U.S. patent application number 10/989054 was filed with the patent office on 2005-07-14 for novel rgs-containing molecules and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Hodge, Martin R., Yowe, David.
Application Number | 20050153414 10/989054 |
Document ID | / |
Family ID | 22922236 |
Filed Date | 2005-07-14 |
United States Patent
Application |
20050153414 |
Kind Code |
A1 |
Hodge, Martin R. ; et
al. |
July 14, 2005 |
Novel RGS-containing molecules and uses thereof
Abstract
Novel RGS polypeptides, proteins, and nucleic acid molecules are
disclosed. In addition to isolated, full-length RGS proteins, the
invention further provides isolated RGS fusion proteins, antigenic
peptides, and anti-RGS antibodies. The invention also provides RGS
nucleic acid molecules, recombinant expression vectors containing a
nucleic acid molecule of the invention, host cells into which the
expression vectors have been introduced, and nonhuman transgenic
animals in which an RGS gene has been introduced or disrupted.
Diagnostic, screening, and therapeutic methods utilizing
compositions of the invention are also provided.
Inventors: |
Hodge, Martin R.;
(Arlington, MA) ; Yowe, David; (North Quincy,
MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
22922236 |
Appl. No.: |
10/989054 |
Filed: |
November 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10989054 |
Nov 15, 2004 |
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09894749 |
Jun 27, 2001 |
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6830914 |
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09894749 |
Jun 27, 2001 |
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09244314 |
Feb 4, 1999 |
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6274362 |
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Current U.S.
Class: |
435/196 |
Current CPC
Class: |
C07K 2319/00 20130101;
Y02A 50/30 20180101; C07K 14/4702 20130101; A61K 38/00 20130101;
Y02A 50/411 20180101 |
Class at
Publication: |
435/196 |
International
Class: |
C12Q 001/68; C12N
009/16 |
Claims
That which is claimed:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2
or SEQ ID NO:4; b) a polypeptide comprising an amino acid sequence
encoded by the cDNA insert of the plasmid deposited with ATCC as
Accession Number 207048 or an amino acid sequence encoded by the
DNA sequence obtained from the overlapping clones deposited with
ATCC as Accession 207049 and 207050; c) a polypeptide having RGS
activity, wherein the polypeptide comprises a fragment of the amino
acid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein the fragment
comprises at least 30 contiguous amino acids of SEQ ID NO:2 or SEQ
ID NO:4; d) a polypeptide having RGS activity, wherein the
polypeptide is encoded by a nucleic acid molecule comprising a
nucleotide sequence that is at least 75% identical to the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, nucleotides
160-864 of SEQ ID NO:1, nucleotides 134-838 of SEQ ID NO:3, or a
complement thereof; e) a polypeptide having RGS activity, wherein
the polypeptide is encoded by a nucleic acid molecule that
hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, SEQ
ID NO:3, nucleotides 160-864 of SEQ ID NO:1, nucleotides 134-838 of
SEQ ID NO:3, or a complement thereof under stringent conditions,
said stringent conditions comprising hybridization in 6.times. SSC
at 42.degree. C., followed by washing with 1.times.SSC at
55.degree. C.; and e) a polypeptide having RGS activity, wherein
the polypeptide is at least 75% identical to the amino acid
sequence of SEQ ID NO:2 or SEQ ID NO:4, an amino acid sequence
encoded by the cDNA insert of the plasmid deposited with ATCC as
Accession Number 207048, or an amino acid sequence encoded by the
DNA sequence obtained from the overlapping clones deposited with
ATCC as Accession 207049 and 207050.
2. The isolated polypeptide of claim 1 further comprising
heterologous amino acid sequences.
3. The isolated polypeptide of claim 2, wherein said polypeptide is
a fusion protein.
4. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:2 or SEQ ID NO:4, an amino acid sequence encoded by the
cDNA insert of the plasmid deposited with ATCC as Accession Number
207048, or an amino acid sequence encoded by the DNA sequence
obtained from the overlapping clones deposited with ATCC as
Accession 207049 and 207050.
5. The isolated polypeptide of claim 4 comprising the amino acid
sequence of SEQ ID NO:2 or SEQ ID NO:4.
6. The isolated polypeptide of claim 4 further comprising
heterologous amino acid sequences.
7. The isolated polypeptide of claim 6, wherein said polypeptide is
a fusion protein.
8. An isolated polypeptide having RGS activity, wherein the
polypeptide comprises a fragment of the amino acid sequence of SEQ
ID NO:2 or SEQ ID NO:4, wherein the fragment comprises at least 30
contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:4.
9. The isolated polypeptide of claim 8 further comprising
heterologous amino acid sequences.
10. The isolated polypeptide of claim 9, wherein said polypeptide
is a fusion protein.
11. An isolated polypeptide having RGS activity, wherein the
polypeptide is encoded by a nucleic acid molecule comprising a
nucleotide sequence that is at least 75% identical to the
nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, nucleotides
160-864 of SEQ ID NO:1, nucleotides 134-838 of SEQ ID NO:3, or a
complement thereof.
12. The isolated polypeptide of claim 11 further comprising
heterologous amino acid sequences.
13. The isolated polypeptide of claim 12, wherein said polypeptide
is a fusion protein.
14. An isolated polypeptide having RGS activity, wherein the
polypeptide is encoded by a nucleic acid molecule that hybridizes
to a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3,
nucleotides 160-864 of SEQ ID NO:1, nucleotides 134-838 of SEQ ID
NO:3, or a complement thereof under stringent conditions, said
stringent conditions comprising hybridization in 6.times. SSC at
42.degree. C., followed by washing with 1.times. SSC at 55.degree.
C.
15. The isolated polypeptide of claim 14 further comprising
heterologous amino acid sequences.
16. The isolated polypeptide of claim 15, wherein said polypeptide
is a fusion protein.
17. An isolated polypeptide having RGS activity, wherein the
polypeptide is at least 75% identical to the amino acid sequence of
SEQ ID NO:2 or SEQ ID NO:4, an amino acid sequence encoded by the
cDNA insert of the plasmid deposited with ATCC as Accession Number
207048, or an amino acid sequence encoded by the DNA sequence
obtained from the overlapping clones deposited with ATCC as
Accession 207049 and 207050.
18. The isolated polypeptide of claim 17 further comprising
heterologous amino acid sequences.
19. The isolated polypeptide of claim 18, wherein said polypeptide
is a fusion protein.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 09/244,314, filed Feb. 4, 1999, herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to novel RGS (regulators of G-protein
signaling) nucleic acids and proteins. Also provided are vectors,
host cells, and recombinant methods for making and using the novel
molecules.
BACKGROUND OF THE INVENTION
[0003] Regulators of G-protein signaling (RGS) accelerate guanosine
triphosphate (GTP) hydrolysis by G.sub.i, but not by G.sub.s class
.alpha.-subunits (Popov et al. (1997) Proc. Natl. Acad. Sci. USA
94: 7216-20). RGS proteins were first identified in genetic screens
in fungi and nematodes as negative regulators of G-protein
signaling (Dolhman et al. (1995) Mol. Cell. Biol. 15: 3635-43). RGS
proteins have been shown to-function as GTPase-activating proteins.
It has additionally been proposed that RGS proteins may act as
effector antagonists by occluding the effector-binding sites on
G-protein .alpha.-subunits (Helper et al. (1997) Proc. Natl. Acad.
Sci. USA 94: 428-432).
[0004] RGS has been implicated in a distinct molecular mechanism
with the potential to modulate G-protein responses. Proteins
containing the RGS domain can directly control aspects of G-protein
stimulated signaling pathways. RGS proteins appear to enhance the
endogenous GTPase activity of G-proteins, thus decreasing the
half-life of the active GTP-bound state and limiting the duration
of G.alpha..sub.i signaling.
[0005] The glucose-dependent insulinotropic peptide receptor
(GIP-R) is a member of the G-protein coupled receptors. GIP was
first isolated from porcine small intestine and was described as a
member of the secretin family of gastrointestinal regulatory
peptides (Tseng and Zhang (1998) Endocrin. 139: 4470-75). In the
presence of glucose, GIP is a potent stimulator of insulin release
by pancreatic islet .beta.-cells. GIP may represent an important
hormonal mediator in the entero-insular axis. Insulinotropic
properties of GIP in diabetic patients have been shown to be
diminished despite elevated serum levels of GIP. While the precise
mechanism for the decline in insulinotropic activity of GIP in
diabetic patients has not been defined, agonist-induced
desensitization of G-protein-coupled receptors is well documented
(Premont et al. (1995) FASEB J 9: 175-182).
[0006] Recently, an interaction of the G-protein with members of
RGS proteins has been demonstrated to mediate a desensitization
mechanism. RGS proteins act as GTPase activating proteins to
decrease the half-life of the activated G .alpha.-subunit (Koelle
et al. (1996) Cell 84: 115-125; Druey et al. (1996) Nature 379:
742-46).
[0007] Additionally, RGS proteins may be involved in cell
migration. Cell migration is a required behavior in the development
and maintenance of multicellular organisms. Generally, cells
migrate in response to various chemoattractants and chemorepellents
in the environment (Bowman et al. (1998) J. Biol. Chem. 273:
28040-48). Chemoattractants provide a directional signal to cells
leading to migration of the cells towards the source of the
chemoattractant (Butcher et al. (1996) Science 272: 60-66; Mackay,
C. R. (1996) J. Exp. Med. 184: 799-802). Chemoattractants also
direct the rapid, integrin-dependent adhesion of leukocytes to
various cell associated or extracellular proteins if the
corresponding chemoattractant receptor is expressed at high levels.
RGS proteins appear to be involved as most leukocyte
chemoattractants mediate their activity by binding and stimulating
specific Ga-coupled receptors.
[0008] RGS proteins constitute a family of proteins characterized
by an RGS domain. A number of RGS proteins have been identified and
several have been shown to function as GTPase-activating proteins
(Chatterjce et al. (1997) Genomics 45: 429-33). Identification of
other members of the RGS family are needed.
[0009] Because of the complexity of the immune response and
regulation of heterotrimeric G-protein signaling, additional
mechanisms are needed to modulate such functions. Additionally,
methods are needed to regulate an immune response, and provide
therapies for a range of diseases.
SUMMARY OF THE INVENTION
[0010] Isolated nucleic acid molecules corresponding to regulators
of G-protein signaling (RGS) nucleic acid sequences are provided.
Additionally amino acid sequences corresponding to the
polynucleotides are encompassed. In particular, the present
invention provides for isolated nucleic acid molecules comprising
nucleotide sequences encoding the amino acid sequences shown in SEQ
ID NOS:2 and 4 or the nucleotide sequences encoding the DNA
sequence deposited in a bacterial host as ATCC Accession Number
207048, or the DNA sequence obtained from the overlapping clones
deposited as ATCC Accession Numbers 207049 and 207050. By "DNA
sequence obtained from the overlapping clones" is intended that the
DNA sequence of the human sequence can be obtained by sequencing of
the two individual clones which together comprise the entire human
sequence. Further provided are RGS polypeptides having an amino
acid sequence encoded by a nucleic acid molecule described
herein.
[0011] The present invention also provides vectors and host cells
for recombinant expression of the nucleic acid molecules described
herein, as well as methods of making such vectors and host cells
and for using them for production of the polypeptides or peptides
of the invention by recombinant techniques.
[0012] The RGS molecules of the present invention are useful for
modulating the phenotype of immune and respiratory responses,
particularly for regulating an immune response. The molecules are
useful for the diagnosis and treatment of immune and respiratory
disorders, including, but not limited to, atopic conditions, such
as asthma and allergy, including allergic rhinitis, psoriasis, the
effects of pathogen infection, chronic inflammatory diseases,
organ-specific autoimmunity, graft rejection, and graft versus host
disease. Accordingly, in one aspect, this invention provides
isolated nucleic acid molecules encoding RGS proteins or
biologically active portions thereof, as well as nucleic acid
fragments suitable as primers or hybridization probes for the
detection of RGS-encoding nucleic acids.
[0013] Another aspect of this invention features isolated or
recombinant RGS proteins and polypeptides. Preferred RGS proteins
and polypeptides possess at least one biological activity possessed
by naturally occurring RGS proteins.
[0014] Variant nucleic acid molecules and polypeptides
substantially homologous to the nucleotide and amino acid sequences
set forth in the sequence listings are encompassed by the present
invention. Additionally, fragments and substantially homologous
fragments of the nucleotide and amino acid sequences are
provided.
[0015] Antibodies and antibody fragments that selectively bind the
RGS polypeptides and fragments are provided. Such antibodies are
useful in detecting the RGS polypeptides as well as in regulating
G-protein signaling.
[0016] In another aspect, the present invention provides a method
for detecting the presence of RGS activity or expression in a
biological sample by contacting the biological sample with an agent
capable of detecting an indicator of RGS activity such that the
presence of RGS activity is detected in the biological sample.
[0017] In yet another aspect, the invention provides a method for
modulating RGS activity comprising contacting a cell with an agent
that modulates (inhibits or stimulates) RGS activity or expression
such that RGS activity or expression in the cell is modulated. In
one embodiment, the agent is an antibody that specifically binds to
RGS protein. In another embodiment, the agent modulates expression
of RGS protein by modulating transcription of an RGS gene, splicing
of an RGS mRNA, or translation of an RGS mRNA. In yet another
embodiment, the agent is a nucleic acid molecule having a
nucleotide sequence that is antisense to the coding strand of the
RGS mRNA or the RGS gene.
