vitro with RNA polymerases in accordance with standard procedures.
In vitro transcripts are suspended in water at a final
concentration of 0.2 mg/ml. Ovarian lobes are removed from adult
female toads, Stage V defolliculated oocytes are obtained, and RNA
transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a
microinjection apparatus. Two electrode voltage clamps are used to
measure the currents from individual Xenopus oocytes in response to
agonist exposure. Recordings are made in Ca2+ free Barth's medium
at room temperature. The Xenopus system can be used to screen known
ligands and tissue/cell extracts for activating ligands.
Example 5
Microphysiometric Assays
[0150] Activation of a wide variety of secondary messenger systems
results in extrusion of small amounts of acid from a cell. The acid
formed is largely as a result of the increased metabolic activity
required to fuel the intracellular signaling process. The pH
changes in the media surrounding the cell are very small but are
detectable by the CYTOSENSOR microphysiometer (Molecular Devices
Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of
detecting the activation of a receptor which is coupled to an
energy utilizing intracellular signaling pathway such as the
G-protein coupled receptor of the present invention.
Example 6
Extract/Cell Supernatant Screening
[0151] A large number of mammalian receptors exist for which there
remains, as yet, no cognate activating ligand (agonist). Thus,
active ligands for these receptors may not be included within the
ligands banks as identified to date. Accordingly, the 7TM receptor
of the invention is also functionally screened (using calcium,
cAMP, microphysiometer, oocyte electrophysiology, etc., functional
screens) against tissue extracts to identify natural ligands.
Extracts that produce positive functional responses can be
sequentially subfractionated until an activating ligand is isolated
identified.
Example 7
Calcium and cAMP Functional Assays
[0152] 7TM receptors which are expressed in HEK 293 cells have been
shown to be coupled functionally to activation of PLC and calcium
mobilization and/or cAMP stimulation or inhibition. Basal calcium
levels in the HEK 293 cells in receptor-transfected or vector
control cells were observed to be in the normal, 100 nM to 200 nM,
range. HEK 293 cells expressing recombinant receptors are loaded
with fura 2 and in a single day>150 selected ligands or
tissue/cell extracts are evaluated for agonist induced calcium
mobilization. Similarly, HEK 293 cells expressing recombinant
receptors are evaluated for the stimulation or inhibition of cAMP
production using standard cAMP quantitation assays. Agonists
presenting a calcium transient or cAMP fluctuation are tested in
vector control cells to determine if the response is unique to the
transfected cells expressing receptor.
Example 8
S-1-P-induced Ca2+ mobilization in untransfected HEK 293 cells
[0153] HEK 293 cells respond to S-1-P in a concentration-dependent
manner with a robust calcium mobilization response, indicating that
the cells contain endogenous receptors that respond toS-1-P. FIG. 3
shows the concentration response curves for S-1-P against
untransfected HEK 293 cells. The data were generated with the 96
well Fluorescent Imaging Plate Reader (FLIPR). Each point is the
mean of 6-8 wells read on FLIPR.
Example 9
S-1-P-induced Reporter Gene Expression in Yeast
[0154] Human EDG-1c receptor was expressed in yeast strains
containing endogenous yeast G-proteins and/or co-expressed
yeast/human chimeric G proteins, and/or human G-proteins. The yeast
strain(s) used contain mutations in genes in the pheromone response
pathway, e.g., (i) deletion of the STE2 or STE3 gene encoding the
endogenous G-protein coupled pheromone receptors; (ii) deletion of
the FAR1 gene encoding a protein that normally associates with
cyclin-dependent kinases leading to cell cycle arrest; and (iii)
construction of reporter genes fused to the FUS1 gene promoter
(where FUS1 encodes a membrane-anchored glycoprotein required for
cell fusion). The downstream reporter (FUS1-LacZ) permits a
colorimetric or fluorimetric readout in response to ligand.
FUS1-LacZ cells expressing human EDG-1c demonstrated a
receptor-dependent response to S-1-P as determined by the
expression of ?-galactosidase. This response, which is shown in
FIG. 5, indicates functional coupling of the human EDG-1c receptor
to yeast or yeast/human chimeric G-proteins.
Example 10
S-1-P-induced a Dose Dependent Cellular Hypertrophy in Rat Neonatal
Myocytes in Culture
[0155] S-1-P was tested in its ability to induce hypertrophy in an
in vitro neonatal cardiomyocyte model. The assessment of
cardiomyocyte hypertrophy is measured using four different
parameters: protein synthesis (tritiated phenylalanine
incorporation and protein content increase), tritiated thymidine
incorporation (evaluation of fibroblast contamination), Brain
Natriuretic Peptide (BNP) release and morphological parameters.
