U.S. patent application number 11/228364 was filed with the patent office on 2006-01-12 for promiscuous g-protein compositions and their use.
This patent application is currently assigned to Invitrogen Corporation, a Delaware corporation. Invention is credited to Paul Negulescu, Stefan Offermanns, Melvin Simon, Charles Zuker.
Application Number | 20060008855 11/228364 |
Document ID | / |
Family ID | 21797473 |
Filed Date | 2006-01-12 |
United States Patent
Application |
20060008855 |
Kind Code |
A1 |
Negulescu; Paul ; et
al. |
January 12, 2006 |
Promiscuous G-protein compositions and their use
Abstract
Disclosed are compositions and methods for their use, such as in
identifying G-protein coupled receptors and ligands and compounds
that modulate signal transduction. The compositions and methods
employ promicuous G-proteins. Activation of the promiscous
G-protein can be detected in a variety of assays, including assays
in which activation is indicated by a change in fluorescence
emission of a sample that contains the composition.
Inventors: |
Negulescu; Paul; (Del Mar,
CA) ; Offermanns; Stefan; (Berlin, DE) ;
Simon; Melvin; (San Marino, CA) ; Zuker; Charles;
(San Diego, CA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
PO BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Invitrogen Corporation, a Delaware
corporation
|
Family ID: |
21797473 |
Appl. No.: |
11/228364 |
Filed: |
September 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09468002 |
Dec 20, 1999 |
|
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|
11228364 |
Sep 16, 2005 |
|
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|
08878801 |
Jun 19, 1997 |
6004808 |
|
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09468002 |
Dec 20, 1999 |
|
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60020234 |
Jun 21, 1996 |
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Current U.S.
Class: |
435/7.2 ;
530/350 |
Current CPC
Class: |
C12N 15/85 20130101;
C12N 2830/003 20130101; G01N 33/5008 20130101; G01N 33/5041
20130101; C12N 2830/002 20130101; C07K 14/705 20130101 |
Class at
Publication: |
435/007.2 ;
530/350 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/567 20060101 G01N033/567; C07K 14/705 20060101
C07K014/705 |
Claims
1. A stable, isolated cell comprising a construct comprising
promoter operably linked to a polynucleotide encoding a polypeptide
having a biological activity of a promiscuous G.alpha. protein.
2. The cell of claim 1, wherein said promoter is an inducible
promoter.
3. The cell of claim 2, wherein said construct permits expression
in a mammalian cell.
4. The cell of claim 1, wherein said polynucleotide has a
nucleotide sequence with at least 70% sequence identity to a
nucleotide sequence selected from the group consisting of the
nucleotide sequence of G.alpha..sub.16 (SEQ ID NO: 1), and the
nucleotide sequence of G.alpha..sub.15 (SEQ ID NO: 2).
5.-7. (canceled)
8. The cell of claim 1, further comprising a second construct
comprising a reporter gene operably linked to a second promoter,
wherein said second promoter is modulated by a promiscuous G.alpha.
protein.
9. The cell of claim 8, wherein the reporter gene encodes a
reporter selected from the group consisting of luciferase, green
fluorescent protein, chloramphenicol acetyl transferase,
.beta.-galactosidase, alkaline phosphatase, .beta.-lactamase, and
human growth hormone.
10.-11. (canceled)
12. The cell of claim 8, wherein the reporter gene encodes
.beta.-lactamase.
13. The cell of claim 8, wherein said second promoter comprises a
protein kinase C-responsive promoter.
14.-26. (canceled)
27. A method of identifying a GPCR for a given ligand, the method
comprising: (i) expressing a putative GPCR in a cell of claim 1;
(ii) contacting said cell with a ligand; and (iii) detecting
reporter gene expression.
28. The method of claim 27, wherein said detecting comprises
fluorescence detection.
29. The method of claim 28, further comprising contacting said cell
with a reporter gene substrate.
30. The method of claim 27, further comprising contacting said cell
with a compound that increases calcium levels inside said cell.
31. The method of claim 30, wherein said compound is selected from
the group consisting of ionomycin and thapsigargin.
32. The method of claim 30, further comprising contacting said cell
with phorbol myristate acetate or an analog thereof.
33. A method of a identifying of a ligand for a given GPCR, the
method comprising: a) expressing a GPCR in a cell of claim 1; b)
contacting said cell with a test chemical; and c) detecting a
signal with a signal transduction detection system.
34.-36. (canceled)
37. The method of claim 33, wherein said detecting comprises
reporter gene detection.
38. A method for identifying a modulator of signal transduction in
a cell, the method comprising: a) contacting a cell of claim 1 with
a test chemical; b) contacting said cell with a ligand that, in the
absence of the test chemical, activates signal transduction in said
cell, and c) detecting a signal with a signal transduction
detection system.
39.-42. (canceled)
43. A kit comprising assay reagents and a container containing a
cell of claim 1.
44.-62. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. Section 119
to provisional patent application 60/020,234 filed on Jun. 21,
1996, by Negulescu et al., which is herein incorporated by
reference and of which this application is a continuation in
part.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods for
identifying G-protein coupled receptors (GPCRs) and compounds that
modulate activity of G-proteins or their receptors.
BACKGROUND
[0003] Many physiological signals (e.g., sensory, hormonal and
neurotransmitter signals) are transduced from extracellular to
intracellular environments by cell surface receptors termed
G-protein coupled receptors (GPCRs) (for a review, see Neer, 1995,
Cell 80:249-257). Typically, GPCRs contain seven transmembrane
domains. Putative GPCRs can be identified on the basis of sequence
homology to known GPCRs.
[0004] GPCRs mediate signal transduction across a cell membrane
upon the binding of a ligand to an extracellular portion of a GPCR.
The intracellular portion of a GPCR interacts with a G-protein to
modulate signal transduction from outside to inside a cell. A GPCR
is therefore said to be "coupled" to a G-protein. G-proteins are
composed of three polypeptide subunits: an .alpha. subunit, which
binds and hydolyzes GTP, and a dimeric .beta..gamma. subunit. In
the basal, inactive state, the G-protein exists as a heterotrimer
of the .alpha. and .beta..gamma. subunits. When the G-protein is
inactive, guanosine diphosphate (GDP) is associated with the
.alpha. subunit of the G-protein. When a GPCR is bound and
activated by a ligand, the GPCR binds to the G-protein heterotrimer
and decreases the affinity of the G.alpha. subunit for GDP. In its
active state, the G submit exchanges GDP for guanine triphosphate
(GTP) and active G.alpha. subunit disassociates from both the
receptor and the dimeric .beta..gamma. subunit. The disassociated,
active G.alpha. subunit transduces signals to effectors that are
"downstream" in the G-protein signalling pathway within the cell.
Eventually, the G-protein's endogenous GTPase activity returns
active G subunit to its inactive state, in which it is associated
with GDP and the dimeric .beta..gamma. subunit.
[0005] Numerous members of the heterotrimeric G-protein family have
been cloned, including more than 20 genes encoding various G.alpha.
subunits. The various G subunits have been categorized into four
families, on the basis of amino acid sequences and functional
homology. These four families are termed G.alpha..sub.s,
G.alpha..sub.i, G.alpha..sub.q, and G.alpha..sub.12. Functionally,
these four families differ with respect to the intracellular
signaling pathways that they activate and the GPCR to which they
couple.
[0006] For example, certain GPCRs normally couple with
G.alpha..sub.s. and, through G.alpha..sub.s, these GPCRs stimulate
adenylyl cyclase activity. Other GPCRs normally couple with
G.alpha..sub.q, and through G.alpha..sub.q, these GPCRs can
activate phospholipase C (PLC), such as the .beta. isoform of
phospholipase C (PLC.beta.) (Stemweis and Smrcka, 1992, Trends in
Biochem. Sci. 17:502-506).
[0007] Certain G-proteins are considered "promiscuous" G-proteins
because their G subunits allow them to couple with GPCRs that
normally couple with G-proteins of other families. For example, two
members of the G.alpha..sub.q family, human G.alpha..sub.16 and its
murine homolog G.alpha..sub.15, have been shown in transient
cell-based systems to possess promiscuous receptor coupling.
Although G-proteins having these G subunits are promiscuous with
respect to the GPCR with which they couple, these G-proteins retain
the ability to couple with a specific downstream effector. In other
words, regardless of which receptor is used to activate these
G-proteins, the active promiscuous G subunit nonetheless activates
PLC.beta..
SUMMARY OF THE INVENTION
[0008] The invention provides for the first time, a stable,
isolated cell that expresses, from a construct, a G.alpha. subunit
of a promiscuous G-protein (e.g., G.alpha..sub.15, or
G.alpha..sub.16). In a preferred embodiment, a polynucleotide
encoding a promiscuous G subunit is linked to an inducible promoter
on the construct. To detect activation of the promiscuous
G-protein, the cell can include an additional construct that
includes a reporter gene operably linked to a promoter that is
activated (usually indirectly) by an active G subunit of a
promiscuous G-protein. For the first time, these cells allow
occupation of any G-protein coupled receptor (GPCR) by a ligand to
be detected using a signal transduction detection system, such as
expression of a reporter gene. Other signal transduction detection
systems include detecting changes in intracellular activity, such
as methods of detecting G-protein activation from changes in
calcium levels in the cell. Preferred methods for detecting
expression of the reporter gene involve detecting a change in
fluorescence emission from a sample that includes the cell
containing the reporter gene.
[0009] Another key aspect of the invention is functional selection
of stable cell lines. Stable cell lines can be functionally
selected using a signal transduction detection system as described
herein. Stable cells are generated that tolerate the expression of
a target protein (such as an ion channel, kinase, phospholipase,
phosphatase, transcription factors or GPCR) or a signal
transduction coupling protein (e.g. G protein) or both.
[0010] The cells of the invention can be employed in methods for
(i) determining whether a polypeptide is a GPCR for a given ligand;
(ii) determining whether a "test" ligand is a ligand for a given
GPCR; (iii) functionally characterizing the ability of a ligand to
activate various GPCRs; and (iv) determining whether a compound
modulates signal transduction in a cell (e.g., as an agonist or
antagonist).
[0011] Another aspect of the invention includes, a method of a
identifying of a ligand for a GPCR, the method comprising: [0012]
a) contacting a cell with a test chemical, wherein said cell is
expressing a GPCR and arises from a cell line subjected to
functional cell analysis with a signal transduction detection
system; and [0013] b) detecting a signal with a signal transduction
detection system.
[0014] A related aspect of the invention includes, a method for
identifying modulators of signal transduction in a cell, the method
comprising: [0015] c) contacting a cell with a compound that
directly or indirectly activates a G.alpha. protein encoded by a
polynucleotide, wherein said cell arises from a cell line subjected
to functional cell analysis with a signal transduction detection
system, [0016] d) contacting said cell with a test chemical, and
[0017] e) detecting a signal with a signal transduction detection
system. The invention also includes, a method for identifying a
GPCR for a given ligand or method of identifying a modulator of a
GPCR, the method comprising: [0018] f) expressing a putative GPCR
or a GPCR of known function in a cell, wherein said cell arises
from a cell line subjected to functional cell analysis with a
signal transduction detection system; [0019] g) contacting
contacting said cell with a test chemical or a ligand known to be a
GPCR ligand; and [0020] h) detecting a calcium level within said
cell.
[0021] Also included within the invention, are kits that components
for signal transduction detection systems and cells of the
invention.
[0022] The invention also includes methods of identifying
modulators that do not employ a GPCR, but which employ direct
activators of G-proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows uses of promiscuous G.alpha.-protein to detect
activation of a variety of GPCRs. Three major classes of GPCRs are
diagrammed coupling to their endogenous signaling cascade.
Promiscuous G-protein expression will allow various classes to
couple to the PLC cascade.
[0024] FIG. 2 shows one embodiment of the invention that can be
used for screening for Gq type G-protein activation. Modulation of
GPCR activity initiates signaling cascade via PLC. PLC signals can
be detected using NFAT responsive element linked to a
transcriptional readout (e.g. reporter gene).
[0025] FIG. 3 is a copy of a photograph of a Western blot. Lanes
corresponding to samples that lacked or contained doxycyclin are
indicated by "-" and "+" respectively. Lanes 1-4 show inducible
expression of G.alpha..sub.15 in two distinct clones of COS-7
cells. Lanes 5-8 show inducible expression of G.alpha..sub.16 in
two distinct clones. G.alpha..sub.15 and G.alpha..sub.16 each
appear as a species having a molecular weight of approximately 43
kDA in each of the "+" lanes. As a negative control, COS-7 cells
were analyzed in lane 9. The predicted molecular weight of G.alpha.
subunit is 43-45 kDa
[0026] FIG. 4A is a graph showing the ionomycin dose response, as
measured by fluorescence emission of living cells that express
.beta.-lactamase reporter gene, and which were contacted with a
fluorogenic .beta.-lactamase substrate. Because the NFAT response
element usually requires both a calcium increase and protein kinase
C activation, these cells were also treated with 10 nm PMA FIG. 4B
is a graph showing the PMA dose response of living cells that
express a .beta.-lactamase reporter gene, and which were contacted
with a fluorogenic .beta.-lactamase substrate. In this case, all
samples were also treated with 2 .mu.M ionomycin.
[0027] FIG. 5 is a graphic representation of the emission spectrum
of the .beta.-lactamase substrate CCF2 before and after it is
cleaved by .beta.-lactamase.
[0028] FIG. 6 shows the results of an NFAT .beta.-lactamase
transcription based assay using a heterologouly express GPCR (Gq
subtype) in the presence of agonist, agonist and antagonist or
solvent for agonist ("non-stimulated" control).
[0029] FIG. 7 shows activation of a G.alpha.s subtype GPCR (panels
A-C) and a G.alpha.i subtype GPCR (panels D-F) using promiscuous
G.alpha. protein in a cell-based (transient transfection of all
constructs) calcium indicator assay (FURA-PE3).
[0030] Panel A: 60 seconds after starting of the experiment, 10
.mu.M agonist solution was added to the cells transfected by
pCIS/G.alpha. 16 and Gs-receptor expression plasmids.
[0031] Panel B: 60 seconds after starting of the experiment, 10
.mu.M agoinst solution was added to the cells transfected by both
pCIS/G.alpha. 16 alone.
[0032] Panel C: 60 seconds after starting of the experiment, 10
.mu.M agonist solution was added to the cells transfected by
G.alpha.s receptor expression plasmid alone.
[0033] Panel D: 60 seconds after starting of the experiment, 10
.mu.M agonist solution was added to the cells transfected by
pCIS/G.alpha.16 and G.alpha.i-receptor expression plasmids.
[0034] Panel E: 60 seconds after starting of the experiment, 10
.mu.M agonist solution was added to the cells transfected by
pCIS/G.alpha. 16 alone.
[0035] Panel F: 60 seconds after starting of the experiment, 10
.mu.M agonist solution was added to the cells transfected by
G.alpha.i-receptor expression plasmid alone.
[0036] FIG. 8 shows activation of a Gas subtype GPCR using
promiscuous G.alpha. protein in a cell-based (stably transfected
constructs for both the promiscuous G.alpha.-protein and GPCR)
calcium indicator assay.
[0037] Panel A: Calcium imaging of the G.alpha.15G.alpha.s-receptor
dual stable pool-2. 10 .mu.m agonist was added 40 seconds after the
starting of the experiment.
[0038] Panel B: Calcium imaging of the G.alpha.s-receptor stable
pool-2. 10 .mu.M agonist was added 40 seconds after the starting of
the experiment.