[0018] In one embodiment, the methods of the present invention are
used to treat a subject having a disorder characterized by aberrant
RGS protein activity or nucleic acid expression by administering an
agent that is an RGS modulator to the subject. In one embodiment,
the RGS modulator is an RGS protein. In another embodiment, the RGS
modulator is an RGS nucleic acid molecule. In other embodiments,
the RGS modulator is a peptide, peptidomimetic, or other small
molecule.
[0019] The present invention also provides a diagnostic assay for
identifying the presence or absence of a genetic lesion or mutation
characterized by at least one of the following: (1) aberrant
modification or mutation of a gene encoding an RGS protein; (2)
misregulation of a gene encoding an RGS protein; and (3) aberrant
post-translational modification of an RGS protein, wherein a
wild-type form of the gene encodes a protein with an RGS
activity.
[0020] In another aspect, the invention provides a method for
identifying a compound that binds to or modulates the activity of
an RGS protein. In general, such methods entail measuring a
biological activity of an RGS protein in the presence and absence
of a test compound and identifying those compounds that alter the
activity of the RGS protein.
[0021] The invention also features methods for identifying a
compound that modulates the expression of RGS genes by measuring
the expression of the RGS sequences in the presence and absence of
the compound.
[0022] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 provides the amino sequences for the h16395 and m1975
proteins. FIGS. 1A and B provide the human and murine sequences,
respectively; the RGS domain is underlined. FIG. 1C provides the
alignment of the human sequence with the murine orthologue.
[0024] FIG. 2 shows the amino acid sequence alignment for the
proteins encoded by h16395 with human RGS2, RGS4, and RGS5. The RGS
proteins share closest homology to human RGS5 protein (about 44%
sequence identity for the human sequence) as compared to 38% and
39% for RGS2 and RGS4, respectively. The sequence identity was
determined by the Clustal method. The region of homology is
observed for the RGS domains, amino acids 82-201, with little
homology observed for the N-terminal, 1-81, and C-terminal,
202-235, amino acids.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides isolated nucleic acid
molecules comprising a nucleotide sequence encoding the RGS
polypeptides whose amino acid sequence is given in SEQ ID NO:2 or 4
respectively, or a variant or fragment of the polypeptide.
Nucleotide sequences encoding the RGS proteins of the invention are
set forth in SEQ ID NOS:1 and 3.
[0026] The present invention relates to methods and compositions
for the modulation, diagnosis, and treatment of immune and
respiratory disorders, especially RGS related disorders. Such
immune disorders include, but are not limited to, chronic
inflammatory diseases and disorders, such as Crohn's disease,
reactive arthritis, including Lyme disease, insulin-dependent
diabetes, organ-specific autoimmunity, including multiple
sclerosis, rheumatoid arthritis, inflammatory bowel disease,
Hashimoto's thyroiditis and Grave's disease, contact dermatitis,
psoriasis, graft rejection, graft versus host disease, sarcoidosis,
atopic conditions, such as asthma and allergy, including allergic
rhinitis, gastrointestinal allergies, including food allergies,
eosinophilia, conjunctivitis, glomerular nephritis, certain
pathogen susceptibilities such as helminthic (e.g., leishmaniasis),
certain viral infections, including HIV, and bacterial infections,
including tuberculosis and lepromatous leprosy.
[0027] Respiratory disorders include, but are not limited to,
apnea, asthma, particularly bronchial asthma, berillium disease,
bronchiectasis, bronchitis, bronchopneumonia, cystic fibrosis,
diphtheria, dyspnea, emphysema, chronic obstructive pulmonary
disease, allergic bronchopulmonary aspergillosis, pneumonia, acute
pulmonary edema, pertussis, pharyngitis, atelectasis, Wegener's
granulomatosis, Legionnaires disease, pleurisy, rheumatic fever,
and sinusitis.
[0028] Two novel genes, human clone h16395 and its corresponding
murine orthologue m1975, that are differentially expressed in
spleen and in various cells of hematopoietic origin are provided. A
Northern blot analysis of h16395 revealed expression in the
following tissues in order of highest to lowest expression:
peripheral blood leukocytes, spleen, liver, colon, placenta, and
heart. Expression of m1975 was greater in the spleen followed by
the heart. The sequences were detected in T-cells, monocytes, and
granulocytes by RT-PCR. 3'UTR probes were used to avoid
cross-hybridization with other RGS proteins. Such sequences are
referred to as "RGS" indicating that the genes encode an RGS
protein comprising an RGS domain.
[0029] The sequences of the invention find use in modulating an
immune response as well as other cellular activities. By
"modulating" is intended the upregulating or downregulating of a
response, particularly a G-protein-mediated signaling response.
[0030] The proteins in the RGS-containing protein family act to
inhibit G-protein-mediated signaling at the level of the
receptor/G-protein interaction or the G-protein .alpha. subunit.
G.alpha..sub.i-linked receptors support rapid adhesion and directed
migration of leukocytes and other cell types. RGS proteins regulate
G.alpha..sub.i-stimulated pathways. Thus, the compositions of the
invention (proteins, polynucleotides, fragments and variants
thereof, as well as agonists and antagonists) can be used to
modulate cell adhesion and chemotaxis. Movement of fibroblasts into
areas of injury plays an important role in wound repair. Further,
the migration of endothelial cells performs a paramount role during
angiogenesis. Leukocytes are recruited to sites of inflammation,
and lymphocytes are recruited to lymphatic organs to promote an
immune response. Chemoattractants mediate their activity by binding
and stimulating specific G.alpha..sub.i-coupled receptors. RGS
proteins enhance the endogenous GTPase activity of G-proteins,
decreasing the half-life of the active GTP-bound state and limiting
the duration of G.alpha..sub.i signaling. Thus, RGS compositions of
the invention can be used to modulate (stimulate or inhibit)
cellular migratory and proadhesive responses to chemoattractants.
Thus, nucleic acid molecules or antisense nucleic acid molecules of
the invention may find use in suppressing or enhancing an immune
and/or inflammatory response. Proteins and/or antibodies of the
invention are also useful in modulating an immune and/or
inflammatory response.
[0031] The RGS genes, clones h16395 and m1975, were identified in a
human spleen cDNA library and a mouse spleen cDNA library,
respectively. The first of these genes, clone h16395, encodes a 2.2
Kb RNA transcript having the corresponding cDNA set forth in SEQ ID
NO:1. This transcript encodes a 235 aminoacid protein (SEQ ID NO:2)
having a molecular weight of approximately 27.5 kDa.
[0032] The second of these genes, clone m1975, encodes a 2.2 Kb RNA
transcript having the corresponding cDNA set forth in SEQ ID NO:3.
This transcript also encodes a 235 amino acid protein (SEQ ID NO:4)
having a molecular weight of approximately 27.5 kDa. This mouse RGS
protein shares 84% identity with the human RGS protein disclosed in
SEQ ID NO:2 as determined by the Clustal method.
[0033] Both of these RGS proteins have N-terminal (amino acids
1-81) and C-terminal (amino acids 202-235) sequences that appear to
be unique. The proteins comprise an RGS domain that spans amino
acids 82-201. The RGS domain of each of these clones contains 10/11
RGS4 residues (amino acids 107, 109, 111, 112, 152, 154, 183, 187,
188, and 191 of SEQ ID NOS:2 and 4) that make direct contact with
G.alpha..sub.i and 18/23 RGS4 residues (amino acids 83, 90, 100,
103, 104, 115, 116, 138, 139, 142, 143, 151, 152, 184, 189, 192,
193, and 198 of SEQ ID NOS:2 and 4) that form the hydrophobic core
of the RGS domain. N-terminal ends of the proteins (amino acids
0-15) are hydrophobic in nature and are important for targeting to
the cellular location of G.alpha.proteins. These RGS proteins share
closest homology to human RGS5 protein (about 44% sequence identity
for the human sequence) (see FIG. 2).
[0034] Two plasmids containing overlapping clones, designated
Eph16395A and Eph16395B, for the h16395 DNA were deposited with
American Type Culture Collection (ATCC), 10801 University Blvd.,
Manassas, Va., on Jan. 14, 1999, and assigned Accession Numbers
207049 and 207050, respectively. Eph16395A comprises nucleotides 1
to 801 of h16395 and Eph16395B comprises nucleotides 802 to 1355 of
h16395. It is noted, however, that clones Eph16395A and Eph16395B
contain common sequences at the regions where they overlap.
Eph16395B overlaps Eph16395A from nucleotide 595 to nucleotide 801.
One of skill in the art by sequencing the clones and aligning the
overlap may obtain the entire sequence of h16395.
[0035] A plasmid containing the insert for the m1975, designated
Epm1975, was deposited with American Type Culture-Collection
(ATCC), 10801. University Blvd., Manassas, Va., on Jan. 14, 1999,
and assigned Accession Number 207048.
[0036] These deposits will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. These deposits
were made merely as a convenience for those of skill in the art and
are not an admission that a deposit is required under 35 U.S.C.
112.
[0037] The RGS sequences of the invention are members of a family
of molecules (the "RGS family") having conserved functional
features. As described above, the members of the family comprise an
RGS domain. The term "family" when referring to the proteins and
nucleic acid molecules of the invention is intended to mean two or
more proteins or nucleic acid molecules having sufficient amino
acid or nucleotide sequence identity as defined herein. Such family
members can be naturally occurring and can be from either the same
or different species. For example, a family can contain a first
protein of murine origin and a homologue of that protein of human
origin, as well as a second, distinct protein of human origin and a
murine homologue of that protein. Members of a family may also have
common functional characteristics.
[0038] Preferred RGS polypeptides of the present invention have an
amino acid sequence sufficiently identical to the amino acid
sequence of SEQ ID NO:2. The term "sufficiently identical" is used
herein to refer to a first amino acid or nucleotide sequence that
contains a sufficient or minimum number of identical or equivalent
(e.g., with a similar side chain) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences have a
common structural domain and/or common functional activity. For
example, amino acid or nucleotide sequences that contain a common
structural domain having at least about 45%, 55%, or 65% identity,
preferably 75%, 80% identity, more preferably 85%, 90%, 95%, or 98%
identity are defined herein as sufficiently identical.
[0039] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes. The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e., percent identity=number of identical
positions/total number of positions (e.g., overlapping
positions).times.100). In one embodiment, the two sequences are the
same length. The percent identity between two sequences can be
determined using techniques similar to those described below, with
or without allowing gaps. In calculating percent identity, only
exact matches are counted.
[0040] The determination of percent identity between two sequences
can be accomplished using a mathematical algorithm. A preferred,
nonlimiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:
5873-5877. Such an algorithm is incorporated into the NBLAST and
XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215: 403.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12, to obtain nucleotide sequences homologous
to RGS nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3, to obtain amino acid sequences homologous to RGS
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively,
PSI-Blast can be used to perform an iterated search that detects
distant relationships between molecules. See Altschul et al. (1997)
supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller et al. (1988) CABIOS 4: 11-17. Such an algorithm is
incorporated into the ALIGN program (version 2.0), which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used.
[0041] Accordingly, another embodiment of the invention features
isolated RGS proteins and polypeptides having an RGS protein
activity. As used interchangeably herein, a "RGS protein activity",
"biological activity of an RGS protein", or "functional activity of
an RGS protein" refers to an activity exerted by an RGS protein,
polypeptide, or nucleic acid molecule on an RGS responsive cell as
determined in vivo, or in vitro, according to standard assay
techniques. An RGS activity can be a direct activity, such as an
association with or an enzymatic activity on a second protein, or
an indirect activity, such as a cellular signaling activity
mediated by interaction of the RGS protein with a second protein.
In a preferred embodiment, an RGS activity includes at least one or
more of the following activities: (1) modulating (stimulating
and/or enhancing or inhibiting) cellular proliferation,
differentiation, and/or function, particularly immune cells, for
example leukocytes; (2) modulating immune and inflammatory
responses, particularly T-lymphocyte responses; (3) modulating
chemoattractant-induced cell migration and adhesion; (4) modulating
G-protein signaling; (5) regulating G.alpha..sub.i-stimulated
pathways; (6) acting as GTPase-activating proteins; (7) mediating
desensitization process of receptors, particularly G-protein
coupled receptors; (8) binding an RGS ligand; and (9) inducing
and/or maintaining tolerance in both transplant and autoimmune
diseases.
[0042] An "isolated" or "purified" RGS nucleic acid molecule or
protein, or biologically active portion thereof, is substantially
free of other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
Preferably, an "isolated" nucleic acid is free of sequences
(preferably protein encoding sequences) that naturally flank the
nucleic acid (i.e., sequences located at the 5' and 3' ends of the
nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For purposes of the invention, "isolated"
when used to refer to nucleic acid molecules, excludes isolated
chromosomes. For example, in various embodiments, the isolated RGS
nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb,
2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that
naturally flank the nucleic acid molecule in genomic DNA of the
cell from which the nucleic acid is derived. An RGS protein that is
substantially free of cellular material includes preparations of
RGS protein having less than about 30%, 20%, 10%, or 5% (by dry
weight) of non-RGS protein (also referred to herein as a
"contaminating protein"). When the RGS protein or biologically
active portion thereof is recombinantly produced, preferably,
culture medium represents less than about 30%, 20%, 10%, or 5% of
the volume of the protein preparation. When RGS protein is produced
by chemical synthesis, preferably the protein preparations have
less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical
precursors or non-RGS chemicals.