Phenylephrine (PE) at 100 .mu.M concentration is used as internal
control experiments. S-1-P was applied at 10 nM, 100 nM and 1 .mu.M
(n=3). At each concentration S-1-P induced a cellular hypertrophy
with an increase of protein content, phenylalanine incorporation
and BNP secretion to the control cell values. FIG. 6 shows the
concentration response for S-1-P against rat neonatal
cardiomyocytes. Cardiomyocytes in culture display features of
myocyte hypertrophy observed in vivo, such as changes in morphology
(vizualised using light microscopy after staining with crystal
violet), protein content, and pattern of gene expression. For
example, at 1 .mu.M (n=3), S-1-P induced a cellular hypertrophy
with an 35.6%.+-.6.3; 30.1%.+-.9.2 and 11.4%.+-.1.8 increase of
protein content, phenylalanine and thymidine incorporation
respectively to the control cell values. BNP secretion was four
fold higher in S-1-P treated cardiomyocyte vs. control.
Example 11
Human EDG1-c mRNA Expression in Human Cardiac Pathologies
[0156] Northern blot analysis was done either on 2 .mu.g of poly A+
RNA of each sample fractionated on 1% formaldehyde-agarose gel,
blotted on to a nylon membrane (Hybond N+, Amersham) and
subsequently hybridized using standard methods (Sambrook, et al.,
1989) with a DNA fragment containing the human EDG1-c gene. The DNA
probe was labelled using (?-32P)-dCTP and the Ready-prime labeling
system (Amersham). Northern blots were hybridized overnight at
65.degree. C. and subsequently washed with 0.1.times.SSC, 0.1% SDS
at 55.degree. C. and exposed to X-ray film for 2-12 h. Northern
blot experiments on cardiac human pathological blot membranes
indicated reproducible overexpression of EDG-1 receptor mRNA in
dilated cardiomyopathy and ischemic samples.
Example 12
Functional Effects of S-1-P on Isolated Perfused Heart
[0157] The functional effects of S-1-P have been examined in
isolated perfused rabbit heart. S-1-P (10 nM) produced a slight
negative inotropic effect as the values were at 10 min of drug
administration 96.09.+-.6 mm Hg and 84.7.+-.6.3 mm Hg in controls
versus S-1-P-treated group, respectively. An increased in AoP
(aortic pressure), reflecting a vasoconstrictor effect was
observed, i.e., about 30% of increase at 5 min of treatment
compared to the vehicle (methanol 0.001%). A marked reduction of
LVEDP (left ventricular end-diastolic pressure) was also noted.
Example 13
S-1-P- induced Calcium Mobilation Response in RBL 2H3 Cells Stably
Transfected with Human EDG-1c Receptor
[0158] Based on the results generated in FIG. 3, it was shown that
an S-1-P exhibiteded an endogenous response in HEK 293 cells. A
number of cell lines were examined to identify one that would not
respond to S-1-P through an endogenous receptor. The cell line that
we identified was RBL 2H3 cells. Stable cell lines of the EDG1-c
receptor were prepared in RBL 2H3 cell line. The expression of
functionally active clones were followed using Fluorescent imaging
plate reader (FLIPR). The responses for several clones to S-1-P is
presented in FIG. 7. As can be seen from this figure, the best
clones respond in a concentration-dependent manner with EC.sub.50s
about 10-20 nM. The best clones were characterized further, and
that is shown in FIG. 8. The cells responded with high potency
through the EDG1-c receptor to S-1-P and dihydro-S-1-P with similar
EC.sub.50s in the 10-20 nM range and weakly to sphingosine
phosphorylcholine (SPPC) with EC.sub.50 in the uM range. The cells
did not respond to lysophosphatidic acid (LPA, an EDG2 receptor
ligand). Included in the figure are the responses to endogenous
receptors, namely, leukotriene D.sub.4 (LTD.sub.4) and ATP to
demonstrate that the cells were functionally in good shape. These
endogenous ligands gave the expected EC.sub.50 values for these
cells. Muscarine and endogenous ligand for HEK 293, cells but not
RBL 2H3 cells, did not respond.
[0159] All publications including, but not limited to, patents and
patent applications, cited in this specification, are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
[0160] The above description fully discloses the invention,
including preferred embodiments thereof. Modifications and
improvements of the embodiments specifically disclosed herein are
within the scope of the following claims. Without further
elaboration, it is believed that one skilled in the art can, using
the preceding description, utilize the present invention to its
fullest extent. Therefore, the examples provided herein are to be
construed as merely illustrative and are not a limitation of the
scope of the present invention in any way. The embodiments of the
invention in which an exclusive property or privilege is claimed
are defined as follows.