[0039] Panel C: Clacium imaging of the G.alpha.15 stable pool-H. 10
.mu.M agoinst was added 40 seconds after the starting of the
experiment.
DEFINITIONS
[0040] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Generally, the nomenclature used herein and the laboratory
procedures in spectroscopy, drug discovery, cell culture, and
molecular genetics, described below are those well known and
commonly employed in the art. Standard techniques are typically
used for preparation of signal detection, recombinant nucleic acid
methods, polynucleotide synthesis, and microbial culture and
transformation (e.g., electroporation, and lipofection). The
techniques and procedures are generally performed according to
conventional methods in the art and various general references (see
generally, Sambrook et al. Molecular Cloning: A Laboratory Manual,
2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., and Lakowicz, J. R. Principles of Fluorescence
Spectroscopy, New York: Plenum Press (1983) for fluorescence
techniques, which are incorporated herein by reference) which are
provided throughout this document. Standard techniques are used for
chemical syntheses, chemical analyses, and biological assays. As
employed throughout the disclosure, the following terms, unless
otherwise indicated, shall be understood to have the following
meanings:
[0041] "Fluorescent donor moiety" refers to the radical of a
fluorogenic compound, which can absorb energy and is capable of
transferring the energy to another fluorogenic molecule or part of
a compound. Suitable donor fluorogenic molecules include, but are
not limited to, coumarins and related dyes xanthene dyes such as
fluoresceins, rhodols, and rhodamines, resorufins, cyanine dyes,
bimanes, acridines, isoindoles, dansyl dyes, aminophthalic
hydrazides such as luminol and isoluminol derivatives,
aminophthalimides, aminonaphthalimides, aminobenzofurans,
aminoquinolines, dicyanohydroquinones, and europium and terbium
complexes and related compounds.
[0042] "Quencher" refers to a chromophoric molecule or part of a
compound, which is capable of reducing the emission from a
fluorescent donor when attached to the donor. Quenching may occur
by any of several mechanisms including fluorescence resonance
energy transfer, photoinduced electron transfer, paramagnetic
enhancement of intersystem crossing, Dexter exchange coupling, and
excitation coupling such as the formation of dark complexes.
[0043] "Acceptor" refers to a quencher that operates via
fluorescence resonance energy transfer. Many acceptors can re-emit
the transferred as energy as fluorescence. Examples include
coumarins and related fluorophores, xanthenes such as fluoresceins,
rhodols, and rhodamines, resorufins, cyanines,
difluoroboradiazaindacenes, and phthalocyanines. Other chemical
classes of acceptors generally do not re-emit the transferred
energy. Examples include indigos, benzoquinones, anthraquinones,
azo compounds, nitro compounds, indoanilines, di- and
triphenylmethanes.
[0044] "Binding pair" refers to two moieties (e.g. chemical or
biochemical) that have an affinity for one another. Examples of
binding pairs include antigen/antibodies, lectin/avidin, target
polynucleotide/probe oligonucleotide, antibody/anti-antibody,
receptor/ligand, enzyme/ligand and the like. "One member of a
binding pair" refers to one moiety of the pair, such as an antigen
or ligand.
[0045] "Dye" refers to a molecule or part of a compound that
absorbs specific frequencies of light, including but not limited to
ultraviolet light. The terms "dye" and "chromophore" are
synonymous.
[0046] "Fluorophore" refers to a chromophore that fluoresces.
[0047] "Membrane-permeaiit derivative" refers a chemical derivative
of a compound that has enhanced membrane permeability compared to
an underivativized compound. Examples include ester, ether and
carbamate derivatives. These derivatives are made better able to
cross cell membranes, i.e. membrane permeant, because hydrophilic
groups are masked to provide more hydrophobic derivatives. Also,
masking groups are designed to be cleaved from a precursor (e.g.,
fluorogenic substrate precursor) within the cell to generate the
derived substrate intracellularly. Because the substrate is more
hydrophilic than the membrane permeant derivative it is now trapped
within the cells.
[0048] "Isolated polynucleotide" refers a polynucleotide of
genomic, cDNA, or synthetic origin or some combination there of,
which by virtue of its origin the "isolated polynucleotide" (1) is
not associated with the cell in which the "isolated polynucleotide"
is found in nature, or (2) is operably linked to a polynucleotide
which it is not linked to in nature.
[0049] "Isolated protein" refers a protein, usually of cDNA,
recombinant RNA, or synthetic origin or some combination thereof,
which by virtue of its origin the "isolated protein" (1) is not
associated with proteins that it is normally found with in nature,
(2) is isolated from the cell in which it normally occurs, (3) is
isolated free of other proteins from the same cellular source, e.g.
free of human proteins, (4) is expressed by a cell from a different
species, or (5) does not occur in nature. "Isolated naturally
occurring protein" refers to a protein which by virtue of its
origin the "isolated naturally occurring protein" (1) is not
associated with proteins that it is normally found with in nature,
or (2) is isolated from the cell in which it normally occurs or (3)
is isolated free of other proteins from the same cellular source,
e.g. free of human proteins.
[0050] "Polypeptide" as used herein as a generic term to refer to
native protein, fragments, or analogs of a polypeptide sequence.
Hence, native protein, fragments, and analogs are species of the
polypeptide genus. Preferred G.alpha. polypeptides, include those
with the polypeptide sequence represented in the SEQUENCE ID
LISTING and any other protein having activity similar to such
G.alpha. proteins as measured by one or more of the assays
described herein. SEQ. ID NO.: 1 is G.alpha..sub.16 (murine). SEQ.
ID NO. 2 is G.alpha..sub.15 (human). G.alpha. polypeptides or
proteins can include any protein having sufficient activity for
detection in the assays described herein.
[0051] "Naturally-occurring" as used herein, as applied to an
object, refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0052] "Operably linked" refers to a juxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences, such as when the appropriate
molecules (e.g., inducers and polymerases) are bound to the control
or regulatory sequence(s).
[0053] "Control sequence" refers to polynucleotide sequences which
are necessary to effect the expression of coding and non-coding
sequences to which they are ligated. The nature of such control
sequences differs depending upon the host organism; in prokaryotes,
such control sequences generally include promoter, ribosomal
binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, components whose presence can
influence expression, and can also include additional components
whose presence is advantageous, for example, leader sequences and
fusion partner sequences.
[0054] "Polynucleotide" refers to a polymeric form of nucleotides
of at least 10 bases in length, either ribonucleotides or
deoxynucleotides or a modified form of either type of nucleotide.
The term includes single and double stranded forms of DNA.
[0055] Two amino acid sequences are homologous if there is a
partial or complete identity between their sequences. For example,
85% homology means that 85% of the amino acids are identical when
the two sequences are aligned for maximum matching. Gaps (in either
of the two sequences being matched) are allowed in maximizing
matching; gap lengths of 5 or less are preferred with 2 or less
being more preferred. Alternatively and preferably, two protein
sequences (or polypeptide sequences derived from them of at least
30 amino acids in length) are homologous, as this term is used
herein, if they have an alignment score of at more than 5 (in
standard deviation units) using the program ALIGN with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M.O.,
in Atlas of Protein Sequence and Structure, 1972, Volume 5,
National Biomedical Research Foundation, pp. 101-110, and
Supplement 2 to this volume, pp. 1-10. The two sequences or parts
thereof are more preferably homologous if their amino acids are
greater than or equal to 30% identical when optimally aligned using
the ALIGN program.
[0056] "Corresponds to" refers to a sequence that is homologous
(i.e., is identical, not strictly evolutionarily related) to all or
a portion of a reference sequence.
[0057] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence," "comparison window," "sequence identity," "percentage of
sequence identity" and "substantial identity." A "reference
sequence" is a defined sequence used as a basis for a sequence
comparison; a reference sequence may be a subset of a larger
sequence, for example, as a segment of a full-length cDNA or gene
sequence given in a sequence listing such as a SEQ. ID NO. 1, or
may comprise a complete cDNA or gene sequence. Generally, a
reference sequence is at least 400 nucleotides in length,
frequently at least 600 nucleotides in length, and often at least
800 nucleotides in length. Since two polynucleotides may each (1)
comprise a sequence (i.e., a portion of the complete polynucleotide
sequence) that is similar between the two polynucleotides, and (2)
may further comprise a sequence that is divergent between the two
polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of
the two polynucleotides over a "comparison window" to identify and
compare local regions of sequence similarity. A "comparison
window," as used herein, refers to a conceptual segment of at least
20 contiguous nucleotide positions wherein a polynucleotide
sequence may be compared to a reference sequence of at least 20
contiguous nucleotides and wherein the portion of the
polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) of 20 percent or less as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith and Waterman
(1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm
of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search
for similarity method of Pearson and Lipman (1988) Proc. Natl.
Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection, and the best
alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by the various methods is
selected.. The term "sequence identity" means that two
polynucleotide sequences are identical (i.e., on a
nucleotide-by-nucleotide basis) over the window of comparison. The
term "percentage of sequence identity" is calculated by comparing
two optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical nucleic
acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to
yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the window of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity. The terms
"substantial identity" as used herein denotes a characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a
sequence that has at least 50 percent sequence identity, preferably
at least 60 to 70 percent sequence identity, more usually at least
80 percent sequence identity as compared to a reference sequence
over a comparison window of at least 20nucleotide positions,
frequently over a window of at least 25-50 nucleotides, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the polynucleotide sequence which may include
deletions or additions which total 20 percent or less of the
reference sequence over the window of comparison.
[0058] As applied to proteins, the term "substantial identity"
means that two protein sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, typically
share at least 70 percent sequence identity, preferably at least 80
percent sequence identity, more preferably at least 90 percent
sequence identity, and most preferably at least 95 percent sequence
identity. Preferably, residue positions which are not identical
differ by conservative amino acid substitutions. Conservative amino
acid substitutions refer to the interchangeability of residues
having similar side chains. For example, a group of amino acids
having aliphatic side chains is glycine, alanine, valine, leucine,
and isoleucine; a group of amino acids having aliphatic-hydroxyl
side chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginne, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, glutamic-aspartic, and
asparagine-glutamine.
[0059] "Promiscuous G.alpha. protein" refers to a protein with the
promiscuous coupling activity of one of the G.alpha. proteins of
the SEQ. ID listing. Preferably, the promiscuous G.alpha. protein
can couple to at least one GPCR that normally couples to a G.alpha.
protein other than a promiscuous G.alpha. protein. Examples of
G.alpha. proteins, include G.alpha.q, G.alpha.s, G.alpha.i and
G.alpha.l.sub.12. Promiscuous G.alpha. protein coupling activity
can be measured with an endogenously or heterologously expressed
GPCR using the assays described herein. Preferably, a promiscuous
G.alpha. protein can couple to at least two different types of
GPCRs that normally couple to one of the following G.alpha.
proteins, G.alpha.q, G.alpha.s, G.alpha.i and G.alpha.12. More
preferably, a promiscuous G.alpha. protein can couple to at least
three different types of GPCRs that normally couple to one of the
following G.alpha. proteins, G.alpha.q, G.alpha.s, G.alpha.i and
G.alpha.12. Promiscuous G.alpha. proteins permit coupling under
conditions that would not occur with a G.alpha. protein and a
receptor of a different G.alpha. subtype, unless the G.alpha.
protein was expressed at sufficiently high levels to promote
coupling with a GFCR that is not its normal coupling partner.
Examples of G.alpha..sub.15 include (Wilke, T. M. et al., PNAS,
Vol. 88 pp. 10049-10053, 1991) and G.alpha..sub.16 include
(Amatruda, T. T. et al., PNAS, Vol. 88 pp. 5587-5591, 1991). It is
understood that promiscuous G.alpha. proteins do not include
members of G.alpha.q, G.alpha.s, G.alpha.i and G.alpha.12 proteins
that couple to only one type of GPCR
[0060] "Modulation" refers to the capacity to either enhance or
inhibit a functional property of biological activity or process
(e.g., enzyme activity or receptor binding); such enhancement or
inhibition may be contingent on the occurrence of a specific event,
such as activation of a signal transduction pathway, and/or may be
manifest only in particular cell types.
[0061] The term "modulator" refers to a chemical compound
(naturally occurring or non-naturally occurring), such as a
biological macromolecule (e.g., nucleic acid, protein, non-peptide,
or organic molecule), or an extract made from biological materials
such as bacteria, plants, fungi, or animal (particularly mammalian)
cells or tissues. Modulators are evaluated for potential activity
as inhibitors or activators (directly or indirectly) of a
biological process or processes (e.g., agonist, partial antagonist,
partial agonist, inverse agonist, antagonist, antineoplastic
agents, cytotoxic agents, inhibitors of neoplastic transformation
or cell proliferation, cell proliferation-promoting agents, and the
like) by inclusion in screening assays described herein. The
activity of a modulator may be known, unknown or partially
known.
[0062] "Sequence homology" refers to the proportion of base matches
between two nucleic acid sequences or the proportion amino acid
matches between two amino acid sequences. When sequence homology is
expressed as a percentage, e.g., 50%, the percentage denotes the
proportion of matches over the length of sequence from a desired
sequence (e.g., SEQ. ID NO. 1) that is compared to some other
sequence. Gaps (in either of the two sequences) are permitted to
maximize matching; gap lengths of 15 bases or less are usually
used, 6 bases or less are preferred with 2 bases or less more
preferred.
[0063] The term "test chemical" refers to a chemical to be tested
by one or more screening method(s) of the invention as a putative
modulator.
[0064] The terms "label" or "labeled" refers to incorporation of a
detectable marker, e.g., by incorporation of a radio labeled amino
acid or attachment to a polypeptide of biotinyl moieties that can
be detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical, or colorimetric methods). Various methods of labeling
polypeptides and glycoproteins are known in the art and may be
used. Examples of labels for polypeptides include, but are not
limited to, the following: radioisotopes (e.g., .sup.3H, .sup.14C,
.sup.35S, .sup.125I, .sup.131I, fluorescent labels (e.g., FITC,
rhodamine, lanthanide phosphors), enzymatic labels (or reporter
genes) (e.g., horseradish peroxidase, .beta.-galactosidase,
.beta.-latamase, luciferase, alkaline phosphatase),
chemiluminescent, biotinyl groups, predetermined polypeptide
epitopes recognized by a secondary reporter (e.g., leucine zipper
pair sequences, binding sites for secondary antibodies, metal
binding domains, epitope tags). In some embodiments, labels are
attached by spacer arms of various lengths to reduce potential
steric hindrance.
[0065] "Fluorescent label" refers to incorporation of a detectable
marker, e.g., by incorporation of a fluorescent moiety to a
chemical entity that binds to a target or attachment to a
polypeptide of biotinyl moieties that can be detected by avidin
(e.g., streptavidin containing a fluorescent label or enzymatic
activity that can be detected by fluorescence detection methods).
Various methods of labeling polypeptides and glycoproteins are
known in the art and may be used. Examples of labels for
polypeptides include, but are not limited to dyes (e.g., FITC and
rhodamine), intrinsically fluorescent proteins, and lanthanide
phosphors. In some embodiments, labels are attached by spacer arms
of various lengths to reduce potential steric hindrance.