[0043] Various aspects of the invention are described in further
detail in the following subsections.
[0044] I. Isolated Nucleic Acid Molecules
[0045] One aspect of the invention pertains to isolated nucleic
acid molecules comprising nucleotide sequences encoding RGS
proteins or biologically active portions thereof, as well as
nucleic acid molecules sufficient for use as hybridization probes
to identify RGS-encoding nucleic acids (e.g., RGS mRNA) and
fragments for use as PCR primers for the amplification or mutation
of RGS nucleic acid molecules. As used herein, the term "nucleic
acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., "mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0046] Nucleotide sequences encoding the RGS proteins of the
present invention include sequences set forth in SEQ ID NOS:1 and
3, the nucleotide sequences included in the plasmids deposited with
the ATCC as Accession Numbers (the "cDNA of ATCC 207048" for the
mouse, or "the DNA of ATCC 207049 and 207050" for the human"), and
complements thereof. For purposes of the human sequence the entire
coding sequence for the RGS protein can be obtained from sequencing
the overlapping clones deposited with the ATCC and assigned ATCC
Nos: 207049 and 207050. By "complement" is intended a nucleotide
sequence that is sufficiently complementary to a given nucleotide
sequence such that it can hybridize to the given nucleotide
sequence to thereby form a stable duplex. The corresponding amino
acid sequences for the RGS proteins encoded by these nucleotide
sequences are set forth in SEQ ID NOS:2 and 4, respectively.
[0047] Nucleic acid molecules that are fragments of these RGS
nucleotide sequences are also encompassed by the present invention.
By "fragment" is intended a portion of the nucleotide sequence
encoding an RGS protein of the invention. A fragment of an RGS
nucleotide sequence may encode a biologically active portion of an
RGS protein, or it may be a fragment that can be used as a
hybridization probe or PCR primer using methods disclosed below. A
biologically active portion of an RGS protein can be prepared by
isolating a portion-of one of the RGS nucleotide sequences of the
invention, expressing the encoded portion of the RGS protein (e.g.,
by recombinant expression in vitro), and assessing the activity of
the encoded portion of the RGS protein.
[0048] It is recognized that isolated fragments include any
contiguous sequence not disclosed prior to the invention as well as
sequences that are substantially the same and that are not
disclosed. When a sequence is not disclosed prior to the invention,
fragments of an RGS nucleotide sequence comprise at least 15, 16,
18, 20, or 30 nucleotides in length and hybridize to the nucleotide
sequence of SEQ ID NOS:1 or 3 under stringent conditions. For
example, for h16395, nucleotides 1-23, 275-303, and 732-791 are not
disclosed prior to the invention. Other regions of the nucleotide
sequence may comprise fragments of various sizes, depending upon
potential homology with previously disclosed sequences. For
example, the nucleotide sequence from about 24 to about 274
encompasses fragments greater than 236 or 240 nucleotides, the
nucleotide sequence from about 304 to about 792 encompasses
fragments greater than 19 or 20 nucleotides, the nucleotide
sequence from about 792 to about 1400 encompasses fragments greater
than 537, 540, or 550 nucleotides, and the nucleotide sequence from
about 1400 to the end of the molecule encompasses fragments greater
than 307, 310 or 320 nucleotides. In these embodiments, depending
on the region, the nucleic acid can be at least 15, 20, 30, 40, 50,
75, 100, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,
800, 900, 1,000, or 1,100 nucleotides, or up to the number of
nucleotides present in a full-length RGS nucleotide sequence
disclosed herein (for example, 2,217 or 1,164 nucleotides for SEQ
ID NO:1 or 3, respectively).
[0049] A fragment of an RGS nucleotide sequence that encodes a
biologically active portion of an RGS protein of the invention will
encode at least 15, 25, 30, 50, 100, 125, 150, 175, 200, or 225
contiguous amino acids, or up to the total number of amino acids
present in a full-length RGS protein of the invention (for example,
235 amino acids, SEQ ID NOS:2 and 4). Fragments of an RGS
nucleotide sequence that are useful as hybridization probes for PCR
primers generally need not encode a biologically active portion of
an RGS protein.
[0050] Nucleic acid molecules that are variants of the RGS
nucleotide sequences disclosed herein are also encompassed by the
present invention. "Variants" of the RGS nucleotide sequences
include those sequences that encode the RGS proteins disclosed
herein but that differ conservatively because of the degeneracy of
the genetic code. These naturally occurring allelic variants can be
identified with the use of well-known molecular biology techniques,
such as polymerase chain reaction (PCR) and hybridization
techniques as outlined below. Variant nucleotide sequences also
include synthetically derived nucleotide sequences that have been
generated, for example, by using site-directed mutagenesis but
which still encode the RGS proteins disclosed in the present
invention as discussed below. Generally, nucleotide sequence
variants of the invention will have at least 45%, 55%, 65%, 75%,
85%, 95%, or 98% identity to the nucleotide sequences disclosed
herein. A variant RGS nucleotide sequence will encode an RGS
protein that has an amino acid sequence having at least 45%, 55%,
65%, 75%, 85%, 95%, or 98% identity to an amino acid sequence of an
RGS protein disclosed herein.
[0051] In addition to the RGS nucleotide sequences shown in SEQ ID
NOS:1 and 3, the nucleotide sequence of the cDNA of ATCC 207048,
and the DNA of ATCC 207049 and 207050, it will be appreciated by
those skilled in the art that DNA sequence polymorphisms that lead
to changes in the amino acid sequences of RGS proteins may exist
within a population (e.g., the human population). Such genetic
polymorphism in an RGS gene may exist among individuals within a
population due to natural allelic variation. An allele is one of a
group of genes that occur alternatively at a given genetic locus.
As used herein, the terms "gene" and "recombinant gene" refer to
nucleic acid molecules comprising an open reading frame encoding an
RGS protein, preferably a mammalian RGS protein. As used herein,
the phrase "allelic variant" refers to a nucleotide sequence that
occurs at an RGS locus or to a polypeptide encoded by the
nucleotide sequence. Such natural allelic variations can typically
result in 1-5% variance in the nucleotide sequence of the RGS gene.
Any and all such nucleotide variations and resulting amino acid
polymorphisms or variations in an RGS sequence that are the result
of natural allelic variation and that do not alter the functional
activity of RGS proteins are intended to be within the scope of the
invention.
[0052] Moreover, nucleic acid molecules encoding RGS proteins from
other species (RGS homologues), which have a nucleotide sequence
differing from that of the RGS sequences disclosed herein, are
intended to be within the scope of the invention. Nucleic acid
molecules corresponding to natural allelic variants and homologues
of the RGS DNA sequences of the invention can be isolated based on
their identity to the mouse or human RGS nucleic acids disclosed
herein using the sequences of the invention, or a portion thereof,
as a hybridization probe according to standard hybridization
techniques under stringent hybridization conditions as disclosed
below.
[0053] In addition to naturally-occurring allelic variants of the
RGS sequence that may exist in the population, the skilled artisan
will further appreciate that changes can be introduced by mutation
into the nucleotide sequences of the invention thereby leading to
changes in the amino acid sequence of the encoded RGS protein,
without altering the biological activity of the RGS protein. Thus,
an isolated nucleic acid molecule encoding an RGS protein having a
sequence that differs from that of SEQ ID NO:2 or 4, can be created
by introducing one or more nucleotide substitutions, additions, or
deletions into the nucleotide sequences disclosed herein, such that
one or more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced by
standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis. Such variant nucleotide sequences are
also encompassed by the present invention.
[0054] For example, preferably, conservative amino acid
substitutions may be made at one or more predicted, preferably
nonessential amino acid residues. A "nonessential" amino acid
residue is a residue that can be altered from the wild-type
sequence of an RGS protein (e.g., the sequence of SEQ ID NO:2 or 4)
without altering the biological activity, whereas an "essential"
amino acid residue is required for biological activity. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine tryptophan, histidine). Such
substitutions would not be made for conserved amino acid residues,
such as amino acid residues residing within a conserved motif, such
as the RGS domain, where such residues are essential for protein
activity.
[0055] Alternatively, variant RGS nucleotide sequences can be made
by introducing mutations randomly along all or part of an RGS
coding sequence, such as by saturation mutagenesis, and the
resultant mutants can be screened for RGS biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded protein can be expressed recombinantly, and the activity of
the protein can be determined using standard assay techniques.
[0056] Thus the nucleotide sequences of the invention include those
sequences disclosed herein as well as fragments and variants
thereof. The RGS nucleotide sequences of the invention, and
fragments and variants thereof, can be used as probes and/or
primers to identify and/or clone RGS homologues in other cell
types, e.g., from other tissues, as well as RGS homologues from
other mammals. Such probes can be used to detect transcripts or
genomic sequences encoding the same or identical proteins. These
probes can be used as part of a diagnostic test kit for identifying
cells or tissues that misexpress an RGS protein, such as by
measuring levels of an RGS-encoding nucleic acid in a sample of
cells from a subject, e.g., detecting RGS mRNA levels or
determining whether a genomic RGS gene has been mutated or
deleted.
[0057] In this manner, methods such as PCR, hybridization, and the
like can be used to identify such sequences having substantial
identity to the sequences of the invention. See, e.g., Sambrook et
al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold
Spring Harbor Laboratory Press, Plainview, N.Y.) and Inns, et al.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, NY). RGS nucleotide sequences isolated based on their
sequence identity to the RGS nucleotide sequences set forth herein
or to fragments and variants thereof are encompassed by the present
invention.
[0058] In a hybridization method, all or part of a known RGS
nucleotide sequence can be used to screen cDNA or genomic
libraries. Methods for construction of such cDNA and genomic
libraries are generally known in the art and are disclosed in
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The
so-called hybridization probes may be genomic DNA fragments, cDNA
fragments, RNA fragments, or other oligonucleotides, and may be
labeled with a detectable group such as .sup.32P, or any other
detectable marker, such as other radioisotopes, a fluorescent
compound, an enzyme, or an enzyme co-factor. Probes for
hybridization can be made by labeling synthetic oligonucleotides
based on the known RGS nucleotide sequences disclosed herein.
Degenerate primers designed on the basis of conserved nucleotides
or amino acid residues in a known RGS nucleotide sequence or
encoded amino acid sequence can additionally be used. The probe
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, preferably about
25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250,
300, 350, or 400 consecutive nucleotides of an RGS nucleotide
sequence of the invention or a fragment or variant thereof.
Preparation of probes for hybridization is generally known in the
art and is disclosed in Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Plainview, N.Y.), herein incorporated by reference.
[0059] For example, in one embodiment, a previously unidentified
RGS nucleic acid molecule hybridizes under stringent conditions to
a probe that is a nucleic acid molecule comprising one of the RGS
nucleotide sequences of the invention or a fragment thereof. In
another embodiment, the previously unknown RGS nucleic acid
molecule is at least 300, 325, 350, 375, 400, 425, 450, 500, 550,
600, 650, 700, 800, 900, 1000, 2,000, 3,000, 4,000 or 5,000
nucleotides in length and hybridizes under stringent conditions to
a probe that is a nucleic acid molecule comprising one of the RGS
nucleotide sequences disclosed herein or a fragment thereof.
[0060] Accordingly, in another embodiment, an isolated previously
unknown RGS nucleic acid molecule of the invention is at least 300,
325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900,
1000, 1,100, 1,200, 1,300, or 1,400 nucleotides in length and
hybridizes under stringent conditions to a probe that is a nucleic
acid molecule comprising one of the nucleotide sequences of the
invention, preferably the coding sequence set forth in SEQ ID NO:1
or 3, the cDNA of ATCC 207048, the DNA of ATCC 207049 and 207050,
or a complement, fragment, or variant thereof.
[0061] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences having at least 60%,
65%, 70%, preferably 75% identity to each other typically remain
hybridized to each other. Such stringent conditions are known to
those skilled in the art and can be found in Current Protocols in
Molecular Biology (John Wiley & Sons, New York (1989)),
6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization condition is hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times. SSC, 0.1% SDS at 50-65.degree. C.
In another preferred embodiment, stringent conditions comprise
hybridization in 6.times. SSC at 42.degree. C., followed by washing
with 1.times. SSC at 55.degree. C. Preferably, an isolated nucleic
acid molecule that hybridizes under stringent conditions to an RGS
sequence of the invention corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein).
[0062] Thus, in addition to the RGS nucleotide sequences disclosed
herein and fragments and variants thereof, the isolated nucleic
acid molecules of the invention also encompass homologous DNA
sequences identified and isolated from other cells and/or organisms
by hybridization with entire or partial sequences obtained from the
RGS nucleotide sequences disclosed herein or variants and fragments
thereof.
[0063] The present invention also encompasses antisense nucleic
acid molecules, i.e., molecules that are complementary to a sense
nucleic acid encoding a protein, e.g., complementary to the coding
strand of a double-stranded cDNA molecule, or complementary to an
mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen
bond to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire RGS coding strand, or to only a portion
thereof, e.g., all or part of the protein coding region (or open
reading frame). An antisense nucleic acid molecule can be antisense
to a noncoding region of the coding strand of a nucleotide sequence
encoding an RGS protein. The noncoding regions are the 5' and 3'
sequences that flank the coding region and are not translated into
amino acids.