Sequence CWU 1
1
2 1 1149 DNA Human 1 atggggccca ccagcgtccc gctggtcaag gcccaccgca
gctcggtctc tgactacgtc 60 aactatgata tcatcgtccg gcattacaac
tacacgggaa agctgaatat cagcgcggac 120 aaggagaaca gcattaaact
gacctcggtg gtgttcattc tcatctgctg ctttatcatc 180 ctggagaaca
tctttgtctt gctgaccatt tggaaaacca agaaattcca ccgacccatg 240
tactatttta ttggcaatct ggccctctca gacctgttgg caggagtagc ctacacagct
300 aacctgctct tgtctggggc caccacctac aagctcactc ccgcccagtg
gtttctgcgg 360 gaagggagta tgtttgtggc cctgtcagcc tccgtgttca
gtctcctcgc catcgccatt 420 gagcgctata tcacaatgct gaaaatgaaa
ctccacaacg ggagcaataa cttccgcctc 480 ttcctgctaa tcagcgcctg
ctgggtcatc tccctcatcc tgggtggcct gcctatcatg 540 ggctggaact
gcatcagtgc gctgtccagc tgctccaccg tgctgccgct ctaccacaag 600
cactatatcc tcttctgcac cacggtcttc actctgcttc tgctctccat cgtcattctg
660 tactgcagaa tctactcctt ggtcaggact cggagccgcc gcctgacgtt
ccgcaagaac 720 atttccaagg ccagccgcag ctctgagaag tcgctggcgc
tgctcaagac cgtaattatc 780 gtcctgagcg tcttcatcgc ctgctgggca
ccgctcttca tcctgctcct gctggatgtg 840 ggctgcaagg tgaagacctg
tgacatcctc ttcagagcgg agtacttcct ggtgttagct 900 gtgctcaact
ccggcaccaa ccccatcatt tacactctga ccaacaagga gatgcgtcgg 960
gccttcatcc ggatcatgtc ctgctgcaag tgcccgagcg gagactctgc tggcaaattc
1020 aagcgaccca tcatcgccgg catggaattc agccgcagca aatcggacaa
ttcctcccac 1080 ccccagaaag acgaagggga caacccagag accattatgt
cttctggaaa cgtcaactct 1140 tcttcctag 1149 2 382 PRT Human 2 Met Gly
Pro Thr Ser Val Pro Leu Val Lys Ala His Arg Ser Ser Val 1 5 10 15
Ser Asp Tyr Val Asn Tyr Asp Ile Ile Val Arg His Tyr Asn Tyr Thr 20
25 30 Gly Lys Leu Asn Ile Ser Ala Asp Lys Glu Asn Ser Ile Lys Leu
Thr 35 40 45 Ser Val Val Phe Ile Leu Ile Cys Cys Phe Ile Ile Leu
Glu Asn Ile 50 55 60 Phe Val Leu Leu Thr Ile Trp Lys Thr Lys Lys
Phe His Arg Pro Met 65 70 75 80 Tyr Tyr Phe Ile Gly Asn Leu Ala Leu
Ser Asp Leu Leu Ala Gly Val 85 90 95 Ala Tyr Thr Ala Asn Leu Leu
Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100 105 110 Thr Pro Ala Gln Trp
Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu 115 120 125 Ser Ala Ser
Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile 130 135 140 Thr
Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn Phe Arg Leu 145 150
155 160 Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu Ile Leu Gly
Gly 165 170 175 Leu Pro Ile Met Gly Trp Asn Cys Ile Ser Ala Leu Ser
Ser Cys Ser 180 185 190 Thr Val Leu Pro Leu Tyr His Lys His Tyr Ile
Leu Phe Cys Thr Thr 195 200 205 Val Phe Thr Leu Leu Leu Leu Ser Ile
Val Ile Leu Tyr Cys Arg Ile 210 215 220 Tyr Ser Leu Val Arg Thr Arg
Ser Arg Arg Leu Thr Phe Arg Lys Asn 225 230 235 240 Ile Ser Lys Ala
Ser Arg Ser Ser Glu Lys Ser Leu Ala Leu Leu Lys 245 250 255 Thr Val
Ile Ile Val Leu Ser Val Phe Ile Ala Cys Trp Ala Pro Leu 260 265 270
Phe Ile Leu Leu Leu Leu Asp Val Gly Cys Lys Val Lys Thr Cys Asp 275
280 285 Ile Leu Phe Arg Ala Glu Tyr Phe Leu Val Leu Ala Val Leu Asn
Ser 290 295 300 Gly Thr Asn Pro Ile Ile Tyr Thr Leu Thr Asn Lys Glu
Met Arg Arg 305 310 315 320 Ala Phe Ile Arg Ile Met Ser Cys Cys Lys
Cys Pro Ser Gly Asp Ser 325 330 335 Ala Gly Lys Phe Lys Arg Pro Ile
Ile Ala Gly Met Glu Phe Ser Arg 340 345 350 Ser Lys Ser Asp Asn Ser
Ser His Pro Gln Lys Asp Glu Gly Asp Asn 355 360 365 Pro Glu Thr Ile
Met Ser Ser Gly Asn Val Asn Ser Ser Ser 370 375 380
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