[0066] "Reporter gene" refers to a nucleotide sequence encoding a
protein that is readily detectable either by its presence or
activity, including, but not limited to, luciferase, green
fluorescent protein, chloramphenicol acetyl transferase,
.beta.-galactosidase, secreted placental alkaline phosphatase,
.beta.-lactamase, human growth hormone, and other secreted enzyme
reporters. Generally, reporter genes encode a polypeptide not
otherwise produced by the host cell, which is detectable by
analysis of the cell(s), e.g., by the direct fluorometric,
radioisotopic or spectrophotometric analysis of the cell(s) and
preferably without the need to kill the cells for signal analysis.
Preferably, the gene encodes an enzyme, which produces a change in
fluorometric properties of the host cell, which is detectable by
qualitative, quantitative or semi-quantitative function of
transcriptional activation. Exemplary enzymes include esterases,
phosphatases, proteases (tissue plasminogen activator or urokinase)
and other enzymes whose function can be detected by appropriate
chromogenic or fluorogenic substrates known to those skilled in the
art.
[0067] "Signal transduction detection system" refers to system for
detecting signal transduction across a cell membrane, typically a
cell plasma membrane. Such systems typically detect at least one
activity or physical property directly or indirectly associated
with signal transduction. For example, an activity or physical
property directly associated with signal transduction is the
activity or physical property of either the receptor (e.g.,
GPCR),or a coupling protein (e.g., a G.alpha. protein). Signal
transduction detection systems for monitoring an activity or
physical property directly associated with signal transduction,
include GTPase activity, and conformational changes. An activity or
physical property indirectly associated with signal transduction is
the activity or physical property produced by a molecule other than
by either the receptor (e.g., GPCR), or a coupling protein (e.g., a
G.alpha. protein) and associated with receptor (e.g., GPCR), or a
coupling protein (e.g., a G.alpha. protein). Such indirect
activities and properties include changes in intracellular levels
of molecules (e.g., ions (e.g., Ca, Na or K), second messenger
levels (e.g., cAMP, cGMP and inostol phosphate)), kinase acitvites,
transcriptional activity, enzymatic activity, phospholipase
activities, ion channel activities and phosphatase activites.
Signal transduction detection systems for monitoring an activity or
physical property indirectly associated with signal transduction,
include transcriptional-based assays, enzymatic assays,
intracellular ion assays and second messenger assays.
[0068] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill,
San Francisco, incorporated herein by reference).
A DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0069] The invention provides cells and methods for screening or
characterizing G-protein coupled receptors (GPCRs), ligands for
GPCRs, and compounds that modulate signal transduction (e.g.,
agonists and antagonists). The term "G-protein coupled receptor" is
used herein in accordance with its conventional definition. Such
receptors are cell surface receptors that typically contain seven
transmembrane regions and that transduce signals (e.g., sensory,
hormonal, and neurotransmitter signals) from extracellular
environments to intracellular environments.
[0070] Included within the invention are cells that are useful for
expressing G proteins and practicing methods of the invention. A
preferred cell is a stable, isolated cell that comprises a
promiscuous G.alpha. protein construct comprising a promoter
operably linked to a gene (or polynucleotide) that encodes a
polypeptide with the biological activity of a promiscuous G.alpha.
protein. A "stable isolated cell" of the invention is a cell that
retains a construct typically longer than at least 3 to 4 passages
in tissue culture, preferably longer than 6 to 10 passages and most
preferably longer than about 12 passages. An "isolated" cell refers
to a cell in an in vitro state (e.g., a cell of a mammalian tissue
culture). The cells that are useful in the invention include both
eukaryotic and prokaryotic cells that contain the constructs
described herein. Preferably, the cell is a cell of a mammalian
cell line (e.g., a COS-7 cell); human cells are also preferred
(e.g., a human T lymphocyte). Although not preferred, yeast cells
can also be used.
[0071] A "construct," when used in the context of molecular
biology, is any genetically engineered nucleic acid (e.g., a
plasmid, restriction fragment or an engineered chromosome). As used
herein, a "promoter" is the minimal sequence sufficient to direct
transcription of a gene (including a cDNA encoding a protein) in an
eukaryote. Preferably, the promoter is derived from an eukaryotic
gene or a virus that can direct transcription in an eukaryotic
cell. A promoter can include a TATA box, a CAAT box, and a
transcriptional start site. The term "gene" refers to a
polynucleotide that encodes a protein, such as a cDNA encoding a
protein.
[0072] Polypeptides that have the biological activity of a G.alpha.
protein are those polypeptides that are able to transduce a signal
(including extracellular signals) to an effector(s) in a G-protein
signaling pathway. Typically, such a polypeptide or protein, in its
inactive state, is associated with GDP and the .beta..gamma. dimer
of a G-protein. In its "active" state, the polypeptide typically is
associated with GTP and disassociated from the .beta..gamma. dimer
of a G-protein. The unassociated G.alpha. protein is able to
transduce a signal to an effector in the G-protein signaling
pathway. Examples promiscuous G.alpha. proteins include promiscuous
G.alpha..sub.16 protein and a promiscuous G.alpha..sub.15 protein.
Either promiscuous G.alpha..sub.16 protein or a promiscuous
G.alpha..sub.15 protein can couple to a GPCR that normally couples
to G.sub.i, G.sub.sor G.sub.q (see FIG. 1). Preferably, the
promiscuous G.alpha. protein employed in the invention has the
ability to couple with specificity to an effector in the G-protein
signaling pathway. For example, the promiscuous G.alpha..sub.16 and
G.alpha..sub.15 proteins each retain the ability to specifically
activate the .beta. isoform of phospholipase C.
[0073] Preferably, the nucleotide sequence of a promiscuous
G.alpha. protein has at least 70% (more preferably, at least 80% or
95%) sequence identity to the nucleotide sequence of
G.alpha..sub.16 (SEQ ID NO: 1) and/or G.alpha..sub.15 (SEQ ID NO:
2). Other preferred promiscuous G.alpha. proteins are those that
are encoded by degenerate variants of the nucleotide sequences of
promiscuous G.alpha..sub.16 (SEQ ID NO: 1) and/or G.alpha..sub.15
(SEQ ID NO: 2). A "degenerate variant" of a nucleotide sequence is
a nucleotide sequence that encodes the same amino acid sequence as
a given nucleotide sequence, but in which at least one codon in the
nucleotide sequence is different, because two or more different
codons can encode the same amino acid. Accordingly, numerous
degenerate variants can encode the promiscuous G.alpha. proteins of
SEQ ID NO: 3 and SEQ ID NO: 4. Other preferred promiscuous G.alpha.
protein are those that are encoded by conservative variations of
the nucleotide sequences of G.alpha..sub.16 (SEQ ID NO: 1) and/or
G.alpha..sub.15 (SEQ ID NO: 2). A "conservative variation" denotes
the replacement of an arnino acid residue by another, biologically
similar, residue. Examples of conservative variations include the
substitution of one hydrophobic residue, such as isoleucine,
valine, leucine, or methionine, for another, or the substitution of
one polar residue for similar polar residue, such as the
substitution of arginine for lysine, glutamic acid for aspartic
acid, or glutamine for asparagine, and the like.
[0074] In some embodiments of the invention it will be desirable to
control the level of promiscuous G.alpha. protein expression. High
levels of promiscuous G.alpha. protein in a cell can deleteriously
alter cell metabolism that can result in cell instability. High
levels of promiscuous G.alpha. protein in a cell (or normal
G.alpha. protein) can also produce high basal activities GPCRs that
results in high background activities, which is not desirable for
methods described herein, such as screening chemicals that may
modulate receptor activity. Typically, cells having endogenously
low levels of normal G.alpha. protein are used. Basal activity
levels of GPCRs can be easily tested in a potential cell type to be
used for screening with a signal transduction detection system to
detect the affect of endogenously expressed G-proteins. Basal
activity levels of GPCRs can also be easily tested with either
endogenously expressed or heterologously expressed GPCRs in cells
expressing a promiscuous G.alpha. protein or other G-proteins.
[0075] With GPCRs normally having high basal activity, controlled
levels of promiscuous G.alpha. protein can help reduce background
activity in a cell while achieving suitable coupling for testing
putative modulators of a receptor. The amount promiscuous G.alpha.
protein expressed in a cell can be titrated by using, or selecting
for, either a weak promoter or an inducible promoter. An inducible
promoter offers the advantage, compared to a weak promoter, of
regulatable expression of promiscuous G.alpha. protein. By using an
inducible promoter the amount inducer can be used to optimize the
signal to noise ratio of a screen for GPCR modulators by adjusting
the amount of promiscuous G.alpha. protein expression the cell.
[0076] An "inducible" promoter is a promoter that, in the absence
of an inducer (e.g., doxycyclin) does not direct expression, or
directs low levels of expression (e.g., produces less than 500
proteins per cell at steady state) of an operably linked gene
(including CDNA). In the presence of an inducer, expression
promiscuous G.alpha. protein directed by the inducible promoter is
typically increased at least 3-fold (preferably at least 10-100- or
1,000-fold). Other useful inducible promoters include those that
are inducible by IPTG or ecdysone. If desired, an inducible
promoter can include a first promoter (e.g., a cytomegalovirus
promoter) operably linked to a tet operator to regulate the first
promoter (see, Gossen and Bujard, 1992, Proc. Natl. Acad. Sci.
89:5547-5551).
[0077] Many embodiments of the invention will include a
polynculeotide encoding a GPCR not naturally occurring in the cell
and a promiscuous G.alpha. protein construct. The GPCR will
typically not be under the control of the control sequence
controlling promiscuous G.alpha. protein expression. The GPCR maybe
a GPCR of known function or of protein of unknown function, such as
an orphan GPCR. Promoters known in the art can be used to either
constitutively or inducible express the receptor or putative
receptor.
[0078] If desired, a cell of the invention can contain a
polynucleotide having a control sequence and encoding a protein
useful in signal transduction detection system. The construct is
designed to detect activation of a G.alpha. protein. This second
construct is typically located on a second vector. It can include a
reporter gene that is operably linked to a promoter that is
modulated (directly or indirectly) by an active promiscuous
G.alpha. protein. Preferably, the expression of the reporter gene
can be detected by detecting a change in fluorescence emission of a
sample that contains the cell.
[0079] For instance, the reporter system described in PCT
publication WO96/30540 (Tsien) has significant advantages over
existing reporters for gene integration analysis, as it allows
sensitive detection and isolation of both expressing and
non-expressing single living cells. This assay system uses a
non-toxic, non-polar fluorescent substrate, which is easily loaded
and then trapped intracellularly. Cleavage of the fluorescent
substrate by .beta.-lactamase lactamase yields a fluorescent
emission shift as substrate is converted to product. Because the
.beta.-lactamase reporter readout can be ratiometric, it is unique
among reporter gene assays in that it controls variables such as
the amount of substrate loaded into individual cells. The stable,
easily detected, intracellular readout simplifies assay procedures
by eliminating the need for washing steps, which facilitates
screening with cells using the invention. Preferably, a ratiometric
fluorescent signal transduction detection system can be used with
the invention. Preferred fluorogenic substrates are described in
the Examples.
[0080] Other reporter genes such as polynucleotides encoding a
polypeptide having the biological activity of green fluorescent
protein (GFP) can be used.
[0081] A promoter is considered to be "modulated" by an active,
promiscuous G.alpha. protein when the expression of a reporter gene
to which the promoter is operably linked is either increased or
decreased upon activation of the promiscuous G.alpha. protein. It
is not necessary that the active, promiscuous G.alpha. protein
directly modulate reporter gene expression.
[0082] For example, embodiments of the invention presume that
activation of G.alpha..sub.15 or G.alpha..sub.16 can, through a
G-protein signaling pathway, activate PLC.beta., which in turn
increases intracellular calcium levels. An increase in calcium
levels can lead to modulation of a "calcium-responsive" promoter
that is part of a signal transduction detection system, i.e., a
promoter that is activated (e.g., a NFAT promoter) or inhibited by
a change in calcium levels. One example of an NFAT-DNA binding site
is found in Shaw, et al. Science 291:202-205 1988. Likewise, a
promoter that is responsive to changes in protein kinase C levels
(i.e., a "protein kinase C-responsive promoter") can be modulated
by an active G.alpha. protein through G-protein signaling pathway.
The cells described above can also include a G-protein coupled
receptor. Genes encoding numerous GPCRs have been cloned (Simon et
al., 1991, Science 252:802-808), and conventional molecular biology
techniques can be used to express a GPCR on the surface of a cell
of the invention. Preferably, the sum responsive promoter allows
only a relatively short lag (e.g., less than 90 minutes) between
engagement of the GPCR and transcriptional activation. A preferred
responsive promoter includes the nuclear factor of activated T-cell
promoter (Flanagan et al., 1991, Nature 352:803-807).
[0083] Many cells can be used in the invention, particularly for
heterologous expression of a GPCR. Such cells include, but are not
limited to; baby hamster kidney (BHK) cells (ATCC No. CCL10), mouse
L cells (ATCC No. CCLI.3), Jurkats (ATCC No. TIB 152) and 153 DG44
cells [see, Chasin (1986) Cell. Molec. Genet. 12: 555] human
embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese hamster
ovary (CHO) cells (ATCC Nos. CRL9618, CCL61, CRL9096), PC12 cells
(ATCC No. CRL17.21) and COS-7 cells (ATCC No. CRL1 651). Preferred
cells for heterologous cell surface protein expression are those
that can be readily and efficiently transfected. Preferred cells
include Jurkat cells CHO cells and HEK 293 cells, such as those
described in U.S. Pat. No. 5,024,939 and by Stillman et al. (1 985)
Mol. Cell. Biol. 5: 2051-2060.
[0084] GPCRs that can be used with the invention include, but are
not limited to, muscarinic receptors, e.g., human M2 (GenBank
accession #M16404); rat M3 (GenBank accession #M16407); human M4
(GenBank accession #M16405); human M5 (Bonner, et al., (1988)
Neuron 1, pp. 403-410); and the like; neuronal nicotinic
acetylcholine receptors, e.g., the human .alpha..sub.2,
.alpha..sub.3, and .beta..sub.2, subtypes disclosed in U.S. Ser.
No. 504,455 (filed Apr. 3, 1990, which is hereby expressly
incorporated by reference 0herein in its entirety); the human
.alpha..sub.5 subtype (Chini, et al. (1992) Proc. Natl. Acad. Sci.
U.S.A. 89: 1572-1576), the rat .alpha..sub.2 subunit (Wada, et al.
(1988) Science 240, pp. 330-334); the rat .alpha..sub.3 subunit
(Boulter, et al. (1986) Nature 319, pp. 368-374); the rat
.alpha..sub.4 subunit (Goldman, et al. (1987) Cell 48, pp.
965-973); the rat .alpha..sub.5 subunit (Boulter, et al. (1990) I.
Biol. Chem. 265, pp. 4472-4482); the chicken .alpha..sub.7 subunit
(Couturier et. al. (1990) Neuron 5: 847-856); the rat .beta..sub.2
subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54) the rat
.beta..sub.3 subunit (Deneris, et al. (1989) J. Biol. Chem. 264,
pp. 6268-6272); the rat .beta..sub.4 subunit (Duvoisin, et al.
(1989) Neuron 3, pp. 487-496); combinations of the rat .alpha.
subunits, and s .beta. subunits and a and p subunits; GABA
receptors, e.g., the bovine x, and .beta..sub.1, subunits
(Schofield, et al. (1987) Nature 328, pp.. 221-227); the bovine
X.sub.2, and X.sub.3, subunits (Levitan, et al. (1988) Nature 335,
pp. 76-79); the .gamma.-subunit (Pritchett, et al. (1989) Nature
338, pp. 582-585); the .beta..sub.2, and .beta..sub.3, subunits
(Ymer, et al. (1989) EMBO J. 8, pp. 1665-1670); the 8 subunit
(Shivers, B. D. (1989) Neuron 3, pp. 327-337); and the like;
glutamate receptors, e.g., rat GluR1 receptor (Hollman, et al.