[0064] Given the coding-strand sequences encoding an RGS protein
disclosed herein (e.g., SEQ ID NOS:1 and 3), antisense nucleic
acids of the invention can be designed according to the rules of
Watson and Crick base pairing. The antisense nucleic acid molecule
can be complementary to the entire-coding-region of RGS mRNA, but
more preferably is an oligonucleotide that is antisense to only a
portion of the coding or noncoding region of RGS mRNA. For example,
the antisense oligonucleotide can be complementary to the region
surrounding the translation start site of RGS mRNA. An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid
of the invention can be constructed using chemical synthesis and
enzymatic ligation procedures known in the art.
[0065] For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, including, but not limited to, for example
e.g., phosphorothioate derivatives and acridine substituted
nucleotides. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0066] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding an RGS protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. An
example of a route of administration of antisense nucleic acid
molecules of the invention includes direct injection at a tissue
site. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered
systemically. For example, antisense molecules can be linked to
peptides or antibodies to form a complex that specifically binds to
receptors or antigens expressed on a selected cell surface. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[0067] An antisense nucleic acid molecule of the invention can be
an .alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric
nucleic acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gaultier et al. (1987) Nucleic
Acids Res. 15: 6625-6641). The antisense nucleic acid molecule can
also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987)
Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue
(Inoue et al. (1987) FEBS Lett. 215: 327-330).
[0068] The invention also encompasses ribozymes, which are
catalytic RNA molecules with ribonuclease activity that are capable
of cleaving a single-stranded nucleic acid, such as an mRNA, to
which they have a complementary region. Ribozymes (e.g., hammerhead
ribozymes (described in Haselhofff et al. (1988) Nature 334:
585-591)) can be used to catalytically cleave RGS mRNA transcripts
to thereby inhibit translation of RGS mRNA. A ribozyme having
specificity for an RGS-encoding nucleic acid can be designed based
upon the nucleotide sequence of an RGS cDNA disclosed herein (e.g.,
SEQ ID NO:1 or 3). See, e.g., Cech et al., U.S. Pat. No. 4,987,071;
and Cech et al., U.S. Pat. No. 5,116,742. Alternatively, RGS mRNA
can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel et al. (1993) Science 261: 1411-1418.
[0069] The invention also encompasses nucleic acid molecules that
form triple helical structures. For example, RGS gene expression
can be inhibited by targeting nucleotide sequences complementary to
the regulatory region of the RGS protein (e.g., the RGS promoter
and/or enhancers) to form triple helical structures that prevent
transcription of the RGS gene in target cells. See generally Helene
(1991) Anticancer Drug Des. 6(6): 569; Helene (1992) Ann. N.Y.
Acad. Sci. 660: 27; and Maher (1992) Bioassays 14(12): 807.
[0070] In preferred embodiments, the nucleic acid molecules of the
invention can be modified at the base moiety, sugar moiety, or
phosphate backbone to improve, e.g., the stability, hybridization,
or solubility of the molecule. For example, the deoxyribose
phosphate backbone of the nucleic acids can be modified to generate
peptide nucleic acids (see Hyrup et al. (1996) Bioorganic &
Medicinal Chemistry 4: 5). As used herein, the terms "peptide
nucleic acids" or "PNAs" refer-to nucleic acid mimics, e.g., DNA
mimics, in which the deoxyribose phosphate backbone is replaced by
a pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid-phase peptide synthesis protocols as described in
Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93: 14670.
[0071] PNAs of an RGS molecule can be used in therapeutic and
diagnostic applications. For example, PNAs can be used as antisense
or antigene agents for sequence-specific modulation of gene
expression by, e.g., inducing transcription or translation arrest
or inhibiting replication. PNAs of the invention can also be used,
e.g., in the analysis of single base pair mutations in a gene by,
e.g., PNA-directed PCR clamping; as artificial restriction enzymes
when used in combination with other enzymes, e.g., S1 nucleases
(Hyrup (1996), supra; or as probes or primers for DNA sequence and
hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996),
supra).
[0072] In another embodiment, PNAs of an RGS molecule can be
modified, e.g., to enhance their stability, specificity, or
cellular uptake, by attaching lipophilic or other helper groups to
PNA, by the formation of PNA-DNA chimeras, or by the use of
liposomes or other techniques of drug delivery known in the art.
The synthesis of PNA-DNA chimeras can be performed as described in
Hyrup (1996), supra; Finn et al. (1996) Nucleic Acids Res. 24(17):
3357-63; Mag et al. (1989) Nucleic Acids Res. 17: 5973; and
Peterser et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119.
[0073] II. Isolated RGS Proteins and Anti-RGS Antibodies
[0074] RGS proteins are also encompassed within the present
invention. By "RGS protein" is intended proteins having the amino
acid sequence set forth in SEQ ID NO:2 or 4 as well as fragments,
biologically active portions, and variants thereof.
[0075] "Fragments" or "biologically active portions" include
polypeptide fragments suitable for use as immunogens to raise
anti-RGS antibodies. Fragments include peptides comprising amino
acid sequences sufficiently identical to or derived from the amino
acid sequence of an RGS protein of the invention and exhibiting at
least one activity of an RGS protein, but which include fewer amino
acids than the full-length RGS proteins disclosed herein.
Typically, biologically active portions comprise a domain or motif
with at least one activity of the RGS protein. A biologically
active portion of an RGS protein can be a polypeptide that is, for
example, 10, 25, 50, 100 or more amino acids in length. Such
biologically active portions can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native RGS protein. As used here, a fragment
comprises at least 6 contiguous amino acids, such as from amino
acids 1-110. The invention encompasses other fragments, however,
such as any fragment in the protein greater than 10, 12, 15, or 16
amino acids.
[0076] By "variants" is intended proteins or polypeptides having an
amino acid sequence that is at least about 45%, 55%, 65%,
preferably about 75%, 85%, 95%, or 98% identical to the amino acid
sequence of SEQ ID NO:2 or 4. Variants also include polypeptides
encoded by the cDNA insert of the plasmid deposited with ATCC as
Accession Number 207048 for the mouse, and by the DNA sequence
obtained from the plasmids deposited with the ATCC as Accession
Numbers 207049 and 207050 for the human, or polypeptides encoded by
a nucleic acid molecule that hybridizes to a nucleic acid molecule
of SEQ ID NO:1, 3, or a complement thereof, under stringent
conditions. Such variants generally retain the functional activity
of the RGS proteins of the invention. Variants include polypeptides
that differ in amino acid sequence due to natural allelic variation
or mutagenesis.
[0077] The invention also provides RGS chimeric or fusion proteins.
As used herein, an RGS "chimeric protein" or "fusion protein"
comprises an RGS polypeptide operably linked to a non-RGS
polypeptide. A "RGS polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to an RGS protein, whereas a
"non-RGS polypeptide" refers to a polypeptide having an amino acid
sequence corresponding to a protein that is not substantially
identical to the RGS protein, e.g., a protein that is different
from the RGS protein and which is derived from the same or a
different organism. Within an RGS fusion protein, the RGS
polypeptide can correspond to all or a portion of an RGS protein,
preferably at least one biologically active portion of an RGS
protein. Within the fusion protein, the term "operably linked" is
intended to indicate that the RGS polypeptide and the non-RGS
polypeptide are fused in-frame to each other. The non-RGS
polypeptide can be fused to the N-terminus or C-terminus of the RGS
polypeptide.
[0078] One useful fusion protein is a GST-RGS fusion protein in
which the RGS sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of
recombinant RGS proteins.
[0079] In yet another embodiment, the fusion protein is an
RGS-immunoglobulin fusion protein in which all or part of an RGS
protein is fused to sequences derived from a member of the
immunoglobulin protein family. The RGS-immunoglobulin fusion
proteins of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between an RGS ligand and an RGS protein on the surface
of a cell, thereby suppressing RGS-mediated signal transduction in
vivo. The RGS-immunoglobulin fusion proteins can be used to affect
the bioavailability of an RGS cognate ligand. Inhibition of the RGS
ligand/RGS interaction may be useful therapeutically, both for
treating proliferative and differentiative disorders and for
modulating (e.g., promoting or inhibiting) cell survival. Moreover,
the RGS-immunoglobulin fusion proteins of the invention can be used
as immunogens to produce anti-RGS antibodies in a subject, to
purify RGS ligands, and in screening assays to identify molecules
that inhibit the interaction of an RGS protein with an RGS
ligand.
[0080] Preferably, an RGS chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences may be ligated together in-frame, or the fusion gene can
be synthesized, such as with automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried
out using anchor primers that give rise to complementary overhangs
between two consecutive gene fragments, which can subsequently be
annealed and reamplified to generate a chimeric gene sequence (see,
e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular
Biology) (Greene Publishing and Wiley-Interscience, NY). Moreover,
an RGS-encoding nucleic acid can be cloned into a commercially
available expression vector such that it is linked in-frame to an
existing fusion moiety.
[0081] Variants of the RGS proteins can function as either RGS
agonists (mimetics) or as RGS antagonists. Variants of the RGS
protein can be generated by mutagenesis, e.g., discrete point
mutation or truncation of the RGS protein. An agonist of the RGS
protein can retain substantially the same, or a subset, of the
biological activities of the naturally occurring form of the RGS
protein. An antagonist of the RGS protein can inhibit one or more
of the activities of the naturally occurring form of the RGS
protein by, for example, competitively binding to a downstream or
upstream member of a cellular signaling cascade that includes the
RGS protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. Treatment of a
subject with a variant having a subset of the biological activities
of the naturally occurring form of the protein can have fewer side
effects in a subject relative to treatment with the naturally
occurring form of the RGS proteins.
[0082] Variants of the RGS protein that function as either RGS
agonists or as RGS antagonists can be identified by screening
combinatorial libraries of mutants, e.g., truncation mutants, of
the RGS protein for RGS protein agonist or antagonist activity. In
one embodiment, a variegated library of RGS variants is generated
by combinatorial mutagenesis at the nucleic acid level and is
encoded by a variegated gene library. A variegated library of RGS
variants can be produced by, for example, enzymatically ligating a
mixture of synthetic oligonucleotides into gene sequences such that
a degenerate set of potential RGS sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of RGS
sequences therein. There are a variety of methods that can be used
to produce libraries of potential RGS variants from a degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene
sequence can be performed in an automatic DNA synthesizer, and the
synthetic gene then ligated into an appropriate expression vector.
Use of a degenerate set of genes allows for the provision, in one
mixture, of all of the sequences encoding the desired set of
potential RGS sequences. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang (1983)
Tetrahedron 39: 3; Itakura et al. (1984) Ann. Rev. Biochem. 53:
323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983)
Nucleic Acid Res. 11: 477).
[0083] In addition, libraries of fragments of the RGS protein
coding sequence can be used to generate a variegated population of
RGS fragments for screening and subsequent selection of variants of
an RGS protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double-stranded PCR
fragment of an RGS coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double-stranded DNA, renaturing the DNA to form double-stranded DNA
which can include sense/antisense pairs from different nicked
products, removing single-stranded portions from reformed duplexes
by treatment with S1 nuclease, and ligating the resulting fragment
library into an expression vector. By this method, one can derive
an expression library that encodes N-terminal and internal
fragments of various sizes of the RGS protein.
[0084] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of RGS proteins. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
that enhances the frequency of functional mutants in the libraries,
can be used in combination with the screening assays to identify
RGS variants (Arkin et al. (1992) Proc. Natl. Acad. Sci. USA 89:
7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):
327-331).
[0085] An isolated RGS polypeptide of the invention can be used as
an immunogen to generate antibodies that bind RGS proteins using
standard techniques for polyclonal and monoclonal antibody
preparation. The full-length RGS protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of RGS proteins for use as immunogens. The antigenic peptide of an
RGS protein comprises at least 8, preferably 10, 15, 20, or 30
amino acid residues of the amino acid sequence shown in SEQ ID NO:2
or 4 and encompasses an epitope of an RGS protein such that an
antibody raised against the peptide forms a specific immune complex
with the RGS protein. Preferred epitopes encompassed by the
antigenic peptide are regions of a RGS protein that are located on
the surface of the protein, e.g., hydrophilic regions.
[0086] Accordingly, another aspect of the invention pertains to
anti-RGS polyclonal and monoclonal antibodies that bind an RGS
protein. Polyclonal anti-RGS antibodies can be prepared by
immunizing a suitable subject (e.g., rabbit, goat, mouse, or other
mammal) with an RGS immunogen. The anti-RGS antibody titer in the
immunized subject can be monitored over time by standard
techniques, such as with an enzyme linked immunosorbent assay
(ELISA) using immobilized RGS protein. At an appropriate time after
immunization, e.g., when the anti-RGS antibody titers are highest,
antibody-producing cells can be obtained from the subject and used
to prepare monoclonal antibodies by standard techniques, such as
the hybridoma technique originally described by Kohler et al.