(1989) Nature 342, pp. 643-648); rat GluR2 and GluR3 receptors
(Boulter et al. (1990) Science 249:1033-1037; rat GluR4 receptor
(Keinanen et al. (1990) Science 249: 556-560); rat GluR5 receptor
(Bettler et al. (1990) Neuron 5: 583-595); rat GluR6 receptor
(Egebjerg et al. (1991) Nature 351: 745-748); rat GluR7 receptor
(Bettler et al. (1992) neuron 8:257-265); rat NMDAR1 receptor
(Moriyoshi et al. (1991) Nature 354:31-37 and Sugihara et al.
(1992) Biochem. Biophys. Res. Comm. 185:826-832); mouse NMDA el
receptor (Meguro et al. (1992) Nature 357: 70-74); rat NMDAR2A,
NMDAR2B and NMDAR2C receptors (Monyer et al. (1992) Science 256:
1217-1221); rat metabotropic mGluR1 receptor (Houamed et al. (1991)
Science 252: 1318-1321); rat metabotropic mGluR2, mGluR3 and mGluR4
receptors (Tanabe et al. (1992) Neuron 8:169-179); rat metabotropic
mGluR5 receptor (Abe et al. (1992) I. Biol. Chem. 267:
13361-13368); and the like; adrenergic receptors, e.g., human
.beta.1 (Frielle, et al. (1987) Proc. Natl. Acad. Sci. 84, pp.
7920-7924); human .alpha..sub.2 (Kobilka, et al. (1987) Science
238, pp. 650-656); hamster .beta..sub.2 (Dixon, et al. (1986)
Nature 321, pp. 75-79); and the like; dopamine receptors, e.g.,
human D2 (Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6);
mammalian dopamine D2 receptor (U.S. Pat. No. 5,128,254); rat
(Bunzow, et al. (1988) Nature 336, pp. 783-787); and the like; and
the like; serotonin receptors, e.g., human 5HT1a (Kobilka, et al.
(1987) Nature 329, pp. 75-79); serotonin 5HT1C receptor (U.S. Pat.
No. 4,985,352); human 5HT1D (U.S. Pat. No. 5,155,218); rat 5HT2
(Julius, et al. (1990) PNAS 87, pp.928-932); rat 5HT1c (Julius, et
al. (1988) Science 241, pp. 558-564), and the like.
[0085] If desired (e.g., for commercial purposes), a cell(s) of the
invention can packaged into a container that is packaged within a
kit. Such a kit may also contain any of the various isolated
nucleic acids, antibodies, proteins, signal transduction detection
systems, substrates, and/or drugs described herein, known in the
art or developed in the future. A typical kit also includes a set
of instructions for any or all of the methods described herein.
METHODS OF THE INVENTION
[0086] The invention provides several methods for cloning or
characterizing GPCRs, screening or characterizing ligands (e.g.,
known ligands) of GPCRs, and identifying or characterizing
compounds that modulate signal transduction. For example, the
invention provides a method for determining whether a "target"
polypeptide is a GPCR for a given ligand. The method involves
expressing a target polypeptide in a cell described herein that
comprises a reporter gene construct (e.g., a construct encoding
.beta.-lactamase reporter gene operably linked to a NFAT promoter).
In this method, the test polypeptide is contacted with a chosen
ligand, usually of established activity, and a change in reporter
gene expression is detected. A "target" polypeptide, which is
usually a GPCR, is any polypeptide expressed by a cell that can be
assayed for activity using the present invention.
[0087] Similar methods can be used to test ligands and compounds
using GPCRs of known, partially known and unknown function. A test
ligand is a molecule that can be assayed for its ability to bind to
a GPCR. A test compound is a molecule that can be assayed for its
ability to modulator of signal transduction. Often, such a target
polypeptide, test ligand, or test compound is, because of its
sequence or structure, suspected of being able to function in a
given capacity. Nonetheless, randomly chosen target polypeptides,
test ligands, and test compounds also can be used in the methods
described herein, and with techniques known in the art or developed
in the future. For example, expression of target polypeptides from
nucleic acid libraries, can be used to identify proteins involved
in signal transduction, such as orphan GPCRs. For instance, this
technique can be used to identify physiologically responsive
receptors (e.g., taste-responsive GPCRs) where the ligand
responsible for inducing a physiological event is known (e.g., a
given taste sensation is known).
[0088] The invention also includes enhancement of reporter gene
expression in a signal transduction detection system. This
particularly useful for improving the signal to noise ratio in a
screening assay. It generally involves contacting the cell with a
molecule ("subthreshold regulating molecule") that alters the
activity of a cellular process to a level subthreshold to the
activation of a cellularly responsive control sequence that is
operably linked to the reporter gene. Because the level of cellular
activity is subthreshold, the reporter gene has a low expression
level. The reporter gene system, however, is poised for activation
by a change in cellular process induced by either a test chemical,
test ligand or expression of target protein. Such cellularly
responsive control sequences can be responsive elements known in
the art in other applications. Such response elements, however, do
not need be responsive to their naturally occurring signal, since
the assay may occur in cells lacking the required constituents for
activation by a naturally occurring signal. The subthreshold
regulating molecule can either increase or decrease the activity of
the cellular process. It is understood that the cellular process
may not only be "classic" cellular process, such as an enzymatic
activity, but it also includes levels of cellular entities (e.g.,
ions, metabolites and second messengers) or other measurable
properties of the cell (e.g., cell volume, chromatin density,
etc.). Cells described herein are preferred for this method. Other
cells, however, can be used as well which express G.alpha. proteins
endogenously, or heterologously.
[0089] For example, in order to enhance detection of expression of
a reporter gene, the cell can be contacted with a compound (e.g., a
calcium ionophore) that increases calcium levels inside of the
cell. By increasing calcium levels inside the cell, the probability
that activation of a G-protein will activate expression of a
reporter gene is greatly enhanced. Preferably, the calcium levels
are increased to a level that is just below the threshold level for
activation of a calcium-responsive promoter, such as an NFAT
promoter (see FIG. 2). In practice, ionomycin typically is added at
a concentration of about 0.01 to 3 .mu.M, preferably 0.03 .mu.M.
Cells described herein are preferred for this method. Other cells,
however, can be used as well which express G.alpha. proteins
endogenously, or heterologously.
[0090] In an alternate method of enhancing a signal transduction
detection system, thapsigargin is added to the cell to set
intracellular calcium levels at subthreshold levels to enhance
reporter gene activation. Thapsigargin is added to the cell at a
concentration of about 1 to 50 nM, with the effect of partially
depleting intracellular calcium pools and slowing the re-filling of
such pools (Thastrup et al., 1990, Proc. Natl. Acad. Sci.
87:2466-2470). If desired, thapsigargin can be used at a higher
concentration (e.g., 200 nM to 1 .mu.M) in a "Ca.sup.+2-clamp"
protocol, in which membrane potential is used to set the baseline
calcium concentration (Negulescu et al., 1994, Proc. Natl. Acad.
Sci. 91:2873-2877). This can be applied to screening for modulators
of signal transduction using a reporter gene system with a
calcium-responsive promoter. Cells described herein are preferred
for this method. Other cells, however, can be used as well which
express G.alpha. proteins endogenously, or heterologously.
[0091] In yet another method of the invention, conventional
molecular biology techniques can be used to express a calcium
modulating ligand in cells, and thereby increase calcium levels
(Bram et al., 1994, Nature 371:355-358). This can be applied to
screening for modulators of signal transduction using a reporter
gene system with a calcium-responsive promoter. Cells described
herein are preferred for this method. Other cells, however, can be
used as well which express G.alpha. proteins endogenously, or
heterologously.
[0092] A related method of the invention for enhancing detection of
expression of the reporter gene involves contacting the cell with
an activator of protein kinase C. Typically, this method involves
contacting the cell with 1 to 3 nM of phorbol myristate acetate
(PMA) or another phorbol ester, preferably PMA is used at a
concentration of 0.3 nM. The PMA concentration can be titrated to
achieve sub threshold levels. Various analogs of PMA that retain
this activity are known in the art, and can be used in the
invention. Cells described herein are preferred for this method.
Other cells, however, can be used as well which express G.alpha.
proteins endogenously, or heterologously.
[0093] The invention also provides a method for determining whether
a "test" ligand is a ligand for a given GPCR. In this method, a
selected GPCR is expressed in a cell, such as a cell of the
invention, which contains a construct and encodes a reporter gene.
The cell is contacted with a test ligand, and a change in
expression of the reporter gene is detected. This method is
particularly well suited for identifying a ligand not known to bind
to the receptor and it can also be used to determine receptor
selectivity. In this method, the change in expression of the
reporter gene can be compared for a sample of cells in the
presence, versus in the absence, of the test ligand in order to
identify ligand specific activation. Cells described herein are
preferred for this method. Other cells, however, can be used as
well which express G.alpha. proteins endogenously, or
heterologously..
[0094] The aforementioned methods can readily be adapted to provide
a method for characterizing the ability of a ligand to interact
with a panel of GPCRs of interest. In such an assay, the first GPCR
of interest is expressed in a cell, such as a cell of the
invention, that contains a construct encoding a reporter gene. In a
second cell (in a second, separate sample), a second GPCR of
interest is expressed along with reporter gene system. Additional
GPCRs can be expressed in additional cells with reporter gene
systems. Typically, these cells differ only with respect to the
GPCR that is expressed. Each sample of cells is contacted with the
"test" ligand of interest, and a change in reporter gene expression
is detected for each cell sample. By comparing the changes in
expression of the reporter gene between cell samples, one can
characterize the functional activity of the ligand. This method is
particularly well suited for assaying the ability of a known ligand
to interact with several GPCRs that are known to be related. Thus
the selectivity of the ligand can be determined. For example,
various muscarinic receptors (e.g., M.sub.1, M.sub.2, and M.sub.3)
can be expressed, separately, on a cell. If desired, various
modulators of G-protein activity (e.g., agonists and antagonists)
can be characterized in a variation of this method. Cells described
herein are preferred for this method. Other cells, however, can be
used as well which express G.alpha. proteins endogenously, or
heterologously.
[0095] The invention also provides a general method for determining
whether a test compound modulates signal transduction in a cell.
This method also employs a cell, such as a cell of the invention,
that includes a construct, and that expresses a reporter gene. In
this method, the cell expresses a GPCR, and the cell is contacted
with a ligand that, in the absence of a test compound, activates
signal transduction. The cell is also contacted with a test
compound, and a change in expression of the reporter gene indicates
that the test compound modulates signal transduction in the
cell.
[0096] In a variation of this method, the invention provides a
"receptor-less" method for determining whether a test compound
modulates signal transduction. In this variation, the cell is not
engineered to express a GPCR. In lieu of contacting the cell with a
ligand, the cell is contacted with a compound that directly
activates a G.alpha. protein encoded by a construct within the
cell. Examples of such compounds include mastoparan (Calbiochem)
and aluminum fluoride. These compounds typically are used at
concentrations of 0.5 to 5 mM. A change in expression of a reporter
gene indicates that the test compound modulates signal transduction
in the cell. Such a change also indicates that the compound affects
signaling events that occur subsequent to receptor signaling in the
signaling pathway.
[0097] The invention also provides a method for determining whether
a test polypeptide is a GPCR for a given ligand, without employing
a second genetic construct expressing a reporter gene. In this
method, a test polypeptide is expressed in a stable, isolated cell
that carries a genetic construct that includes a promoter operably
linked to a gene that encodes a polypeptide having the biological
activity of a promiscuous G.alpha. protein. The test polypeptide is
contacted with a ligand, and an increase in calcium levels within
the cell is detected. Any of the art-known methods for detecting a
change in calcium levels can be used in this method (Negulescu and
Machen, 1990, Meth. in Enzymol. 192:38-81). In a preferred method,
the increase is detected by contacting the cell with fura-2
(available from Molecular Probes; Eugene, Oreg.) and detecting a
change in fluorescence emission of a sample that includes the
cell.
[0098] The invention offers several advantages. By employing
promiscuous G-proteins, the invention allows the use of a single
intracellular signaling pathway (e.g., activation of PLC.beta.) to
analyze GPCRs that normally couple specifically to G-proteins of a
single family. By providing methods that employ living cells, the
invention allows a receptor or ligand that is identified in an
assay to be cloned. By employing fluorescent detection methods, the
invention, in various embodiments, allows a practitioner to
characterize a single cell. Accordingly, convenient cell-sorting
methods, such as FACS, can be used to analyze and isolate cells.
The fluorescent assays employed in the invention also provide a
stable, non-labile indicator of G-protein activation. Such a stable
signal (lasting up to twelve hours) allows a practitioner to
analyze numerous samples in parallel, thus rendering the invention
useful for high throughput screening of "test" polypeptides,
ligands, and compounds. The invention provides, for the first time,
an assay for associating occupancy of any GPCR with gene
expression, as detected by a fluorescence emission. In addition, by
providing methods for enhancing detection of G-protein activation,
the invention provides a sensitive assay for detecting low levels,
or brief activation, of a G-protein.
[0099] The kits can be produced to accomplish the methods described
herein. Such kits can include the polynucleotides for GPCR
expression, cells for GPCR expression or G.alpha. protein
expression and signal transduction detection systems, such reporter
gene systems.
EXAMPLES
[0100] The following examples are intended to illustrate but not
limit the invention. While they are typical of those methods that
might be used, other procedures known to those skilled in the are
may alternatively be used.
Example 1
Synthesis of a .beta. Lactamase Substrate (Compound 7b)
[0101] ##STR1##
[0102] For synthesis of 2,4 dihydroxy-5-chlorobenzaldehyde, 21.7 g
(0.15 Mol) 4-chlororesorcinol were dissolved in 150 ml dry diethyl
ether and 27 g finely powdered zinc (II) cyanide and 0.5 g
potassium chloride were added with stirring. The suspension was
cooled on ice. A strong stream of hydrogen chloride gas was blown
into the solution with vigorous stirring. After approximately 30
minutes the reactants were dissolved. The addition of hydrogen
chloride gas was continued until it stopped being absorbed in the
ether solution (approx. 1 hour). During this time a precipitate
formed. The suspension was stirred for one additional hour on ice.
Then the solid was let to settle. The ethereal solution was poured
from the solid. The solid was treated with 100 g of ice and heated
to 100.degree. C. in a water bath. Upon cooling the product
crystallized in shiny plates from the solution. They were removed
by filtration on dried over potassium hydroxide. The yield was 15.9
g (0.092 Mol, 61%). .sup.1H NM (CDCl.sub.3): .delta. 6.23 ppm (s,
1H, phenol), .delta. 6.62 ppm (s, 1H, phenyl), .delta. 7.52 ppm (s,
1H, phenyl), .delta. 9.69 ppm (s, 1H, formyl), .delta. 11.25 ppm
(s, 1H, phenol).
[0103] To prepare 3-carboxy 6-chloro 7-hydroxy coumarin, 5.76 g
(0.033 Mol) 2,4-dihydroxy-5-chlorobenzaldehyde and 7.2 g (0.069
Mol) malonic acid were dissolved in 5 ml warm pydine. 75 .mu.l
Aniline were stirred into the solution and the reaction let to
stand at room temperature for 3 days. The yellow solid that formed
was broken into smaller pieces and 50 ml ethanol was added. The
creamy suspension was filtered through a glass frit and the solid
was washed three times with 1 N hydrochloric acid and then with
water. Then the solid was stirred with 100 ml ethyl acetate, 150 ml
ethanol and 10 ml half concentrated hydrochloric acid. The solvent
volume was reduced in vacuo and the precipitate recovered by
filtration, washed with diethyl ether and dried over phosphorous
pentoxide. 4.97 g (0.021 Mol, 63%) of product was obtained as a
white powder. .sup.1H NMR (dDMSO): .delta. 6.95 ppm (s, 1H),
.delta. 8.02 ppm (s, 1H), .delta. 8.67 ppm (s, 1H).
[0104] To prepare 7-butyryloxy-3-carboxy-6-chlorocoumarin, 3.1 g
(12.9 mMol) 3-carboxy-6-chloro-7-hydroxycoumarin were dissolved in
100 ml dioxane and treated with 5 ml butyric anhydride, 8 ml
pyridine and 20 mg dimethyl aminopyridine at room temperature for
two hours. The reaction solution was added with stirring to 300 ml
heptane upon which a white precipitate formed. It was recovered by
filtration and dissolved in 150 ml ethyl acetate. Undissolved
material was removed by filtration and the filtrate extracted twice
with 50 ml 1 N hydrochloric acid/brine (1:1) and then brine. The
solution was dried over anhydrous sodium sulfate. Evaporation in
vacuo yielded 2.63 g (8.47 mMol, 66%) of product. .sup.1H NMR
(CDCl.sub.3): .delta. 1.08 ppm (t, 3H, J=7.4 Hz, butyric methyl),
.delta. 1.85 ppm (m, 2H, J.sub.1 .delta. J.sub.2=7.4 Hz, butyric
methylene), .delta. 2.68 ppm (t, 2H, J=7.4 Hz, butyric methylene),
.delta. 7.37 ppm (s, 1H, coumarin), .delta. 7.84 ppm (s, 1H,
coumarin), .delta. 8.86 ppm (s, 1H, coumarin).
[0105] Preparation of
7-butyryloxy-3-benzyloxycarbonylmethylaminocarbonyl-6-chlorocoumarin
is effected as follows. 2.5 g (8.06 mMol)
7-Butyryloxy-3-carboxy-6-chlorocoumarin, 2.36 g hydroxybenztriazole
hydrate (16 mMol) and 1.67 g (8.1 mMol) dicyclohexyl carbodiimide
were dissolved in 30 ml dioxane. A toluene solution of
O-benxylglycine [prepared by extraction of 3.4 g (10 mMol)
benzylglycine tosyl salt with ethyl acetate-toluene-saturated
aqueous bicarbonate-water (1:1:1:1, 250 ml), drying of the organic
phase with anhydrous sodium sulfate and reduction of the solvent
volume to 5 ml] was added drop wise to the coumarin solution. The
reaction was kept at room temperature for 20 hours after which the
precipitate was removed by filtration and washed extensively with
ethylacetate and acetone. The combined solvent fractions were
reduced to 50 ml on the rotatory evaporator upon which one volume
of toluene was added and the volume further reduced to 30 ml. The
precipitating product was recovered by filtration and dissolved in
200 ml chloroform-absolute ethanol (1:1). The solution was reduced
to 50 ml on the rotatory evaporator and the product filtered off
and dried in vacuo yielding 1.29 g of the title product. Further
reduction of the solvent volume yielded a second crop (0.64 g).
Total yield: 1.93 g (4.22 mMol, 52%). .sup.1H NMR (CDCl.sub.3):
.delta. 1.08 ppm (t, 3H, J=7.4 Hz, butyric methyl), 1.84 ppm (m,
2H, J.sub.1 .delta. J.sub.2=7.4 Hz, butyric methylene), .delta.
2.66 ppm (t, 2H, J 7.4 Hz, butyric methylene), .delta. 4.29 ppm (d,
2H, J 5.5 Hz, glycine methylene), .delta. 5.24 ppm (s, 2H, benzyl),
.delta. 7.36 ppm (s, 1H, coumarin), .delta. 7.38 ppm (s, 5H,
phenyl), .delta. 7.77 ppm (s, 1H, coumarin), .delta. 8.83 ppm (s,
1H, coumarin), .delta. 9.15 ppm (t, 1H, J 5.5 Hz, amide).
[0106] 7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin
was prepared as follows. 920 mg (2 mMol)
7-butyryloxy-3-benzyloxycarbonylmethylamino-carbonyl-6-chlorocoumarin
were dissolved in 50 ml dioxane. 100 mg Palladium on carbon (10%)
and 100 .mu.l acetic acid were added to the solution and the
suspension stirred vigorously in a hydrogen atmosphere at ambient
pressure. After the uptake of hydrogen seized the suspension was
filtered. The product containing carbon was extracted five times
with 25 ml boiling dioxane. The combined dioxane solutions were let
to cool upon which the product precipitated as a white powder.
Reduction of the solvent to 20 ml precipitates more product. The
remaining dioxane solution is heated to boiling and heptane is
added until the solution becomes cloudy. The weights of the dried
powders were 245 mg, 389 mg and 58 mg, totaling 692 mg (1.88 mMol,
94%) of white product. .sup.1H NMR (dDMSO): .delta. 1.02 ppm (t,
3H, J 7.4 Hz, butyric methyl), .delta. 1.73 ppm (m, 2H, J.sub.1
.delta. J.sub.2=7.3 Hz, butyric methylene), .delta. 2.70 ppm (t,
2H, J 0.67 ppm (s, 1H, coumarin), .delta. 8.35 ppm (s, 1H,
coumarin), .delta. 8.90 ppm (s, 1H, coumarin), .delta. 9.00 ppm (t,
1H, J=5.6 Hz, amide).
[0107] Coupling of
7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin with
7-amino-3'-chlorocephalosporanic acid benzhydryl ester was effected
as follows. 368 mg (1 mMol)
7-Butyryloxy-3-carboxymethylaminocarbonyl-6-chlorocoumarin, 270 mg
hydroxybenztriazole hydrate and 415 mg (1 mMol) 7-amino-3'-chloro
cephalosporanic acid benzhydryl ester were suspended in 40 ml
dioxane -acetonitrile (1:1). 260 mg (1.25 mMol)
dicyclohexylcarbodiimide in 5 ml acetonitrile were added and the
suspension was stirred vigorously for 36 hours. The precipitate was
removed by filtration and the volume of the solution reduced to 20
ml on the rotatory evaporator. 50 ml Toluene was added and the
volume reduced to 30 ml. With stirring 50 ml heptane was added and
the suspension chilled on ice. The precipitate was recovered by
filtration. It was redissolved in 10 ml chloroform and the
remaining undissolved solids were filtered off. Addition of 2
volumes of heptane precipitated the title product which was
collected and dried in vacuo and yielded 468 mg (0.64 mMol, 64%)
off-white powder. .sup.1H NMR (CDCl.sub.3): .delta. 1.08 ppm (t,
3H, J 7.4 Hz, butyric methyl), .delta. 1.84 ppm (m, 2H, J.sub.1
.delta. J.sub.2 7.4 Hz, butyric methylene), .delta. 2.66 ppm (t,
2H, J 7.4 Hz, butyric methylene), .delta. 3.54 ppm (2d, 2H, J 18.3
Hz, cephalosporin C-2), .delta. 4.24 ppm (2d, 2H, J 5.8 Hz,
cephalosporin 3 methylene), .delta. 4.37 ppm (d, 2H, J 3.8 Hz,
glycine methylene), .delta. 5.02 ppm (d, 1H, J 4.9 Hz,
cephalosporin C-6), .delta. 5.89 ppm (dd, 1H, J.sub.1 9.0 Hz,
J.sub.2 5.0 Hz, cephalosporin C-7), .delta. 6.96 ppm (s, 1H,
benzhydryl), .delta. 7.30-7.45 ppm (m, 12H, phenyl, coumarin,
amide), .delta. 7.79 ppm (s, 1H, coumarin), .delta. 8.84 ppm (s,
1H, coumarin), .delta. 9.28 ppm (t, 1H, J 3.7 Hz, amide).
[0108] Coupling of the above product with 5-fluoresceinthiol was
effected as follows. 90 mg (0.2 mMol) 5-mercaptofluorescein
diacetate disulfide dimer were dissolved in 10 ml chloroform and
treated with 25 .mu.l tributyl phosphine and 25 .mu.l water in an
argon atmosphere. The solution was kept for 2 hours at ambient
temperature and was then added to a solution of 20 mg sodium
bicarbonate, 25 mg sodium iodide and 110 mg (0.15 mMol) of the
above compound in 10 ml dimethylformamide. After 4 hours the
solvents were removed in vacuo and the residue triturated with
diethylether. The solid was dissolved in ethyl acetate-acetonitrile
(1:1). After removal of the solvents the residue was triturated
once more with diethylether yielding 157 mg (0.13 mMol, 88%) of a
cream-colored powder product.
[0109] A sample of the above compound was treated with a large
access of trifluoroacetic acid-anisole (1:1) at room temperature
for 20 minutes. The reagents are removed in vacuo and the residue
triturated with ether. High performance liquid chromatography of
the solid in 45% aqueous acetonitrile containing 0.5% acetic acid
gives a product in which the butyrate and the diphenylmethyl esters
have been cleaved. It was purified by high performance liquid
chromatography on a reverse phase C.sub.18-column using 45% aqueous
acetonitrile containing 5% acetic acid as the eluent. ##STR2##
[0110] Deprotection of the fluorescein acetates in compound 27 was
accomplished with sodium bicarbonate in methanol (room temperature,
30 minutes) to provide the fluorescent enzyme substrate CCF2. It
was purified by high performance liquid chromatography on a reverse
phase C.sub.18-column using 35% aqueous acetonitrile containing
0.5% acetic acid as the eluent. ##STR3##
[0111] Stirring of compound 27 with excess acetoxymethyl bromide in
dry lutidine produced the menembrane permeable derivative of the
substrate (CCF2/ac.sub.2AM.sub.2). It was purified by high
performance liquid chromatography on a reverse phase C.sub.18
-column using 65% aqueous acetonitrile containing 0.5% acetic acid
as the eluent. CCF2/ac.sub.2AM.sub.2 is readily converted to CCF2
in the cells' cytoplasm.
[0112] The donor and accepter dyes in substrate CCF2 do not stack.
The substrate is fully fluorescent in phosphate buffer and there is
no formation of the "dark complex" (i.e., addition of methanol does
not change the fluorescence spectrum of CCF2, except for the effect
of dilution). This is due to the much smaller and more polar nature
of the 7-hydroxycoumarin compared to that of the xanthene dyes
(eosin, rhodarnine, rhodol and resorufin).
[0113] The emission spectrum of compound CCF2 in 50 mmolar
phosphate buffer pH 7.0 before and after .beta.-lactamase cleavage
of the .beta.-lactam ring. In the intact substrate, efficient
energy transfer occurs from the 7-hydroxycoumarin moiety to the
fluorescein moiety. Excitation of the substrate at 405 nm results
in fluorescence emission at 515 nm (green) from the acceptor dye
fluorescein. The energy transfer is disrupted when .beta.-lactamase
cleaves the .beta.-lactam ring, thereby severing the link between
the two dyes. Excitation of the products at 405 nm now results
entirely in donor fluorescence emission at 448 nm (blue). The
fluorescence emission from the donor moiety increases 25 fold upon
.beta.-lactam cleavage. The fluorescence at 515 nm is reduced by
3.5-fold, all of the remaining fluorescence originating from the
7-hydroxycoumarin as its emission spectrum extends into the green.
Twenty-five-fold quenching of the donor in the substrate is
equivalent to an efficiency of fluorescence energy transfer of 96%.
This large fluorescence change upon .beta.-lactam cleavage can
readily be used to detect .beta.-lactamase in the cytoplasm of
living mammalian cells.
[0114] The 7-hydroxycoumarin moiety in the cephalosporin was
determined to have a fluorescence quantum efficiency in the absence
of the acceptor of 98-100%. This value was determined by comparing
the integral of the corrected fluorescence emission spectrum of the
dye with that of a solution of 9-aminoacridine hydrochloride in
water matched for absorbance at the excitation wavelength. It
follows that 7-hydroxycoumarin is an ideal donor dye, as virtually
every photon absorbed by the dye undergoes fluorescence energy
transfer to the acceptor.
Example 2
Use of a .beta. Lactamase Substrate
[0115] Cells of the T-cell lymphoma line Jurkat were suspended in
an isotonic saline solution (Hank's balanced salt solution)
containing approximately 10.sup.12 .beta.-lactamase enzyme
molecules per milliliter (approximately 1.7 nM; Penicillinase 205
TEM R.sup.+, from Sigma) and 1 mg/ml rhodamine conjugated to
dextran (40 kd) as a marker of loading. The suspension was passed
through a syringe needle (30 gauge) four times. This causes
transient, survivable disruptions of the cells' plasma membrane and
allows entry of labeled dextran and .beta.-lactamase. Cells that
had been successfully permeabilized contained .beta.-lactamase and
were red fluorescent when illuminated at the rhodamine excitation
wavelength on a fluorescent microscope. The cells were incubated
with 5 .mu.M fluorogenic .beta.-lactamase substrate,
CCF2/ac.sub.2AM.sub.2, at room temperature for 30 minutes.
Illumination with violet light (405 nm) revealed blue fluorescent
and green fluorescent cells. All cells that had taken up the marker
rhodamine-dextran appeared fluorescent blue, while cells devoid the
enzyme appeared fluorescent green.
Example 3
Use of a .beta. Lactamase Substrate
[0116] Cells from cell lines of various mammalian origin were
transiently transfected with a plasmid containing the RTEM
.beta.-lactamase gene under the control of a mammalian promoter.
The gene encodes cytosolic .beta.-lactamase lacking any signal
sequence and is listed as SEQ. D. 1.10 to 48 hours after
transfection cells were exposed to 5 micromolar
CCF2/ac.sub.2AM.sub.2 for 1 to 6 hours. In all cases fluorescent
blue cells were detected on examination with a fluorescence
microscope. Not a single blue fluorescent cell was ever detected in
non transfected control cells. To quantitate the fluorescence
measurements the cells were first viewed through coumarin (450 DF
65) and then fluorescein (515 EFLP) emission filters and pictures
were recorded with a charge couple device camera. The average pixel
intensities of CCF2 loaded transfected cells (blue) and controls
(green) at coumarin and fluorescein wavelength in COS-7 (Table 2)
and CHO (Table 3) cells are summarized; values for 4 representative
cells for each population are given. Thus, the substrate CCF2
revealed gene expression in single living mammalian cells.