(1975) Nature 256: 495-497, the human B cell hybridoma technique
(Kozbor et al. (1983) Immunol. Today 4: 72), the EBV-hybridoma
technique (Cole et al. (1985) in Monoclonal Antibodies and Cancer
Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,
N.Y.), pp. 77-96) or trioma techniques. The technology for
producing hybridomas is well known (see generally Coligan et al.,
eds. (1994) Current Protocols in Immunology (John Wiley & Sons,
Inc., New York, N.Y.); Galfre et al. (1977) Nature 266: 550-52;
Kenneth (1980) in Monoclonal Antibodies: A New Dimension In
Biological Analyses (Plenum Publishing Corp., NY; and Lerner (1981)
Yale J. Biol. Med. 54: 387-402).
[0087] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-RGS antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with an RGS
protein to thereby isolate immunoglobulin library members that bind
the RGS protein. Kits for generating and screening phage display
libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, U.S. Pat. No. 5,223,409; PCT
Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO
92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et
al. (199.1) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246:
1275-1281; Griffiths et al. (1993) EMBO J. 12: 725-734.
[0088] Additionally, recombinant anti-RGS antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and nonhuman portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. Such
chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using
methods described in PCT Publication Nos. WO 86101533 and WO
87/02671; European Patent Application Nos. 184,187, 171,496,
125,023, and 173,494; U.S. Pat. Nos. 4,816,567 and 5,225,539;
European Patent Application 125,023; Better et al. (1988) Science
240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:
3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al.
(1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al.
(1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314:
446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559);
Morrison (1985) Science 229: 1202-1207; Oi et al. (1986)
Bio/Techniques 4: 214; Jones et al. (1986) Nature 321: 552-525;
Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al.
(1988) J. Immunol. 141: 4053-4060.
[0089] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice that are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. See, e.g., Lonberg
et al. (1995) Int. Rev. Immunol. 13: 65-93); and U.S. Pat. Nos.
5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.), can
be engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0090] Completely human antibodies that recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide the selection
of a completely human antibody recognizing the same epitope. This
technology is described by Jespers et al. (1994) Bio/Technology 12:
899-903).
[0091] An anti-RGS antibody (e.g., monoclonal antibody) can be used
to isolate RGS proteins by standard techniques such as affinity
chromatography or immunoprecipitation. An anti-RGS antibody can
facilitate the purification of natural RGS protein from cells and
of recombinantly produced RGS protein expressed in host cells.
Moreover, an anti-RGS antibody can be used to detect RGS protein
(e.g., in a cellular lysate or cell supernatant) in order to
evaluate the abundance and pattern of expression of the RGS
protein. Anti-RGS antibodies can be used diagnostically to monitor
protein levels in tissue as part of a clinical testing procedure,
e.g., to, for example, determine the efficacy of a given treatment
regimen. Detection can be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S, or .sup.3H.
[0092] III. Recombinant Expression Vectors and Host Cells
[0093] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an RGS protein (or a portion thereof). "Vector" refers to a nucleic
acid molecule capable of transporting another nucleic acid to which
it has been linked, such as a "plasmid", a circular double-stranded
DNA loop into which additional DNA segments can be ligated, or a
viral vector, where additional DNA segments can be ligated into the
viral genome. The vectors are useful for autonomous replication in
a host cell or may be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome (e.g., nonepisomal mammalian vectors).
Expression vectors are capable of directing the expression of genes
to which they are operably linked. In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids (vectors). However, the invention is intended to include
such other forms of expression vectors, such as viral vectors
(e.g., replication defective retroviruses, adenoviruses, and
adeno-associated viruses), that serve equivalent functions.
[0094] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell. This means that the recombinant
expression vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for expression,
operably linked to the nucleic acid sequence to be expressed.
"Operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
that allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers, and other
expression control elements (e.g., polyadenylation signals). See,
e.g., Goeddel (1990) in Gene Expression Technology: Methods in
Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory
sequences include those that direct constitutive expression of a
nucleotide sequence in many types of host cell and those that
direct expression of the nucleotide sequence only in certain host
cells (e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., RGS proteins, mutant forms of RGS proteins,
fusion proteins, etc.).
[0095] The recombinant expression vectors of the invention can be
designed for expression of RGS protein in prokaryotic or eukaryotic
host cells. Expression of proteins in prokaryotes is most often
carried out in E. coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
nonfusion proteins. Fusion vectors add a number of amino acids to a
protein encoded therein, usually to the amino terminus of the
recombinant protein. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67: 31-40),
pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein. Examples of suitable inducible
nonfusion E. coli expression vectors include pTrc (Amann et al.
(1988) Gene 69: 301-315) and pET 11d (Studier et al. (1990) in Gene
Expression Technology: Methods in Enzymology 185 (Academic Press,
San Diego, Calif.), pp. 60-89). Strategies to maximize recombinant
protein expression in E. coli can be found in Gottesman (1990) in
Gene Expression Technology: Methods in Enzymology 185 (Academic
Press, CA), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res.
20: 2111-2118. Target gene expression from the pTrc vector relies
on host RNA polymerase transcription from a hybrid trp-lac fusion
promoter.
[0096] Suitable eukaryotic host cells include insect cells
(examples of Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and
the pVL series (Lucklow et al. (1989) Virology 170: 31-39)); yeast
cells (examples of vectors for expression in yeast S. cereivisiae
include pYepSec1 (Baldari et al. (1987) EMBO J. 6: 229-234), pMFa
(Kujan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et
al. (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San
Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego,
Calif.)); or mammalian cells (mammalian expression vectors include
pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman et al.
(1987) EMBO J. 6: 187: 195)). Suitable mammalian cells include
Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells,
the expression vector's control functions are often provided by
viral regulatory elements. For example, commonly used promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian
Virus 40. For other suitable expression systems for both
prokaryotic and eukaryotic cells, see chapters 16 and 17 of
Sambrook et al. (1989) Molecular cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See,
Goeddel (1990) in Gene Expression Technology: Methods in Enzymology
185 (Academic Press, San Diego, Calif.). Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0097] The terms "host cell" and "recombinant host cell" are used
interchangeably herein. It is understood that such terms refer not
only to the particular subject cell but to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to either mutation or environmental
influences, such progeny may not, in fact, be identical to the
parent cell but are still included within the scope of the term as
used herein.
[0098] In one embodiment, the expression vector is a recombinant
mammalian expression vector that comprises tissue-specific
regulatory elements that direct expression of the nucleic acid
preferentially in a particular cell type. Suitable tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert et
al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters
(Calame and Eaton (1988) Adv. Immunol. 43: 235-275), in particular
promoters of T-cell receptors (Winoto and Baltimore (1989) EMBO J.
8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:
729-740; Queen et al. (1983) Cell 33: 741-748), neuron-specific
promoters (e.g., the neurofilament promoter; Byrne et al. (1989)
Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific
promoters (Edlund et al. (1985) Science 230: 912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Patent Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel et al. (1990) Science 249:
374-379), the .alpha.-fetoprotein promoter (Campes et al. (1989)
Genes Dev. 3: 537-546), and the like.
[0099] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operably linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to RGS mRNA. Regulatory
sequences operably linked to a nucleic acid cloned in the antisense
orientation can be chosen to direct the continuous expression of
the antisense RNA molecule in a variety of cell types, for instance
viral promoters and/or enhancers, or regulatory sequences can be
chosen to direct constitutive, tissue-specific, or
cell-type-specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid, or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub et al. (1986)
Reviews--Trends in Genetics 1: 1.
[0100] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.) and other laboratory manuals.
[0101] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those which confer resistance to drugs, such as
G418, hygromycin, and methotrexate. Nucleic acid encoding a
selectable marker can be introduced into a host cell on the same
vector as that encoding an RGS protein or can be introduced on a
separate vector. Cells stably transfected with the introduced
nucleic acid can be identified by drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0102] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) RGS protein. Accordingly, the invention further provides
methods for producing RGS protein using the host cells of the
invention. In one embodiment, the method comprises culturing the
host cell of the invention, into which a recombinant expression
vector encoding an RGS protein has been introduced, in a suitable
medium such that RGS protein is produced. In another embodiment,
the method further comprises isolating RGS protein from the medium
or the host cell.
[0103] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which RGS-coding sequences have been introduced. Such
host cells can then be used to create nonhuman transgenic animals
in which exogenous RGS sequences have been introduced into their
genome or homologous recombinant animals in which endogenous RGS
sequences have been altered. Such animals are useful for studying
the function and/or activity of RGS genes and proteins and for
identifying and/or evaluating modulators of RGS activity. As used
herein, a "transgenic animal" is a nonhuman animal, preferably a
mammal, more preferably a rodent such as a rat or mouse, in which
one or more of the cells of the animal includes a transgene. Other
examples of transgenic animals include nonhuman primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A transgene is
exogenous DNA that is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a nonhuman animal, preferably a mammal, more preferably
a mouse, in which an endogenous RGS gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0104] A transgenic animal of the invention can be created by
introducing RGS-encoding nucleic acid into the male pronuclei of a
fertilized oocyte, e.g., by microinjection, retroviral infection,
and allowing the oocyte to develop in a pseudopregnant female
foster animal. The RGS cDNA sequence can be introduced as a
transgene into the genome of a nonhuman animal. Alternatively, a
homologue of the mouse RGS gene can be isolated based on
hybridization and used as a transgene. Intronic sequences and
polyadenylation signals can also be included in the transgene to
increase the efficiency of expression of the transgene. A
tissue-specific regulatory sequence(s) can be operably linked to
the RGS transgene to direct expression of RGS protein to particular
cells. Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866, 4,870,009, and 4,873,191 and in Hogan
(1986) Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the RGS
transgene in its genome and/or expression of RGS mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding RGS gene can
further be bred to other transgenic animals carrying other
transgenes.
[0105] To create a homologous recombinant animal, one prepares a
vector containing at least a portion of an RGS gene or a homologue
of the gene into which a deletion, addition, or substitution has
been introduced to thereby alter, e.g., functionally disrupt, the
RGS gene. In a preferred embodiment, the vector is designed such
that, upon homologous recombination, the endogenous RGS gene is
functionally disrupted (i.e., no longer encodes a functional
protein; also referred to as a "knock out" vector). Alternatively,
the vector can be designed such that, upon homologous
recombination, the endogenous RGS gene is mutated or otherwise
altered but still encodes functional protein (e.g., the upstream
regulatory region can be altered to thereby alter the expression of
the endogenous RGS protein). In the homologous recombination
vector, the altered portion of the RGS gene is flanked at its 5'
and 3' ends by additional nucleic acid of the RGS gene to allow for
homologous recombination to occur between the exogenous RGS gene
carried by the vector and an endogenous RGS gene in an embryonic
stem cell. The additional flanking RGS nucleic acid is of
sufficient length for successful homologous recombination with the
endogenous gene. Typically, several kilobases of flanking DNA (both
at the 5' and 3' ends) are included in the vector (see, e.g.,
Thomas and Capecchi (1987) Cell 51: 503 for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation), and cells in
which the introduced RGS gene has homologously recombined with the
endogenous RGS gene are selected (see, e.g., Li et al. (1992) Cell
69: 915). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse) to form aggregation chimeras (see, e.g.,
Bradley (1987) in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, ed. Robertson (IRL, Oxford, pp. 113-152). A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously recombined DNA in their germ
cells can be used to breed animals in which all cells of the animal
contain the homologously recombined DNA by germline transmission of
the transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley (1991) Current Opinion in Bio/Technology 2: 823-829 and in
PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169.
[0106] In another embodiment, transgenic nonhuman animals
containing selected systems that allow for regulated expression of
the transgene can be produced. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al. (1991) Science 251: 1351-1355). If a
cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[0107] Clones of the nonhuman transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385: 810-813 and PCT Publication Nos. WO
97/07668 and WO 97/07669.
[0108] IV. Pharmaceutical Compositions
[0109] The RGS nucleic acid molecules, RGS proteins, and anti-RGS
antibodies (also referred to herein as "active compounds") of the
invention can be incorporated into pharmaceutical compositions
suitable for administration. Such compositions typically comprise
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0110] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes, or multiple dose vials made of glass
or plastic.
[0111] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF; Parsippany, N.J.), or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride, in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and
gelatin.
[0112] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an RGS protein or anti-RGS
antibody) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying, which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0113] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth, or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring. For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser that contains a suitable propellant, e.g., a
gas such as carbon dioxide, or a nebulizer.
[0114] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art. The compounds can also be prepared in
the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0115] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0116] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated with each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. Depending on the type and severity of the
disease, about 1 .mu.g/kg to about 15 mg/kg (e.g., 0.1 to 20 mg/kg)
of antibody is an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to about 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. However, other dosage regimens may be
useful. The progress of this therapy is easily monitored by
conventional techniques and assays. An exemplary dosing regimen is
disclosed in WO 94/04188. The specification for the dosage unit
forms of the invention are dictated by and directly dependent on
the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and the limitations
inherent in the art of compounding such an active compound for the
treatment of individuals.