Substrate can be loaded using Pluronic formulations (see Molecular
Probes Catalog) using polyethylene glycol. TABLE-US-00001 TABLE 1
COS-7 (origin: SV40 transformed African green monkey kidney cells)
coumarin emission Fluorescein emission Table of pixel intensities
filter filter Blue cell #1 27 20 #2 34 23 #3 31 31 #4 22 33 Green
cell #1 4 43 #2 4 42 #3 5 20 #4 3 24
[0117] TABLE-US-00002 TABLE 2 CHO (origin: Chinese hamster ovary
cells) coumarin emission Fluorescein emission Table of pixel
intensities filter filter Blue cell #1 98 112 #2 70 113 #3 76 92 #4
56 67 Green cell #1 9 180 #2 9 102 #3 7 101 #4 9 83
Example 4
Expression of G.alpha..sub.15 and G.alpha..sub.16 in Cells
[0118] This example illustrates that, although constitutive
expression of G.alpha..sub.15 or G.alpha..sub.16 at high levels is
toxic to cells, expression of G.alpha..sub.15 or G.alpha..sub.16
from a gene that is controlled by an inducible promoter, is
tolerated by the cells. For constitutive or inducible expression of
G.alpha..sub.15 or G.alpha..sub.16, the genes encoding each of
these subunits were placed, separately, under the control of a
cytomegalovirus promoter in the plasmids pcDNA3G.alpha.15 ,
pcDNA3G.alpha.16 (Vector pcDNA3 available from Invitrogen, Inc.,
Del Mar, Calif.), pdEF-BOSG.alpha.15 and pdE F-BOSG.alpha.16 (For
pd BOSG, see Gossen and Bujard, 1992, Proc. Natl. Acad. Sci.
89:5547-5551, also available from Clontech). To construct pdEF-BOSG
15 and pdEF-BOSG 16, sequences encoding the G subunit were inserted
into pdEFBOS at its EcoRI and NotI sites. The plasmid pdEFBOS was
derived from pEFBOS by removing the HindIII fragment containing the
SV40 Ori (see Mizushima and Nagata, 1990, Nucl. Acids. Res. 18).
Each of these plasmids was used to transfect COS-7 cells, according
to conventional protocols, and each plasmid carried a neo gene,
which confers resistance to G418. As is summarized in Table 3,
approximately 150 G418-resistant clones were generated, yet none of
the clones was able to express a promiscuous G-protein. The ability
of a cell to express a promiscuous G-protein was determined by
Western blot analysis using an antibody that binds a peptide having
the amino acid sequence RPSVLARYLDEINLL (SEQ ID NO: 5) (Amatruda et
al., 1991, Proc. Natl. Acad. Sci. 88:5587-5591). These data show
that constitutive expression of a promiscuous G-protein under the
control of a strong promoter is not tolerated by COS-7 cells.
Constitutive expression of promiscuous G proteins at high levels
may lead to constant accumulation of inositol phosphates or
metabolites, which may be toxic to cells. TABLE-US-00003 TABLE 3
High-Level Constitutive Expression of Promiscuous G-Proteins is not
Tolerated G418-resistant Clones expressing Construct Selection
clones picked G.alpha..sub.15/16 PcDNA3G.alpha.15 G418 53 0
PcDNA3G.alpha.16 G418 48 0 pdEF-BOSG.alpha.15 G418 36 0
pdEF-BOSG.alpha.16 G418 19 0
[0119] The data summarized herein indicate that, although cells may
not tolerate constitutive expression of promiscuous G.alpha.
proteins at high levels, they can tolerate expression of
promiscuous G.alpha. proteins from an inducible promoter. In this
case, the genes for G.alpha..sub.15 and G.alpha..sub.16 were placed
under the control of a cytomegalovirus (CMV) promoter that was
operably linked to a heptamerized tet operator (Gossen and Bujard,
1992, Proc. Natl. Acad. Sci. 89:5547-5551). The plasmid encoding
G.alpha. and; the plasmid encoding G.alpha..sub.16 are identical,
except sequences encoding G.alpha..sub.16 in lieu of
G.alpha..sub.15. These plasmids were used to transfect COS-7 cells.
These cells were co-transfected with a tetracyclin-dependent
transactivator, rtTA, that is operably linked to a CMV promoter of
a plasmid that carries a neomycin resistance gene (Gossen et al.,
1995, Science 268:1766-1769).
[0120] Expression of the G.alpha. genes was induced by contacting
the cells with doxycyclin, a tetracyclin analog. In these
experiments, the doxycyclin concentration was 3 g/ml, although
doxycyclin concentrations ranging from 0.01 to 10 .mu.ml can be
used in order to regulate the level of gene expression. Of 17
hygromycin-resistant clones that were analyzed, 2 clones showed
doxycyclin-dependent expression of G.alpha..sub.15 or
G.alpha..sub.16 by Western blot analysis as described above. FIG. 3
illustrates that, in the presence of doxycyclin, expression of
G.alpha..sub.15 or G.alpha..sub.16 is detectable as a band of
approximately 43 kDa. This expression system provides low levels of
constitutive expression of G.alpha..sub.15 or G.alpha..sub.16
(e.g., less than 100 G.alpha. proteins/cell), yet expression of the
G.alpha. protein is highly inducible. Up to 10,000 G.alpha.
proteins/cell are produced upon induction of gene expression. As a
control, COS-7 cells that lacked the G.alpha. gene were analyzed,
and Western blot analysis indicated that the control cells did not
express G.alpha..sub.15 or G.alpha..sub.16. In sum, these
experiments demonstrate that stable cells can be produced by
employing an inducible promoter that provides (a) low levels of
constitutive expression (i.e., producing less than approximately
100 G.alpha. proteins/cell), and (b) high levels of induced
expression (i.e., producing approximately 10,000 G.alpha.
proteins/cell).
Example 5
Detection of G.alpha. Protein Activity by Detection of Fluorescence
Emission
[0121] These examples demonstrate that activation of a G.alpha.
protein in a cell, and a change in expression of a reporter gene,
can be detected by a detecting a change in fluorescence emission of
a sample that includes the cell. These examples employ Jurkat T
lymphocytes that were transfected with a genetic construct that
expresses a reporter gene. The genetic construct includes a NFAT
promoter, which is responsive to increased calcium levels and
protein kinase C activation that result from activation of G.alpha.
protein. The NFAT promoter was operably linked to a
.beta.-lactamase reporter gene. To detect expression of the
reporter gene, and thereby detect activation of G.alpha., the cells
were contacted with the .beta.-lactamase substrate
CCF2ac.sub.2/AM.sub.2 (described herein), and fluorescence emission
was detected according to previously described methods (Tsien et
al., 1993, Trends in Cell Biology 3:242-245).
[0122] Two different compounds, ionomycin and phorbol myristate
acetate (PMA), were used to optimize detection of expression of the
reporter gene in these examples. In the first example, the dose
response to ionomycin was measured. In this example, a set of
samples of cells were contacted with PMA (at 3 nM) and the calcium
ionophore ionomycin (at various concentrations, ranging from 0 to
3.0 .mu.M). Ionomycin increases calcium levels inside of the cells,
and thereby increases the probability that activation of a
G-protein, and a G-protein-mediated increase in calcium levels,
will activate expression of a reporter gene (e.g., a
.beta.-lactamase gene) that is operably linked to a
calcium-responsive promoter (e.g., a NFAT promoter).
[0123] In practicing these methods, it is preferable to add the
ionophore to a level that is just below the threshold level for
activation of the calcium-responsive promoter (e.g., the NFAT
promoter). Expression of the reporter gene then is activated by
activation of the G.alpha. protein, and the subsequent rise in
intracellular calcium levels. As is illustrated in FIG. 4A,
fluorescence emission from a sample of the aforementioned cells can
be measured by FRET. In this example, fluorescence emission was
measured approximately 90 minutes after stimulation. Because the
fluorogenic .beta.-lactamase substrate undergoes a shift in
fluorescence emission, fluorescence emission is measured as an
emission ratio (450/530) when exciting at 400 nm. This figure also
illustrates that an ionomycin concentration of approximately 0.3
.mu.M is preferable for increasing the intracellular calcium level
to a level that is just below the threshold level for activation of
the calcium-responsive promoter.
[0124] In a second example, the dose response of PMA required to
stimulate NFAT-driven expression was measured. Although PMA does
not, by itself, affect NFAT-regulated gene expression, it
potentiates a cell's response to an increase in calcium levels. In
this example, a set of cells was treated with ionomycin (at 1 .mu.M
ionomycin) and PMA (at various concentrations, ranging from 0 to 30
nM). As above, fluorescence emission was measured 90 minutes after
stimulation. As is illustrated in FIG. 4B, increasing
concentrations of PMA increased fluorescence emission from the cell
sample. Thus, treating the cells with PMA enhances detection of
expression of the reporter gene. This example also illustrates that
a PMA concentration of approximately 3 nM is preferable for
enhancing detection of expression of a reporter gene.
Example 6
Monitoring Activation and Inhibition of GPCR Activity with an NFAT
.beta.-Lactamase Assay
[0125] This example demonstrates that activation of a GPCR (Gq
receptor subtype) can be detected with an NFAT .beta.-lactamase
assay, which is an example of signal transduction detection system
based on a calcium-responsive promoter transcription based assay.
Stable cell line (production of described herein) containing
Gq-type GPCR receptor expresses .beta.-lactamase in response to the
addition of the agonist. The G.alpha.q protein was endogenously
expressed. This response is inhibited by an antagonist. Jurkat
clones expressing NFAT-.beta.1a were transfected with expression
vectors containing the Gq receptor and neomycin resistance gene
(double transfection). The transfected population was neo-selected
and sorted by FACS for clones responding to the GPCR agonist. For
the experiments shown, cells were stimulated for three hours with
the indicated ligands. Cells were then loaded with .beta.-lactamase
substrate CCF2/ac2AM for 1 hour, washed, dispensed into wells of a
microtiter plate (100,000 cells/well) and the blue/green ratio was
recorded by a plate reader.
[0126] FIG. 6 demonstrates a twenty-fold change in signal upon
receptor activation with an agonist (saturating dose 100 .mu.M). A
receptor antagonist (10 .mu.M) completely inhibited the agonist
activation of the receptor.
Example 7
Monitoring Activation of GPCRs with a Calcium Dye (Transiently
Transfected Cells)
[0127] This example demonstrates that activation of GPCRs (Gs and
Gi receptor subtypes) can be detected with an intracellular calcium
indicator transiently transfected cells, which is an example of a
signal transduction detection system based on changes in
intracellular ions. The cells used were transiently transfected
with two constructs. 4.times.10.sup.5 CHO-K1 cells were seeded on
35 mm petri dishes one day before transfection. 5 .mu.g plasmid DNA
and 12 .mu.l lipofectamine were added for each dish using the stand
method. In some cases, pBluescripts (-) or KS plasmid was used to
keep the amount of DNA consistent between each transfection. 20 h
later, the cells were stained with 10 mM Fura-PE3 (Molecular Probe)
for 3 h. Imaging analysis of calcium was performed to measure the
[Ca.sup.2+] signal mediated by the agonists addition.
[0128] Following imaging data show that the promiscuous G.alpha. 16
couples a Gs-receptor and a Gi-receptor in CHO-K1 cells following
transient transfections. The data also show that promiscuous
G.alpha. protein can change the effector downstream of the GPCR.
Panel A: 60 seconds after starting of the experiment, 10 .mu.M
agonist solution was added to the cells transfected by
pCIS/G.alpha. 16 (CMV promoter) and Gs-receptor (CMV promoter)
expression plasrnids. Panel B: 60 seconds after starting of the
experiment, 10 .mu.M agonist solution was added to the cells
transfected by pCIS/G.alpha. 16 alone. Panel C: 60 seconds after
starting of the experiment, 10 .mu.M agonist solution was added to
the cells transfected by Gs receptor expression plasmid alone.
Panel D: 60 seconds after starting of the experiment, 10 .mu.M
agonist solution was added to the cells transfected by
pCIS/G.alpha.16 and Gi-receptor expression plasnids. Panel E: 60
seconds after starting of the experiment, 10 .mu.M agonist solution
was added to the cells transfected by pCIS/G.alpha. 16 alone. Panel
F: 60 seconds after starting of the experiment, 10 .mu.M agonist
solution was added to the cells transfected by Gi-receptor
expression plasmid alone.
Example 8
Monitoring Activation of a GPCR with a Calcium Dye (Stably
Transfected Cells)
[0129] This example demonstrates that activation of a GPCR (Gs
receptor subtype) can be detected with an intracellular calcium
indicator in stably transfected cells, which is an example of a
signal transduction detection system based on changes in
intracellular ions. The cells used were transiently stably with two
constructs. Although many cells do not tolerate stable expression
of promiscuous G.alpha. protein, such as described herein,
surprisingly even cells thought not to tolerate stable expression
of promiscuous G.alpha. protein can be sorted using a signal
transduction detection system. Such sorting can be performed with a
high throughput sorting system, such as a FACS or 96 well imaging
system. Typically, the frequency of usable stable cells is about 1
to 2 percent of those cells screened. Functional assay selection of
promiscuous G.alpha. protein/GPCR double transfected cells is a
preferred method of identifying cells that either tolerate, or
express the proper amounts, of promiscuous G.alpha. protein and a
GPCR.
[0130] Stable CHO-K1 cell lines expressing G.alpha. 15-Hyg alone,
the Gs-receptor-Neo alone and both the G.alpha.15 (CMV promoter)
and the Gs receptor (CMV promoter) (double transfection), were
generated. 48 h after transfection (described herein for the method
of lipofectamine-mediated transfection), media containing
Hygromycin (0.5 mg/ml), Neomycin (1 mg/ml) or both were added on to
the cells to select the stable transformants. 12-15 days after
selection, the stable clones were examined using the calcium
imaging assays.
[0131] The following imaging data show that the promiscuous
G.alpha.15 couples a Gs-receptor in CHO-K1 cells following stable
cell line generation. The data also show that promiscuous G.alpha.
protein can change the effector downstream of the GPCR Panel A:
Calcium imaging of the G.alpha.15/Gs-receptor dual stable clone-2.
10 .mu.M agonist was added 40 seconds after the starting of the
experiment. Panel B: Calcium imaging of the Gs-receptor stable
clone-2. 10 .mu.M agonist was added 40 seconds after the starting
of the experiment. Panel C: Calcium imaging of the G.alpha.5 stable
clone-H. 10 .mu.M agonist was added 40 seconds after the starting
of the experiment.
[0132] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
REFERENCES
[0133] Bram, R. J. and G. R. Crabtree. (1994) Nature 371:355-358.
[0134] Offermanns, S. and M. I. Simon. (1995) J. Biol. Chem.
270(25): 15175-15180. [0135] Fiering, S., Northrop, J. P., Nolan,
G. P., Mattila, P. S., Crabtree, G. R., and Herzenberg, L. A.
(1990) Genes Dev. 4, 1823-1834. [0136] Flanagan, W. F., Corthesy,
B., Bram, R. J. and Crabtree, G. R. (1991) Nature 352:803-807.
[0137] Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen,
W., and H. Bujard (1995) Science 268: 1766-1769. [0138]
Grinkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem.
260:3440-3450. [0139] Neer, E. J. (1995). Cell, 80:249-257. [0140]
Negulescu, P. A., Shastri, N., Cahalan, Michael D. (1994). Proc.
Nat. Acad Sci. 91:2873-2877. [0141] Sternweis, P. C. and A. V.
Smrcka (1992) Trends Biochem. Sci. 17:502-506. [0142] Thastrup, O.,
Cullen, P. J., Drobak, B. K., Hanley, M. R., and Dawson, A. P.
(1990) Proc. Natl. Acad. Sci. 87:2466-2470. [0143] Tsien, R. Y.,
Backsai, B. J., and Adams, S. R. (1993) Trends Cell Biol.
3:242-245.
[0144] All publications, including patent documents and scientific
articles, referred to in this application are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication were individually incorporated by
reference.
Sequence ID Listing
[0145] Nucleotide and Amino Acid Sequences of G.alpha.15
[0146] (SEQ ID NO: 2 and SEQ ID NO: 4, respectively) TABLE-US-00004
9 18 27 36 45 54 ATG GCC CGG TCC CTG ACT TGG GGC TGC TGT CCC TGG
TGC CTG ACA GAG GAG GAG --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- Met Ala Arg Ser Leu Thr Trp Gly Cys Cys
Pro Trp Cys Leu Thr Glu Glu Glu 63 72 81 90 99 108 AAG ACT GCC GCC
AGA ATC GAC CAG GAG ATC AAC AGG ATT TTG TTG GAA CAG AAA --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Lys Thr
Ala Ala Arg Ile Asp Gln Glu Ile Asn Arg Ile Leu Leu Glu Gln Lys 117
126 135 144 153 162 AAA CAA GAG CGC GAG GAA TTG AAA CTC CTG CTG TTG
GGG CCT GGT GAG AGC GGG --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- Lys Gln Glu Arg Glu Glu Leu Lys Leu Leu
Leu Leu Gly Pro Gly Glu Ser Gly 171 180 189 198 207 216 AAG AGT ACG
TTC ATC AAG CAG ATG CGC ATC ATT CAC GGT GTG GGC TAC TCG GAG --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Lys
Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly Val Gly Tyr Ser Glu
225 234 243 252 261 270 GAG GAC CGC AGA GCC TTC CGG CTG CTC ATC TAC
CAG AAC ATC TTC GTC TCC ATG --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- Glu Asp Arg Arg Ala Phe Arg Leu Leu
Ile Tyr Gln Asn Ile Phe Val Ser Met 279 288 297 306 315 324 CAG GCC
ATG ATA GAT GCG ATG GAC CGG CTG CAG ATC CCC TTC AGC AGG CCT GAC ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
Gln Ala Met Ile Asp Ala Met Asp Arg Leu Gln Ile Pro Phe Ser Arg Pro
Asp 333 342 351 360 369 378 AGC AAG CAG CAC GCC AGC CTA GTG ATG ACC
CAG GAC CCC TAT AAA GTG AGC ACA --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- Ser Lys Gln His Ala Ser Leu Val
Met Thr Gln Asp Pro Tyr Lys Val Ser Thr 387 396 405 414 423 432 TTC
GAG AAG CCA TAT GCA GTG GCC ATG CAG TAC CTG TGG CGG GAC GCG GGC ATC
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- Phe Glu Lys Pro Tyr Ala Val Ala Met Gln Tyr Leu Trp Arg Asp Ala
Gly Ile 441 450 459 468 477 486 CGT GCA TGC TAC GAG CGA AGG CGT GAA
TTC CAC CTT CTG GAC TCC GCG GTG TAT --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- Arg Ala Cys Tyr Glu Arg Arg
Arg Glu Phe His Leu Leu Asp Ser Ala Val Tyr 495 504 513 522 531 540
TAC CTG TCA CAC CTG GAG CGC ATA TCA GAG GAC AGC TAC ATC CCC ACT GCG
CAA --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- Tyr Leu Ser His Leu Glu Arg Ile Ser Glu Asp Ser Tyr Ile Pro
Thr Ala Gln 549 558 567 576 585 594 GAC GTG CTG CGC AGT CGC ATG CCC
ACC ACA GGC ATC AAT GAG TAC TGC TTC TCC --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- Asp Val Leu Arg Ser Arg
Met Pro Thr Thr Gly Ile Asn Glu Tyr Cys Phe Ser 603 612 621 630 639
648 GTG AAG AAA ACC AAA CTG CGC ATC GTG GAT GTT GGT GGC CAG AGG TCA
GAG CGT --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- Val Lys Lys Thr Lys Leu Arg Ile Val Asp Val Gly Gly Gln
Arg Ser Glu Arg 657 666 675 684 693 702 AGG AAA TGG ATT CAC TGT TTC
GAG AAC GTG ATT GCC CTC ATC TAC CTG GCC TCC --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- Arg Lys Trp Ile His
Cys Phe Glu Asn Val Ile Ala Leu Ile Tyr Leu Ala Ser 711 720 729 738
747 756 CTG AGC GAG TAT GAC CAG TGC CTA GAG GAG AAC GAT CAG GAG AAC
CGC ATG GAG --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- Leu Ser Glu Tyr Asp Gln Cys Leu Glu Glu Asn Asp Gln
Glu Asn Arg Met Glu 765 774 783 792 801 810 GAG AGT CTC GCT CTG TTC
AGC ACG ATC CTA GAG CTG CCC TGG rrC AAG AGC ACC --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- Glu Ser Leu Ala
Leu Phe Ser Thr Ile Leu Glu Leu Pro Trp Phe Lys Ser Thr 819 828 837
846 855 864 TCG GTC ATC CTC TTC CTC AAC AAG ACG GAC ATC CTG GAA GAT
AAG ATT CAC ACC --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- Ser Val Ile Leu Phe Leu Asn Lys Thr Asp Ile Leu
Glu Asp Lys Ile His Thr 873 882 891 900 909 918 TCC CAC CTG GCC ACA
TAC TTC CCC AGC TTC CAG GGA CCC CGG CGA GAC GCA GAG --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- Ser His Leu
Ala Thr Tyr Phe Pro Ser Phe Gln Gly Pro Arg Arg Asp Ala Glu 927 936
945 954 963 972 GCC GCC AAG AGC TTC ATC TTG GAC ATG TAT GCG CGC GTG
TAC GCG AGC TGC GCA --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- Ala Ala Lys Ser Phe Ile Leu Asp Met Tyr Ala
Arg Val Tyr Ala Ser Cys Ala 981 990 999 1008 1017 1026 GAG CCC CAG
GAC GGT GGC AGG AAA GGC TCC CGC GCG CGC CGC TTC TTC GCA CAC --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Glu
Pro Gln Asp Gly Gly Arg Lys Gly Ser Arg Ala Arg Arg Phe Phe Ala His
1035 1044 1053 1062 1071 1080 TTC ACC TGT GCC ACG GAC ACG CAA AGC
GTC CGC AGC GTG TTC AAG GAC GTG CGG --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- Phe Thr Cys Ala Thr Asp Thr
Gln Ser Val Arg Ser Val Phe Lys Asp Val Arg 1089 1098 1107 1116
1125 GAC TCG GTG CTG GCC CGG TAC CTG GAC GAG ATC AAC CTG CTG TGA
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Asp Ser
Val Leu Ala Arg Tyr Leu Asp Glu Ile Asn Leu Leu ***
[0147] Nucleotide and Amino Acid Sequences of G 16
[0148] (SEQ ID NO: 1 and SEQ ID NO: 3, respectively) TABLE-US-00005
9 18 27 36 45 54 ATG GCC CGC TCG CTG ACC TGG CGC TGC TGC CCC TGG
TGC CTG ACG GAG GAT GAG --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- Met Ala Arg Ser Leu Thr Trp Arg Cys Cys
Pro Trp Cys Leu Thr Glu Asp Glu 63 72 81 90 99 108 AAG GCC GCC GCC
CGG GTG GAC CAG GAG ATC AAC AGG ATC CTC TTG GAG CAG AAG --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Lys Ala
Ala Ala Arg Val Asp Gln Glu Ile Asn Arg Ile Leu Leu Glu Gln Lys 117
126 135 144 153 162 AAG CAG GAC CGC GGG GAG CTG AAG CTG CTG CTT TTG
GGC CCA GGC GAG AGC GGG --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- Lys Gln Asp Arg Gly Glu Leu Lys Leu Leu
Leu Leu Gly Pro Gly Glu Ser Gly 171 180 189 198 207 216 AAG AGC ACC
TTC ATC AAG CAG ATG CGG ATC ATC CAC GGC GCC GGC TAC TCG GAG --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Lys
Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly Ala Gly Tyr Ser Glu
225 234 243 252 261 270 GAG GAG CGC AAG GGC TTC CGG CCC CTG GTC TAC
CAG AAC ATC TTC GTG TCC ATG --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- Glu Glu Arg Lys Gly Phe Arg Pro Leu
Val Tyr Gln Asn Ile Phe Val Ser Met 279 288 297 306 315 324 CGG GCC
ATG ATC GAG GCC ATG GAG CGG CTG CAG ATT CCA TTC AGC AGG CCC GAG ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
Arg Ala Met Ile Glu Ala Met Glu Arg Leu Gln Ile Pro Phe Ser Arg Pro
Glu 333 342 351 360 369 378 AGC AAG CAC CAC GCT AGC CTG GTC ATG AGC
CAG GAC CCC TAT AAA GTG ACC ACG --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- Ser Lys His His Ala Ser Leu Val
Met Ser Gln Asp Pro Tyr Lys Val Thr Thr 387 396 405 414 423 432 TTT
GAG AAG CGC TAC GCT GCG 0CC ATG CAG TGG CTG TGG AGG GAT GCC GGC ATC
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- Phe Glu Lys Arg Tyr Ala Ala Ala Met Gln Trp Leu Trp Arg Asp Ala
Gly Ile 441 450 459 468 477 486 CGG GCC TGC TAT GAG CGT CGG CGG GAA
TTC CAC CTG CTC GAT TCA GCC GTG TAC --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- Arg Ala Cys Tyr Glu Arg Arg
Arg Glu Phe His Leu Leu Asp Ser Ala Val Tyr 495 504 513 522 531 540
TAC CTG TCC CAC CTG GAG CGC ATC ACC GAG GAG GGC TAC GTC CCC ACA GCT
CAG --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- Tyr Leu Ser His Leu Glu Arg Ile Thr Glu Glu Gly Tyr Val Pro
Thr Ala Gln 549 558 567 576 585 594 GAC GTG CTC CGC AGC CGC ATG CCC
ACC ACT GGC ATC AAC GAG TAC TGC TTC TCC --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- Asp Val Leu Arg Ser Arg
Met Pro Thr Thr Gly Ile Asn Glu Tyr Cys Phe Ser 603 612 621 630 639
648 GTG CAG AAA ACC AAC CTG CGG ATC GTG GAC GTC 000 GGC CAG AAG TCA
GAG CGT --- --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- Val Gln Lys Thr Asn Leu Arg Ile Val Asp Val Gly Gly Gln
Lys Ser Glu Arg 657 666 675 684 693 702 AAG AAA TGG ATC CAT TGT TTC
GAG AAC GTG ATC GCC CTC ATC TAC CTG GCC TCA --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- Lys Lys Trp Ile His
Cys Phe Glu Asn Val Ile Ala Leu Ile Tyr Leu Ala Ser 711 720 729 738
747 756 CTG AGT GAA TAC GAC CAG TGC CTG GAG GAG AAC AAC CAG GAG AAC
CGC ATG AAG --- --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- Leu Ser Glu Tyr Asp Gln Cys Leu Glu Glu Asn Asn Gln
Glu Asn Arg Met Lys 765 774 783 792 801 810 GAG AGC CTC GCA TTG TTT
GGG ACT ATG CTG GAA CTA CCC TGG TTC AAA AGC ACA --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- Glu Ser Leu Ala
Leu Phe Gly Thr Ile Leu Glu Leu Pro Trp Phe Lys Ser Thr 819 828 837
846 855 864 TGC GTC ATC CTC TTT CTC AAC AAA ACC GAC ATC CTG GAG GAG
AAA ATC CCC ACC --- --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- Ser Val Ile Leu Phe Leu Asn Lys Thr Asp Ile Leu
Glu Glu Lys Ile Pro Thr 873 882 891 900 909 918 TCC CAC CTG GCT ACC
TAT TTC CCC AGT TTC CAG GGC CCT AAG CAG GAT GCT GAG --- --- --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- Ser His Leu
Ala Thr Tyr Phe Pro Ser Phe Gln Gly Pro Lys Gln Asp Ala Glu 927 936
945 954 963 972 GCA GCC AAG AGG TTC ATC CTG GAC ATG TAC ACG AGG ATG
TAC ACC GGG TGC GTG --- --- --- --- --- --- --- --- --- --- --- ---
--- --- --- --- --- --- Ala Ala Lys Arg Phe Ile Leu Asp Met Tyr Thr
Arg Met Tyr Thr Gly Cys Val 981 990 999 1008 1017 1026 GAC GGC CCC
GAG GGC AGC AAG AAG GGC GCA CGA TCC CGA CGC CTT TTC AGC CAC --- ---
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Asp
Gly Pro Glu Gly Ser Lys Lys Gly Ala Arg Ser Arg Arg Leu Phe Ser His
1035 1044 1053 1062 1071 1080 TAC ACA TGT GCC ACA GAC ACA CAG AAC
ATC CGC AAG GTC TTC AAG GAC GTG CGG --- --- --- --- --- --- --- ---
--- --- --- --- --- --- --- --- --- --- Tyr Thr Cys Ala Thr Asp Thr
Gln Asn Ile Arg Lys Val Phe Lys Asp Val Arg 1089 1098 1107 1116
1125 GAC TCG GTG CTC GCC CGC TAC CTG GAC GAG ATC AAC CTG CTG TGA
--- --- --- --- --- --- --- --- --- --- --- --- --- --- --- Asp Ser
Val Leu Ala Arg Tyr Leu Asp Glu Ile Asn Leu Leu ***
[0149]
Sequence CWU 1
1
4 1 1125 DNA Mus musculus CDS (1)...(1122) 1 atg gcc cgc tcg ctg
acc tgg cgc tgc tgc ccc tgg tgc ctg acg gag 48 Met Ala Arg Ser Leu
Thr Trp Arg Cys Cys Pro Trp Cys Leu Thr Glu 1 5 10 15 gat gag aag
gcc gcc gcc cgg gtg gac cag gag atc aac agg atc ctc 96 Asp Glu Lys
Ala Ala Ala Arg Val Asp Gln Glu Ile Asn Arg Ile Leu 20 25 30 ttg
gag cag aag aag cag gac cgc ggg gag ctg aag ctg ctg ctt ttg 144 Leu