[0117] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (U.S. Pat. No. 5,328,470), or by
stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of
the gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0118] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0119] V. Uses and Methods of the Invention
[0120] The nucleic acid molecules, proteins, protein homologues,
and antibodies described herein can be used in one or more of the
following methods: (a) screening assays; (b) detection assays
(e.g., chromosomal mapping, tissue typing, forensic biology); (c)
predictive medicine (e.g., diagnostic assays, prognostic assays,
monitoring clinical trials, and pharmacogenomics); and (d) methods
of treatment (e.g., therapeutic and prophylactic). The isolated
nucleic acid molecules of the invention can be used to express RGS
protein (e.g., via a recombinant expression vector in a host cell
in gene therapy applications), to detect RGS mRNA (e.g., in a
biological sample) or a genetic lesion in an RGS gene, and to
modulate RGS activity. In addition, the RGS proteins can be used to
screen drugs or compounds that modulate the immune response as well
as to treat disorders characterized by insufficient or excessive
production of RGS protein or production of RGS protein forms that
have decreased or aberrant activity compared to RGS wild type
protein. In addition, the anti-RGS antibodies of the invention can
be used to detect and isolate RGS proteins and modulate RGS
activity.
[0121] A. Screening Assays
[0122] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules, or other drugs) that bind to RGS proteins or have a
stimulatory or inhibitory effect on, for example, RGS expression or
RGS activity.
[0123] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including biological libraries, spatially
addressable parallel solid phase or solution phase libraries,
synthetic library methods requiring deconvolution, the "one-bead
one-compound" library method, and synthetic library methods using
affinity chromatography selection. The biological library approach
is limited to peptide libraries, while the other four approaches
are applicable to peptide, nonpeptide oligomer, or small molecule
libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145).
Examples of methods for the synthesis of molecular libraries can be
found in the art, for example in: DeWitt et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci.
USA 91: 11422; Zuckermann et al. (1994) J. Med. Chem. 37: 2678; Cho
et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed.
Engl. 33: 2061; and Gallop et al. (1994) J. Med. Chem. 37:
1233.
[0124] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Bio/Techniques 13: 412-421), or on beads (Lam
(1991) Nature 354: 82-84), chips (Fodor (1993) Nature 364:
555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat.
Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al.
(1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869), or phage (Scott
et al. (1990) Science 249: 386-390; Devlin (1990) Science 249:
404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:
6378-6382; and Felici (1991) J. Mol. Biol. 222: 301-310).
[0125] Determining the ability of the test compound to bind to the
RGS protein can be accomplished, for example, by coupling the test
compound with a radioisotope or enzymatic label such that binding
of the test compound to the RGS protein or biologically active
portion thereof can be determined by detecting the labeled compound
in a complex. For example, test compounds can be labeled with
.sup.125I, .sup.35S, 14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively, test
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product.
[0126] In a similar manner, one may determine the ability of the
RGS protein to bind to or interact with an RGS target molecule. By
"target molecule" is intended a molecule with which an RGS protein
binds or interacts in nature. In a preferred embodiment, the
ability of the RGS protein to bind to or interact with an RGS
target molecule can be determined by monitoring the activity of the
target molecule. For example, the activity of the target molecule
can be monitored by detecting induction of a cellular second
messenger of the target (e.g., intracellular Ca.sup.2+,
diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity
of the target on an appropriate substrate, detecting the induction
of a reporter gene (e.g., an RGS-responsive regulatory element
operably linked to a nucleic acid encoding a detectable marker,
e.g., luciferase), or detecting a cellular response, for example,
cellular differentiation or cell proliferation.
[0127] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting an RGS protein or
biologically active portion thereof with a test compound and
determining the ability of the test compound to bind to the RGS
protein or biologically active portion thereof. Binding of the test
compound to the RGS protein can be determined either directly or
indirectly as described above. In a preferred embodiment, the assay
includes contacting the RGS protein or biologically active portion
thereof with a known compound that binds RGS protein to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to preferentially
bind to RGS protein or biologically active portion thereof as
compared to the known compound.
[0128] In another embodiment, an assay is a cell-free assay
comprising contacting RGS protein or biologically active portion
thereof with a test compound and determining the ability of the
test compound to modulate (e.g., stimulate or inhibit) the activity
of the RGS protein or biologically active portion thereof.
Determining the ability of the test compound to modulate the
activity of an RGS protein can be accomplished, for example, by
determining the ability of the RGS protein to bind to an RGS target
molecule as described above for determining direct binding. In an
alternative embodiment, determining the ability of the test
compound to modulate the activity of an RGS protein can be
accomplished by determining the ability of the RGS protein to
further modulate an RGS target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as previously
described.
[0129] In yet another embodiment, the cell-free assay comprises
contacting the RGS protein or biologically active portion thereof
with a known compound that binds an RGS protein to form an assay
mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to preferentially bind
to or modulate the activity of an RGS target molecule.
[0130] In the above-mentioned assays, it may be desirable to
immobilize either an RGS protein or its target molecule to
facilitate separation of complexed from uncomplexed forms of one or
both of the proteins, as well as to accommodate automation of the
assay. In one embodiment, a fusion protein can be provided that
adds a domain that allows one or both of the proteins to be bound
to a matrix. For example, glutathione-S-transferase/- RGS fusion
proteins or glutathione-S-transferase/target fusion proteins can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione-derivatized microtitre plates, which are
then combined with the test compound or the test compound and
either the nonadsorbed target protein or RGS protein, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads or microtitre plate wells are washed to
remove any unbound components and complex formation is measured
either directly or indirectly, for example, as described above.
Alternatively, the complexes can be dissociated from the matrix,
and the level of RGS binding or activity determined using standard
techniques.
[0131] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention For example,
either RGS protein or its target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated RGS
molecules or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96-well plates
(Pierce Chemicals). Alternatively, antibodies reactive with an RGS
protein or target molecules but which do not interfere with binding
of the RGS protein to its target molecule can be derivatized to the
wells of the plate, and unbound target or RGS protein trapped in
the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the RGS protein or target molecule,
as well as enzyme-linked assays that rely on detecting an enzymatic
activity associated with the RGS protein or target molecule.
[0132] In another embodiment, modulators of RGS expression are
identified in a method in which a cell is contacted with a
candidate compound and the expression of RGS mRNA or protein in the
cell is determined relative to expression of RGS mRNA or protein in
a cell in the absence of the candidate compound. When expression is
greater (statistically significantly greater) in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of RGS mRNA or protein expression.
Alternatively, when expression is less (statistically significantly
less) in the presence of the candidate compound than in its
absence, the candidate compound is identified as an inhibitor of
RGS mRNA or protein expression. The level of RGS mRNA or protein
expression in the cells can be determined by methods described
herein for detecting RGS mRNA or protein.
[0133] In yet another aspect of the invention, the RGS proteins can
be used as "bait proteins" in a two-hybrid assay or three-hybrid
assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993)
Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268:
12046-12054; Bartel et al. (1993) Bio/Techniques 14: 920-924;
Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and PCT Publication
No. WO 94/10300), to identify other proteins, which bind to or
interact with RGS protein ("RGS-binding proteins" or "RGS-bp") and
modulate RGS activity. Such RGS-binding proteins are also likely to
be involved in the propagation of signals by the RGS proteins as,
for example, upstream or downstream elements of the RGS
pathway.
[0134] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0135] B. Detection Assays
[0136] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (1) map their respective genes on a
chromosome; (2) identify an individual from a minute biological
sample (tissue typing); and (3) aid in forensic identification of a
biological sample. These applications are described in the
subsections below.
[0137] 1. Chromosome Mapping
[0138] The isolated complete or partial RGS gene sequences of the
invention can be used to map their respective RGS genes on a
chromosome, thereby facilitating the location of gene regions
associated with genetic disease. Computer analysis of RGS sequences
can be used to rapidly select PCR primers (preferably 15-25 bp in
length) that do not span more than one exon in the genomic DNA,
thereby simplifying the amplification process. These primers can
then be used for PCR screening of somatic cell hybrids containing
individual human chromosomes. Only those hybrids containing the
human gene corresponding to the RGS sequences will yield an
amplified fragment.
[0139] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow (because they lack a
particular enzyme), but in which human cells can, the one human
chromosome that contains the gene encoding the needed enzyme will
be retained. By using various media, panels of hybrid cell lines
can be established. Each cell line in a panel contains either a
single human chromosome or a small number of human chromosomes, and
a full set of mouse chromosomes, allowing easy mapping of
individual genes to specific human chromosomes (D'Eustachio et al.
(1983) Science 220: 919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[0140] Other mapping strategies that can similarly be used to map
an RGS sequence to its chromosome include in situ hybridization
(described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:
6223-27), pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries. Furthermore, fluorescence in situ hybridization (FISH)
of a DNA sequence to a metaphase chromosomal spread can be used to
provide a precise chromosomal location in one step. For a review of
this technique, see Verma et al. (1988) Human Chromosomes: A Manual
of Basic Techniques (Pergamon Press, NY). The FISH technique can be
used with a DNA sequence as short as 500 or 600 bases. However,
clones larger than 1,000 bases have a higher likelihood of binding
to a unique chromosomal location with sufficient signal intensity
for simple detection. Preferably 1,000 bases, and more preferably
2,000 bases will suffice to get good results in a reasonable amount
of time.
[0141] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0142] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, e.g.,
Egeland et al. (1987) Nature 325: 783-787.
[0143] Moreover, differences in the DNA sequences between
individuals affected and a unaffected with a disease associated
with the RGS gene can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0144] 2. Tissue Typing
[0145] The RGS sequences of the present invention can also be used
to identify individuals from minute biological samples. The United
States military, for example, is considering the use of restriction
fragment length polymorphism (RFLP) for identification of its
personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes and probed on a
Southern blot to yield unique bands for identification. The
sequences of the present invention are useful as additional DNA
markers for RFLP (described in U.S. Pat. No. 5,272,057).
[0146] Furthermore, the sequences of the present invention can be
used to provide an alternative technique for determining the actual
base-by-base DNA sequence of selected portions of an individual's
genome. Thus, the RGS sequences of the invention can be used to
prepare two PCR primers from the 5' and 3' ends of the sequences.
These primers can then be used to amplify an individual's DNA and
subsequently sequence it.
[0147] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The RGS sequences of the
invention uniquely represent portions of the human genome. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. It is
estimated that allelic variation between individual humans occurs
with a frequency of about once per each 500 bases. Each of the
sequences described herein can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes. The noncoding sequences of SEQ ID NO:1 or
3 can comfortably provide positive individual identification with a
panel of perhaps 10 to 1,000 primers that each yield a noncoding
amplified sequence of 100 bases. If predicted coding sequences,
such as those in SEQ ID NO:1 or 3 are used, a more appropriate
number of primers for positive individual identification would be
500 to 2,000.
[0148] 3. Use of Partial RGS Sequences in Forensic Biology
[0149] DNA-based identification techniques can also be used in
forensic biology. In this manner, PCR technology can be used to
amplify DNA sequences taken from very small biological samples such
as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0150] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" that is unique to a
particular individual. As mentioned above, actual base sequence
information can be used for identification as an accurate
alternative to patterns formed by restriction enzyme generated
fragments. Sequences targeted to noncoding regions of SEQ ID NO:1
or 3 are particularly appropriate for this use as greater numbers
of polymorphisms occur in the noncoding regions, making it easier
to differentiate individuals using this technique. Examples of
polynucleotide reagents include the RGS sequences or portions
thereof, e.g., fragments derived from the noncoding regions of SEQ
ID NO:1 or 3 having a length of at least 20 or 30 bases.
[0151] The RGS sequences described herein can further be used to
provide polynucleotide reagents, e.g., labeled or labelable probes
that can be used in, for example, an in situ hybridization
technique, to identify a specific tissue. This can be very useful
in cases where a forensic pathologist is presented with a tissue of
unknown origin. Panels of such RGS probes, can be used to identify
tissue by species and/or by organ type.
[0152] In a similar fashion, these reagents, e.g., RGS primers or
probes can be used to screen tissue culture for contamination
(i.e., screen for the presence of a mixture of different types of
cells in a culture).
[0153] C. Predictive Medicine
[0154] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. These applications are described in the
subsections below.
[0155] 1. Diagnostic Assays
[0156] One aspect of the present invention relates to diagnostic
assays for detecting RGS protein and/or nucleic acid expression as
well as RGS activity, in the context of a biological sample. An
exemplary method for detecting the presence or absence of RGS
proteins in a biological sample involves obtaining a biological
sample from a test subject and contacting the biological sample
with a compound or an agent capable of detecting RGS protein or
nucleic acid (e.g., mRNA, genomic DNA) that encodes RGS protein
such that the presence of RGS protein is detected in the biological
sample. Results obtained with a biological sample from the test
subject may be compared to results obtained with a biological
sample from a control subject.
[0157] A preferred agent for detecting RGS mRNA or genomic DNA is a
labeled nucleic acid probe capable of hybridizing to RGS mRNA or
genomic DNA. The nucleic acid probe can be, for example, a
full-length RGS nucleic acid, such as the nucleic acid of SEQ ID
NO:1 or 3, or a portion thereof, such as a nucleic acid molecule of
at least 15, 30, 50, 100, 250, or 500 nucleotides in length and
sufficient to specifically hybridize under stringent conditions to
RGS mRNA or genomic DNA. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
[0158] A preferred agent for detecting RGS protein is an antibody
capable of binding to RGS protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab).sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a fluorescently
labeled secondary antibody and end-labeling of a DNA probe with
biotin such that it can be detected with fluorescently labeled
streptavidin.