Glu Gln Lys Lys Gln Asp Arg Gly Glu Leu Lys Leu Leu Leu Leu 35 40
45 ggc cca ggc gag agc ggg aag agc acc ttc atc aag cag atg cgg atc
192 Gly Pro Gly Glu Ser Gly Lys Ser Thr Phe Ile Lys Gln Met Arg Ile
50 55 60 atc cac ggc gcc ggc tac tcg gag gag gag cgc aag ggc ttc
cgg ccc 240 Ile His Gly Ala Gly Tyr Ser Glu Glu Glu Arg Lys Gly Phe
Arg Pro 65 70 75 80 ctg gtc tac cag aac atc ttc gtg tcc atg cgg gcc
atg atc gag gcc 288 Leu Val Tyr Gln Asn Ile Phe Val Ser Met Arg Ala
Met Ile Glu Ala 85 90 95 atg gag cgg ctg cag att cca ttc agc agg
ccc gag agc aag cac cac 336 Met Glu Arg Leu Gln Ile Pro Phe Ser Arg
Pro Glu Ser Lys His His 100 105 110 gct agc ctg gtc atg agc cag gac
ccc tat aaa gtg acc acg ttt gag 384 Ala Ser Leu Val Met Ser Gln Asp
Pro Tyr Lys Val Thr Thr Phe Glu 115 120 125 aag cgc tac gct gcg gcc
atg cag tgg ctg tgg agg gat gcc ggc atc 432 Lys Arg Tyr Ala Ala Ala
Met Gln Trp Leu Trp Arg Asp Ala Gly Ile 130 135 140 cgg gcc tgc tat
gag cgt cgg cgg gaa ttc cac ctg ctc gat tca gcc 480 Arg Ala Cys Tyr
Glu Arg Arg Arg Glu Phe His Leu Leu Asp Ser Ala 145 150 155 160 gtg
tac tac ctg tcc cac ctg gag cgc atc acc gag gag ggc tac gtc 528 Val
Tyr Tyr Leu Ser His Leu Glu Arg Ile Thr Glu Glu Gly Tyr Val 165 170
175 ccc aca gct cag gac gtg ctc cgc agc cgc atg ccc acc act ggc atc
576 Pro Thr Ala Gln Asp Val Leu Arg Ser Arg Met Pro Thr Thr Gly Ile
180 185 190 aac gag tac tgc ttc tcc gtg cag aaa acc aac ctg cgg atc
gtg gac 624 Asn Glu Tyr Cys Phe Ser Val Gln Lys Thr Asn Leu Arg Ile
Val Asp 195 200 205 gtc ggg ggc cag aag tca gag cgt aag aaa tgg atc
cat tgt ttc gag 672 Val Gly Gly Gln Lys Ser Glu Arg Lys Lys Trp Ile
His Cys Phe Glu 210 215 220 aac gtg atc gcc ctc atc tac ctg gcc tca
ctg agt gaa tac gac cag 720 Asn Val Ile Ala Leu Ile Tyr Leu Ala Ser
Leu Ser Glu Tyr Asp Gln 225 230 235 240 tgc ctg gag gag aac aac cag
gag aac cgc atg aag gag agc ctc gca 768 Cys Leu Glu Glu Asn Asn Gln
Glu Asn Arg Met Lys Glu Ser Leu Ala 245 250 255 ttg ttt ggg act atc
ctg gaa cta ccc tgg ttc aaa agc aca tcc gtc 816 Leu Phe Gly Thr Ile
Leu Glu Leu Pro Trp Phe Lys Ser Thr Ser Val 260 265 270 atc ctc ttt
ctc aac aaa acc gac atc ctg gag gag aaa atc ccc acc 864 Ile Leu Phe
Leu Asn Lys Thr Asp Ile Leu Glu Glu Lys Ile Pro Thr 275 280 285 tcc
cac ctg gct acc tat ttc ccc agt ttc cag ggc cct aag cag gat 912 Ser
His Leu Ala Thr Tyr Phe Pro Ser Phe Gln Gly Pro Lys Gln Asp 290 295
300 gct gag gca gcc aag agg ttc atc ctg gac atg tac acg agg atg tac
960 Ala Glu Ala Ala Lys Arg Phe Ile Leu Asp Met Tyr Thr Arg Met Tyr
305 310 315 320 acc ggg tgc gtg gac ggc ccc gag ggc agc aag aag ggc
gca cga tcc 1008 Thr Gly Cys Val Asp Gly Pro Glu Gly Ser Lys Lys
Gly Ala Arg Ser 325 330 335 cga cgc ctt ttc agc cac tac aca tgt gcc
aca gac aca cag aac atc 1056 Arg Arg Leu Phe Ser His Tyr Thr Cys
Ala Thr Asp Thr Gln Asn Ile 340 345 350 cgc aag gtc ttc aag gac gtg
cgg gac tcg gtg ctc gcc cgc tac ctg 1104 Arg Lys Val Phe Lys Asp
Val Arg Asp Ser Val Leu Ala Arg Tyr Leu 355 360 365 gac gag atc aac
ctg ctg tga 1125 Asp Glu Ile Asn Leu Leu 370 2 374 PRT Mus musculus
2 Met Ala Arg Ser Leu Thr Trp Arg Cys Cys Pro Trp Cys Leu Thr Glu 1
5 10 15 Asp Glu Lys Ala Ala Ala Arg Val Asp Gln Glu Ile Asn Arg Ile
Leu 20 25 30 Leu Glu Gln Lys Lys Gln Asp Arg Gly Glu Leu Lys Leu
Leu Leu Leu 35 40 45 Gly Pro Gly Glu Ser Gly Lys Ser Thr Phe Ile
Lys Gln Met Arg Ile 50 55 60 Ile His Gly Ala Gly Tyr Ser Glu Glu
Glu Arg Lys Gly Phe Arg Pro 65 70 75 80 Leu Val Tyr Gln Asn Ile Phe
Val Ser Met Arg Ala Met Ile Glu Ala 85 90 95 Met Glu Arg Leu Gln
Ile Pro Phe Ser Arg Pro Glu Ser Lys His His 100 105 110 Ala Ser Leu
Val Met Ser Gln Asp Pro Tyr Lys Val Thr Thr Phe Glu 115 120 125 Lys
Arg Tyr Ala Ala Ala Met Gln Trp Leu Trp Arg Asp Ala Gly Ile 130 135
140 Arg Ala Cys Tyr Glu Arg Arg Arg Glu Phe His Leu Leu Asp Ser Ala
145 150 155 160 Val Tyr Tyr Leu Ser His Leu Glu Arg Ile Thr Glu Glu
Gly Tyr Val 165 170 175 Pro Thr Ala Gln Asp Val Leu Arg Ser Arg Met
Pro Thr Thr Gly Ile 180 185 190 Asn Glu Tyr Cys Phe Ser Val Gln Lys
Thr Asn Leu Arg Ile Val Asp 195 200 205 Val Gly Gly Gln Lys Ser Glu
Arg Lys Lys Trp Ile His Cys Phe Glu 210 215 220 Asn Val Ile Ala Leu
Ile Tyr Leu Ala Ser Leu Ser Glu Tyr Asp Gln 225 230 235 240 Cys Leu
Glu Glu Asn Asn Gln Glu Asn Arg Met Lys Glu Ser Leu Ala 245 250 255
Leu Phe Gly Thr Ile Leu Glu Leu Pro Trp Phe Lys Ser Thr Ser Val 260
265 270 Ile Leu Phe Leu Asn Lys Thr Asp Ile Leu Glu Glu Lys Ile Pro
Thr 275 280 285 Ser His Leu Ala Thr Tyr Phe Pro Ser Phe Gln Gly Pro
Lys Gln Asp 290 295 300 Ala Glu Ala Ala Lys Arg Phe Ile Leu Asp Met
Tyr Thr Arg Met Tyr 305 310 315 320 Thr Gly Cys Val Asp Gly Pro Glu
Gly Ser Lys Lys Gly Ala Arg Ser 325 330 335 Arg Arg Leu Phe Ser His
Tyr Thr Cys Ala Thr Asp Thr Gln Asn Ile 340 345 350 Arg Lys Val Phe
Lys Asp Val Arg Asp Ser Val Leu Ala Arg Tyr Leu 355 360 365 Asp Glu
Ile Asn Leu Leu 370 3 1125 DNA Homo sapiens CDS (1)...(1122) 3 atg
gcc cgg tcc ctg act tgg ggc tgc tgt ccc tgg tgc ctg aca gag 48 Met
Ala Arg Ser Leu Thr Trp Gly Cys Cys Pro Trp Cys Leu Thr Glu 1 5 10
15 gag gag aag act gcc gcc aga atc gac cag gag atc aac agg att ttg
96 Glu Glu Lys Thr Ala Ala Arg Ile Asp Gln Glu Ile Asn Arg Ile Leu
20 25 30 ttg gaa cag aaa aaa caa gag cgc gag gaa ttg aaa ctc ctg
ctg ttg 144 Leu Glu Gln Lys Lys Gln Glu Arg Glu Glu Leu Lys Leu Leu
Leu Leu 35 40 45 ggg cct ggt gag agc ggg aag agt acg ttc atc aag
cag atg cgc atc 192 Gly Pro Gly Glu Ser Gly Lys Ser Thr Phe Ile Lys
Gln Met Arg Ile 50 55 60 att cac ggt gtg ggc tac tcg gag gag gac
cgc aga gcc ttc cgg ctg 240 Ile His Gly Val Gly Tyr Ser Glu Glu Asp
Arg Arg Ala Phe Arg Leu 65 70 75 80 ctc atc tac cag aac atc ttc gtc
tcc atg cag gcc atg ata gat gcg 288 Leu Ile Tyr Gln Asn Ile Phe Val
Ser Met Gln Ala Met Ile Asp Ala 85 90 95 atg gac cgg ctg cag atc
ccc ttc agc agg cct gac agc aag cag cac 336 Met Asp Arg Leu Gln Ile
Pro Phe Ser Arg Pro Asp Ser Lys Gln His 100 105 110 gcc agc cta gtg
atg acc cag gac ccc tat aaa gtg agc aca ttc gag 384 Ala Ser Leu Val
Met Thr Gln Asp Pro Tyr Lys Val Ser Thr Phe Glu 115 120 125 aag cca
tat gca gtg gcc atg cag tac ctg tgg cgg gac gcg ggc atc 432 Lys Pro
Tyr Ala Val Ala Met Gln Tyr Leu Trp Arg Asp Ala Gly Ile 130 135 140
cgt gca tgc tac gag cga agg cgt gaa ttc cac ctt ctg gac tcc gcg 480
Arg Ala Cys Tyr Glu Arg Arg Arg Glu Phe His Leu Leu Asp Ser Ala 145
150 155 160 gtg tat tac ctg tca cac ctg gag cgc ata tca gag gac agc
tac atc 528 Val Tyr Tyr Leu Ser His Leu Glu Arg Ile Ser Glu Asp Ser
Tyr Ile 165 170 175 ccc act gcg caa gac gtg ctg cgc agt cgc atg ccc
acc aca ggc atc 576 Pro Thr Ala Gln Asp Val Leu Arg Ser Arg Met Pro
Thr Thr Gly Ile 180 185 190 aat gag tac tgc ttc tcc gtg aag aaa acc
aaa ctg cgc atc gtg gat 624 Asn Glu Tyr Cys Phe Ser Val Lys Lys Thr
Lys Leu Arg Ile Val Asp 195 200 205 gtt ggt ggc cag agg tca gag cgt
agg aaa tgg att cac tgt ttc gag 672 Val Gly Gly Gln Arg Ser Glu Arg
Arg Lys Trp Ile His Cys Phe Glu 210 215 220 aac gtg att gcc ctc atc
tac ctg gcc tcc ctg agc gag tat gac cag 720 Asn Val Ile Ala Leu Ile
Tyr Leu Ala Ser Leu Ser Glu Tyr Asp Gln 225 230 235 240 tgc cta gag
gag aac gat cag gag aac cgc atg gag gag agt ctc gct 768 Cys Leu Glu
Glu Asn Asp Gln Glu Asn Arg Met Glu Glu Ser Leu Ala 245 250 255 ctg
ttc agc acg atc cta gag ctg ccc tgg ttc aag agc acc tcg gtc 816 Leu
Phe Ser Thr Ile Leu Glu Leu Pro Trp Phe Lys Ser Thr Ser Val 260 265
270 atc ctc ttc ctc aac aag acg gac atc ctg gaa gat aag att cac acc
864 Ile Leu Phe Leu Asn Lys Thr Asp Ile Leu Glu Asp Lys Ile His Thr
275 280 285 tcc cac ctg gcc aca tac ttc ccc agc ttc cag gga ccc cgg
cga gac 912 Ser His Leu Ala Thr Tyr Phe Pro Ser Phe Gln Gly Pro Arg
Arg Asp 290 295 300 gca gag gcc gcc aag agc ttc atc ttg gac atg tat
gcg cgc gtg tac 960 Ala Glu Ala Ala Lys Ser Phe Ile Leu Asp Met Tyr
Ala Arg Val Tyr 305 310 315 320 gcg agc tgc gca gag ccc cag gac ggt
ggc agg aaa ggc tcc cgc gcg 1008 Ala Ser Cys Ala Glu Pro Gln Asp
Gly Gly Arg Lys Gly Ser Arg Ala 325 330 335 cgc cgc ttc ttc gca cac
ttc acc tgt gcc acg gac acg caa agc gtc 1056 Arg Arg Phe Phe Ala
His Phe Thr Cys Ala Thr Asp Thr Gln Ser Val 340 345 350 cgc agc gtg
ttc aag gac gtg cgg gac tcg gtg ctg gcc cgg tac ctg 1104 Arg Ser
Val Phe Lys Asp Val Arg Asp Ser Val Leu Ala Arg Tyr Leu 355 360 365
gac gag atc aac ctg ctg tga 1125 Asp Glu Ile Asn Leu Leu 370 4 374
PRT Homo sapiens 4 Met Ala Arg Ser Leu Thr Trp Gly Cys Cys Pro Trp
Cys Leu Thr Glu 1 5 10 15 Glu Glu Lys Thr Ala Ala Arg Ile Asp Gln
Glu Ile Asn Arg Ile Leu 20 25 30 Leu Glu Gln Lys Lys Gln Glu Arg
Glu Glu Leu Lys Leu Leu Leu Leu 35 40 45 Gly Pro Gly Glu Ser Gly
Lys Ser Thr Phe Ile Lys Gln Met Arg Ile 50 55 60 Ile His Gly Val
Gly Tyr Ser Glu Glu Asp Arg Arg Ala Phe Arg Leu 65 70 75 80 Leu Ile
Tyr Gln Asn Ile Phe Val Ser Met Gln Ala Met Ile Asp Ala 85 90 95
Met Asp Arg Leu Gln Ile Pro Phe Ser Arg Pro Asp Ser Lys Gln His 100
105 110 Ala Ser Leu Val Met Thr Gln Asp Pro Tyr Lys Val Ser Thr Phe
Glu 115 120 125 Lys Pro Tyr Ala Val Ala Met Gln Tyr Leu Trp Arg Asp
Ala Gly Ile 130 135 140 Arg Ala Cys Tyr Glu Arg Arg Arg Glu Phe His
Leu Leu Asp Ser Ala 145 150 155 160 Val Tyr Tyr Leu Ser His Leu Glu
Arg Ile Ser Glu Asp Ser Tyr Ile 165 170 175 Pro Thr Ala Gln Asp Val
Leu Arg Ser Arg Met Pro Thr Thr Gly Ile 180 185 190 Asn Glu Tyr Cys
Phe Ser Val Lys Lys Thr Lys Leu Arg Ile Val Asp 195 200 205 Val Gly
Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 210 215 220
Asn Val Ile Ala Leu Ile Tyr Leu Ala Ser Leu Ser Glu Tyr Asp Gln 225
230 235 240 Cys Leu Glu Glu Asn Asp Gln Glu Asn Arg Met Glu Glu Ser
Leu Ala 245 250 255 Leu Phe Ser Thr Ile Leu Glu Leu Pro Trp Phe Lys
Ser Thr Ser Val 260 265 270 Ile Leu Phe Leu Asn Lys Thr Asp Ile Leu
Glu Asp Lys Ile His Thr 275 280 285 Ser His Leu Ala Thr Tyr Phe Pro
Ser Phe Gln Gly Pro Arg Arg Asp 290 295 300 Ala Glu Ala Ala Lys Ser
Phe Ile Leu Asp Met Tyr Ala Arg Val Tyr 305 310 315 320 Ala Ser Cys
Ala Glu Pro Gln Asp Gly Gly Arg Lys Gly Ser Arg Ala 325 330 335 Arg
Arg Phe Phe Ala His Phe Thr Cys Ala Thr Asp Thr Gln Ser Val 340 345
350 Arg Ser Val Phe Lys Asp Val Arg Asp Ser Val Leu Ala Arg Tyr Leu
355 360 365 Asp Glu Ile Asn Leu Leu 370
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