[0159] The term "biological sample" is intended to include tissues,
cells, and biological fluids isolated from a subject, as well as
tissues, cells, and fluids present within a subject. That is, the
detection method of the invention can be used to detect RGS mRNA,
protein, or genomic DNA in a biological sample in vitro as well as
in vivo. For example, in vitro techniques for detection of RGS mRNA
include Northern hybridizations and in situ hybridizations. In
vitro techniques for detection of RGS protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations,
and immunofluorescence. In vitro techniques for detection of RGS
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of RGS protein include introducing into a
subject a labeled anti-RGS antibody. For example, the antibody can
be labeled with a radioactive marker whose presence and location in
a subject can be detected by standard imaging techniques.
[0160] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a peripheral blood leukocyte sample isolated by conventional
means from a subject.
[0161] The invention also encompasses kits for detecting the
presence of RGS proteins in a biological sample (a test sample).
Such kits can be used to determine if a subject is suffering from
or is at increased risk of developing a disorder associated with
aberrant expression of RGS protein (e.g., an immunological
disorder). For example, the kit can comprise a labeled compound or
agent capable of detecting RGS protein or mRNA in a biological
sample and means for determining the amount of an RGS protein in
the sample (e.g., an anti-RGS antibody or an oligonucleotide probe
that binds to DNA encoding an RGS protein, e.g., SEQ ID NO:1 or 3).
Kits can also include instructions for observing that the tested
subject is suffering from or is at risk of developing a disorder
associated with aberrant expression of RGS sequences if the amount
of RGS protein or mRNA is above or below a normal level.
[0162] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) that binds
to RGS protein; and, optionally, (2) a second, different antibody
that binds to RGS protein or the first antibody and is conjugated
to a detectable agent. For oligonucleotide-based kits, the kit can
comprise, for example: (1) an oligonucleotide, e.g., a detectably
labeled oligonucleotide, that hybridizes to an RGS nucleic acid
sequence or (2) a pair of primers useful for amplifying an RGS
nucleic acid molecule.
[0163] The kit can also comprise, e.g., a buffering agent, a
preservative, or a protein stabilizing agent. The kit can also
comprise components necessary for detecting the detectable agent
(e.g., an enzyme or a substrate). The kit can also contain a
control sample or a series of control samples that can be assayed
and compared to the test sample contained. Each component of the
kit is usually enclosed within an individual container, and all of
the various containers are within a single package along with
instructions for observing whether the tested subject is suffering
from or is at risk of developing a disorder associated with
aberrant expression of RGS proteins.
[0164] 2. Prognostic Assays
[0165] The methods described herein can furthermore be utilized as
diagnostic or prognostic assays to identify subjects having or at
risk of developing a disease or disorder associated with RGS
protein, RGS nucleic acid expression, or RGS activity. Prognostic
assays can be used for prognostic or predictive purposes to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with RGS protein, RGS
nucleic acid expression, or RGS activity.
[0166] Thus, the present invention provides a method in which a
test sample is obtained from a subject, and RGS protein or nucleic
acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of
RGS protein or nucleic acid is diagnostic for a subject having or
at risk of developing a disease or disorder associated with
aberrant RGS expression or activity. As used herein, a "test
sample" refers to a biological sample obtained from a subject of
interest. For example, a test sample can be a biological fluid
(e.g., serum), cell sample, or tissue.
[0167] Furthermore, using the prognostic assays described herein,
the present invention provides methods for determining whether a
subject can be administered a specific agent (e.g., an agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, small
molecule, or other drug candidate) or class of agents (e.g., agents
of a type that decrease RGS activity) to effectively treat a
disease or disorder associated with aberrant RGS expression or
activity. In this manner, a test sample is obtained and RGS protein
or nucleic acid is detected. The presence of RGS protein or nucleic
acid is diagnostic for a subject that can be administered the agent
to treat a disorder associated with aberrant RGS expression or
activity.
[0168] The methods of the invention can also be used to detect
genetic lesions or mutations in an RGS gene, thereby determining if
a subject with the lesioned gene is at risk for a disorder
characterized by aberrant cell proliferation and/or
differentiation. In preferred embodiments, the methods include
detecting, in a sample of cells from the subject, the presence or
absence of a genetic lesion or mutation characterized by at least
one of an alteration affecting the integrity of a gene encoding an
RGS-protein, or the misexpression of the RGS gene. For example,
such genetic lesions or mutations can be detected by ascertaining
the existence of at least one of: (1) a deletion of one or more
nucleotides from an RGS gene; (2) an addition of one or more
nucleotides to an RGS gene; (3) a substitution of one or more
nucleotides of an RGS gene; (4) a chromosomal rearrangement of an
RGS gene; (5) an alteration in the level of a messenger RNA
transcript of an RGS gene; (6) an aberrant modification of an RGS
gene, such as of the methylation pattern of the genomic DNA; (7)
the presence of a non-wild-type splicing pattern of a messenger RNA
transcript of an RGS gene; (8) a non-wild-type level of an
RGS-protein; (9) an allelic loss of an RGS gene; and (10) an
inappropriate post-translational modification of an RGS-protein. As
described herein, there are a large number of assay techniques
known in the art that can be used for detecting lesions in an RGS
gene. Any cell type or tissue, preferably peripheral blood
leukocytes, in which RGS proteins are expressed may be utilized in
the prognostic assays described herein.
[0169] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction (PCR) (see,
e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran et al. (1988) Science 241: 1077-1080; and
Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91: 360-364), the
latter of which can be particularly useful for detecting point
mutations in the RGS-gene (see, e.g., Abravaya et al. (1995)
Nucleic Acids Res. 23: 675-682). It is anticipated that PCR and/or
LCR may be desirable to use as a preliminary amplification step in
conjunction with any of the techniques used for detecting mutations
described herein.
[0170] Alternative amplification methods include self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87: 1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta
Replicase (Lizardi et al. (1988) Bio/Technology 6: 1197), or any
other nucleic acid amplification method, followed by the detection
of the amplified molecules using techniques well known to those of
skill in the art. These detection schemes are especially useful for
the detection of nucleic acid molecules if such molecules are
present in very low numbers.
[0171] In an alternative embodiment, mutations in an RGS gene from
a sample cell can be identified by alterations in restriction
enzyme cleavage patterns of isolated test sample and control DNA
digested with one or more restriction endonucleases. Moreover, the
use of sequence specific ribozymes (see, e.g., U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0172] In other embodiments, genetic mutations in an RGS molecule
can be identified by hybridizing a sample and control nucleic
acids, e.g., DNA or RNA, to high density arrays containing hundreds
or thousands of oligonucleotides probes (Cronin et al. (1996) Human
Mutation 7: 244-255; Kozal et al. (1996) Nature Medicine 2:
753-759). In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the RGS
gene and detect mutations by comparing the sequence of the sample
RGS gene with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxim and Gilbert (1977) Proc. Natl. Acad. Sci. USA
74: 560) or Sanger (1977) Proc. Natl. Acad. Sci. USA 74: 5463). It
is also contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Bio/Techniques 19: 448), including sequencing by mass
spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36: 127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38: 147-159).
[0173] Other methods for detecting mutations in the RGS gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230: 1242). See, also Cotton et al. (1988)
Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba et al. (1992) Methods
Enzymol. 217: 286-295. In a preferred embodiment, the control DNA
or RNA can be labeled for detection.
[0174] In still another embodiment, the mismatch cleavage reaction
employs one or more "DNA mismatch repair" enzymes that recognize
mismatched base pairs in double-stranded DNA in defined systems for
detecting and mapping point mutations in RGS cDNAs obtained from
samples of cells. See, e.g., Hsu et al. (1994) Carcinogenesis 15:
1657-1662. According to an exemplary embodiment, a probe based on
an RGS sequence, e.g., a wild-type RGS sequence, is hybridized to a
cDNA or other DNA product from a test cell(s). The duplex is
treated with a DNA mismatch repair enzyme, and the cleavage
products, if any, can be detected from electrophoresis protocols or
the like. See, e.g., U.S. Pat. No. 5,459,039.
[0175] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in RGS genes. For
example, single-strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild-type nucleic acids (Orita et al. (1989) Proc. Natl. Acad.
Sci. USA 86: 2766; see also Cotton (1993) Mutat. Res. 285: 125-144;
Hayashi (1992) Genet. Anal. Tech. Appl. 9: 73-79). The sensitivity
of the assay may be enhanced by using RNA (rather than DNA), in
which the secondary structure is more sensitive to a change in
sequence. In a preferred embodiment, the subject method utilizes
heteroduplex analysis to separate double-stranded heteroduplex
molecules on the basis of changes in electrophoretic mobility (Keen
et al. (1991) Trends Genet. 7: 5).
[0176] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313: 495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys.
Chem. 265: 12753).
[0177] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions that permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324: 163); Saiki
et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6230). Such
allele-specific oligonucleotides are hybridized to PCR-amplified
target DNA or a number of different mutations when the
oligonucleotides are attached to the hybridizing membrane and
hybridized with labeled target DNA.
[0178] Alternatively, allele-specific amplification technology,
which depends on selective PCR amplification, may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule so that amplification
depends on differential hybridization (Gibbs et al. (1989) Nucleic
Acids Res. 17: 2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent or reduce
polymerase extension (Prossner (1993) Tibtech 11: 238). In
addition, it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6: 1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci. USA 88: 189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0179] The methods described herein may be performed, for example,
by utilizing prepackaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving an RGS gene.
[0180] 3. Pharmacogenomics
[0181] Agents, or modulators that have a stimulatory or inhibitory
effect on RGS activity (e.g., RGS gene expression) as identified by
a screening assay described herein, can be administered to
individuals to treat (prophylactically or therapeutically)
disorders associated with aberrant RGS activity as well as to
modulate the phenotype of an immune response. In conjunction with
such treatment, the pharmacogenomics (i.e., the study of the
relationship between an individual's genotype and that individual's
response to a foreign compound or drug) of the individual may be
considered. Differences in metabolism of therapeutics can lead to
severe toxicity or therapeutic failure by altering the relation
between dose and blood concentration of the pharmacologically
active drug. Thus, the pharmacogenomics of the individual permits
the selection of effective agents (e.g., drugs) for prophylactic or
therapeutic treatments based on a consideration of the individual's
genotype. Such pharmacogenomics can further be used to determine
appropriate dosages and therapeutic regimens. Accordingly, the
activity of RGS protein, expression of RGS nucleic acid, or
mutation content of RGS genes in an individual can be determined to
thereby select appropriate agent(s) for therapeutic or prophylactic
treatment of the individual.
[0182] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, e.g.,
Linder (1997) Clin. Chem. 43(2): 254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body are referred to as "altered drug action." Genetic
conditions transmitted as single factors altering the way the body
acts on drugs are referred to as "altered drug-metabolism". These
pharmacogenetic conditions can occur either as rare defects or as
polymorphisms. For example, glucose-6-phosphate dehydrogenase
deficiency (G6PD) is a common inherited enzymopathy in which the
main clinical complication is haemolysis after ingestion of oxidant
drugs (antimalarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0183] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, a PM will show no therapeutic
response, as demonstrated for the analgesic effect of codeine
mediated by its CYP2D6-formed metabolite morphine. The other
extreme are the so called ultra-rapid metabolizers who do not
respond to standard doses. Recently, the molecular basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
[0184] Thus, the activity of RGS protein, expression of RGS nucleic
acid, or mutation content of RGS genes in an individual can be
determined to thereby select appropriate agent(s) for therapeutic
or prophylactic treatment of the individual. In addition,
pharmacogenetic studies can be used to apply genotyping of
polymorphic alleles encoding drug-metabolizing enzymes to the
identification of an individual's drug responsiveness phenotype.
This knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
an RGS modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0185] 4. Monitoring of Effects During Clinical Trials
[0186] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of RGS genes (e.g., the ability to
modulate aberrant cell proliferation and/or differentiation) can be
applied not only in basic drug screening but also in clinical
trials. For example, the effectiveness of an agent, as determined
by a screening assay as described herein, to increase or decrease
RGS gene expression, protein levels, or protein activity, can be
monitored in clinical trials of subjects exhibiting decreased or
increased RGS gene expression, protein levels, or protein activity.
In such clinical trials, RGS expression or activity and preferably
that of other genes that have been implicated in for example, a
cellular proliferation disorder, can be used as a marker of the
immune responsiveness of a particular cell.
[0187] For example, and not by way of limitation, genes that are
modulated in cells by treatment with an agent (e.g., compound,
drug, or small molecule) that modulates RGS activity (e.g., as
identified in a screening assay described herein) can be
identified. Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of RGS genes and other genes implicated in the disorder.
The levels of gene expression (i.e., a gene expression pattern) can
be quantified by Northern blot analysis or RT-PCR, as described
herein, or alternatively by measuring the amount of protein
produced, by one of the methods as described herein, or by
measuring the levels of activity of RGS genes or other genes. In
this way, the gene expression pattern can serve as a marker,
indicative of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at
various points during, treatment of the individual with the
agent.
[0188] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (1) obtaining a preadministration sample
from a subject prior to administration of the agent; (2) detecting
the level of expression of an RGS protein, mRNA, or genomic DNA in
the preadministration sample; (3) obtaining one or more
postadministration samples from the subject; (4) detecting the
level of expression or activity of the RGS protein, mRNA, or
genomic DNA in the postadministration samples; (5) comparing the
level of expression or activity of the RGS protein, mRNA, or
genomic DNA in the preadministration sample with the RGS protein,
mRNA, or genomic DNA in the postadministration sample or samples;
and (vi) altering the administration of the agent to the subject
accordingly to bring about the desired effect, i.e., for example,
an increase or a decrease in the expression or activity of an RGS
protein.
[0189] C. Methods of Treatment
[0190] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant RGS expression or activity. Additionally, the compositions
of the invention find use in modulating the T-lymphocyte response.
Thus, therapies for immune and respiratory disorders are
encompassed herein.
[0191] 1. Prophylactic Methods
[0192] In one aspect, the invention provides a method for
preventing in a subject a disease or condition associated with an
aberrant RGS expression or activity by administering to the subject
an agent that modulates RGS expression or at least one RGS gene
activity. Subjects at risk for a disease that is caused, or
contributed to, by aberrant RGS expression or activity can be
identified by, for example, any or a combination of diagnostic or
prognostic assays as described herein. Administration of a
prophylactic agent can occur prior to the manifestation of symptoms
characteristic of the RGS aberrancy, such that a disease or
disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of RGS aberrancy, for example,
an RGS agonist or RGS antagonist agent can be used for treating the
subject. The appropriate agent can be determined based on screening
assays described herein.
[0193] 2. Therapeutic Methods
[0194] Another aspect of the invention pertains to methods of
modulating RGS expression or activity for therapeutic purposes. The
modulatory method of the invention involves contacting a cell with
an agent that modulates one or more of the activities of RGS
protein activity associated with the cell. An agent that modulates
RGS protein activity can be an agent as described herein, such as a
nucleic acid or a protein, a naturally-occurring cognate ligand of
an RGS protein, a peptide, an RGS peptidomimetic, or other small
molecule. In one embodiment, the agent stimulates one or more of
the biological activities of RGS protein. Examples of such
stimulatory agents include active RGS protein and a nucleic acid
molecule encoding an RGS protein that has been introduced into the
cell. In another embodiment, the agent inhibits one or more of the
biological activities of RGS protein. Examples of such inhibitory
agents include antisense RGS nucleic acid molecules and anti-RGS
antibodies.
[0195] These modulatory methods can be performed in vitro (e.g., by
culturing the cell with the agent) or, alternatively, in vivo (e.g,
by administering the agent to a subject). As such, the present
invention provides methods of treating an individual afflicted with
a disease or disorder characterized by aberrant expression or
activity of an RGS protein or nucleic acid molecule. In one
embodiment, the method involves administering an agent (e.g., an
agent identified by a screening assay described herein), or a
combination of agents, that modulates (e.g., upregulates or
downregulates) RGS expression or activity. In another embodiment,
the method involves administering an RGS protein or nucleic acid
molecule as therapy to compensate for reduced or aberrant RGS
expression or activity.
[0196] Stimulation of RGS activity is desirable in situations in
which an RGS protein is abnormally downregulated and/or in which
increased RGS activity is likely to have a beneficial effect.
Conversely, inhibition of RGS activity is desirable in situations
in which RGS activity is abnormally upregulated and/or in which
decreased RGS activity is likely to have a beneficial effect.
[0197] This invention is further illustrated by the following
examples, which should not be construed as limiting.
Experimental
[0198] Cloning Strategy
[0199] Using the database mining strategy for expressed sequence
tags (ESTs) with similarity to the Regulator of G-protein Signaling
(RGS) domain ESTs, jthsa069c04t1 (human spleen cDNA library) and
jtmea012d11t1 (mouse megakaryocyte cDNA library) were identified
for human 16395 (h16395) and the mouse orthologue 1975 (m1975),
respectively. 5'-RACE of human or mouse spleen Marathon-ready cDNA
libraries (Clontech) was used to complete full-length cloning.
[0200] h16395 and m1975 cDNA Sequences
[0201] For h16395 a nucleotide sequence of 2156 nucleotides (nt)
was obtained which included the entire open reading frame,
polyadenylation sequence and poly-A tail. The nucleotide sequence
length was in agreement with the mRNA transcript size of
approximately 2.4 kb. An open reading frame of 235 amino acids
(MW=27.6 kDa) was identified for both h16395 and m1975, with the
RGS domain being present between amino acids 82 and 201 (FIGS. 1A
and B). 84% identity was observed between h16395 and m1975 (FIG.
1C). Importantly, this high degree of identity was observed within
and outside the RGS domain of h16395 and m1975, indicating they are
orthologues. Typically, different RGSs only exhibit a high degree
of identity within the RGS domain. Using the Kyte Doolittle
hydrophilicity analysis, hydrophobic NH2-- (approximately 1-15
amino acids (aa)) and COOH-ends (approximately 210-235 aa) were
identified.
[0202] No proteins were found to be identical to h16395 or m1975
sequences in the Nucleotide and Preview Nucleotide, MAPEST, DBEST,
and Patent; or the Patent, PDB, PNU, and Protein databases using
TBlastN or BlastP, respectively. Furthermore, no matches were
identified when the NH2-- (1-81 aa) or COOH-- (202-235 aa) ends
were used to search the protein databases with BlastP. However, RGS
protein matches showing similarity to the RGS domains (82-201 aa)
of h16395 and m1975 were identified in the databases. Of those
identified, the top 6 matches included mRGS2 (59%), hRGS2 (58%),
hRGS5 (55%), mRGS5 (55%), hRGS4 (52%), and mRGS4 (52%), with the
percent identity for the RGS domain shown in parentheses. The RGS
domain of h16395/m1975 is present at the COOH-end, and contains the
majority of residues that have been shown in RGS4 to make direct
contact with G.alpha..sub.i or that form the RGS domain hydrophobic
core.
[0203] Expression Pattern
[0204] An mRNA transcript of approximately 2.4 kb was detected for
h16395 or m1975. h16395 was most abundant in peripheral blood
leukocytes and fetal liver. Lower levels of expression were
detected in the spleen, bone marrow, and liver; and to a lesser
extent in the heart, colon, and placenta. Transcripts were detected
in T-cells, monocytes, and granulocytes by RT-PCR. 3'-UTR probes
were used to avoid cross-hybridization with other RSG proteins.
m1975 exhibited an expression pattern consistent with h16395. The
presence of the h16395/m1975 transcript in non-lymphoid tissues may
be due to blood contamination.
[0205] Discussion
[0206] h16395 and m1975 are novel human and mouse RGS orthologues.
These proteins contain an RGS domain that is most likely functional
due to the presence of key amino acids important for G.alpha.
binding and for forming the RGS domain hydrophobic core.
Furthermore, the carboxyl location of the RGS domain is consistent
with RGSs known to act as GTPase activating proteins (GAPs) for
G.alpha. proteins.
[0207] The hydrophobic amino-end of h16395/m1975 is consistent with
several other "short-form" RGSs including RGS 1, 2, 4, 5, 8, and
RATH/A28-RGS14/RGSr/RGS16. It has been shown that RGSs 4, 5, and 16
contain a plasma membrane signal sequence within this region, which
may be involved in targeting the RGS to the G.alpha. protein
cellular location. Hence, the hydrophobic amino end of h16395/m1975
may be important for localizing these RGSs to their site of
cellular function. Interestingly, "long-form" (RGS3, 6, 7, 9, 11,
12, and 14) and some "short-form" RGSs (GAIP, RGZ1, RGS10 and 13)
lack a hydrophobic amino-end, but usually contain other domains
that may play a role in cellular targeting.
[0208] The relatively high levels of h16395 in lymphoid tissues and
cells derived from these organs is consistent with a role for this
RGS in immune cell function. Hence, h16395 may play a role in
regulating the cellular response to chemoattractant stimulation,
and in doing so effect the deactivation or activation of several
intracellular pathways. This is because the majority of chemokine
receptors couple through G.alpha..sub.i and occasionally
G.alpha..sub.q, both targets of RGS proteins. RGSs accelerate the
slow intrinsic GTPase activity of the G.alpha. subunit of
heterotrimeric G proteins. This leads to the deactivation of the
GPCR signaling pathway due to reassociation of the G.alpha. and
G.beta..gamma. subunits, preventing their ability to interact with
downstream effector molecules. While the invention is not bound by
any mechanism of action, RGSs may activate the GPCR signaling
pathway by increasing the cycling rate from the active G.alpha. and
G.beta..gamma. states to the inactive heterotrimeric subunit state,
thereby increasing the level of G-protein substrate for activation
by the GPCR after ligand interaction.
[0209] The importance of chemokine receptors in a wide range of
biological processes is reflected by their expression on both
leukocyte and non-leukocyte cells. Hence, RGSs h16395/m1975 may be
useful in modulating both immune and non-immune cell function,
particularly, in the deactivation and/or activation of
intracellular pathways, resulting in the directed chemotaxis,
adhesion, localization, and prevention of cells to respond to
further chemoattractant stimulation. Non-inflammatory (e.g., cell
migration during development) or inflammatory stimuli are likely to
be responsible for this leukocyte migration. The "hallmark" of
inflammation is the infiltration of specific leukocyte subsets from
the blood into affected tissues. A variety of chemoattractants
(chemokines and classical chemoattractants including formyl
peptides, C5a, leukotriene B4 and the like) and their receptors
control the directed migration of leukocytes to inflammatory sites.
Most of these chemoattractants mediate their activity by G-protein
coupled receptor (GPCR) stimulation of inflammatory migrating cells
through heterotrimeric G-protein-dependent or independent pathways.
Chemokines can also regulate recruitment of T-lymphocytes in
non-inflammatory situations, e.g., lymphocytes must move through
tissue compartments during their development and differentiation.
Chemokine receptors also play a role in the expression of adhesion
molecules during the chemotactic response. Intracellular pathways
that have been implicated with chemokine receptor signaling during
these cellular responses include calcium mobilization, kinase
activation, tyrosine phosphorylation, low molecular weight
G-protein regulation, and STAT/JAK activation.
[0210] h16395/m1975 are likely to be important in the regulation of
chemokine receptor signaling during T-lymphocyte activation and
differentiation, depending on their expression pattern and receptor
specificity. The precise pattern of chemokine receptor expression
depends on the T-cell activation state. In resting T-cells, the
chemokine receptor CXCR4 is only expressed. In Th1 and Th2 cells,
the chemokine receptors CXCR4, CCR1, and CCR2 are expressed.
Several chemokine receptors are also specifically expressed in Th1
versus Th2 cells including CXCR3 and CCR5 in the former and CCR3,
CCR4, and CCR7 in the latter.
[0211] The low expression levels of h16395 in post-mitotic tissues,
and the relatively high levels in the more mitotic tissues is
consistent with a role for this RGS in cell proliferation. GPCRs
are known to be expressed in proliferating cells and many ligands
acting via these receptors are known to elicit a mitogenic
response. Furthermore, overexpression of the p53-responsive gene
A28-RGS 14 inhibits both G.sub.i and G.sub.q coupled growth factor
receptor mediated activation of the MAPK pathway. Such a pathway
had been implicated in proliferation, transformation, and
oncogenesis.
[0212] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0213] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
4 1 2217 DNA Homo sapiens CDS (160)..(867) 1 gaattcggct tccatcctaa
tacgactcac tatagggctc gagcggccgc ccgggcaggt 60 ataacttttt
attctactat gtatatgtat ggaatagtat taataaatga actagggaag 120
gatgtaataa attagacatc tcttcatttt agagagaag atg gaa aca aca ttg 174
Met Glu Thr Thr Leu 1 5 ctt ttc ttt tct caa ata aat atg tgt gaa tca
aaa gaa aaa act ttt 222 Leu Phe Phe Ser Gln Ile Asn Met Cys Glu Ser
Lys Glu Lys Thr Phe 10 15 20 ttc aag tta ata cat ggt tca gga aaa
gaa gaa aca agc aaa gaa gcc 270 Phe Lys Leu Ile His Gly Ser Gly Lys
Glu Glu Thr Ser Lys Glu Ala 25 30 35 aaa atc aga gct aag gaa aaa
aga aat aga cta agt ctt ctt gtg cag 318 Lys Ile Arg Ala Lys Glu Lys
Arg Asn Arg Leu Ser Leu Leu Val Gln 40 45 50 aaa cct gag ttt cat
gaa gac acc cgc tcc agt aga tct ggg cac ttg 366 Lys Pro Glu Phe His
Glu Asp Thr Arg Ser Ser Arg Ser Gly His Leu 55 60 65 gcc aaa gaa
aca aga gtc tcc cct gaa gag gca gtg aaa tgg ggt gaa 414 Ala Lys Glu
Thr Arg Val Ser Pro Glu Glu Ala Val Lys Trp Gly Glu 70 75 80 85 tca
ttt gac aaa ctg ctt tcc cat aga gat gga cta gag gct ttt acc 462 Ser
Phe Asp Lys Leu Leu Ser His Arg Asp Gly Leu Glu Ala Phe Thr 90 95
100 aga ttt ctt aaa act gaa ttc agt gaa gaa aat att gaa ttt tgg ata
510 Arg Phe Leu Lys Thr Glu Phe Ser Glu Glu Asn Ile Glu Phe Trp Ile
105 110 115 gcc tgt gaa gat ttc aag aaa agc aag gga cct caa caa att
cac ctt 558 Ala Cys Glu Asp Phe Lys Lys Ser Lys Gly Pro Gln Gln Ile
His Leu 120 125 130 aaa gca aaa gca ata tat gag aaa ttt ata cag act
gat gcc cca aaa 606 Lys Ala Lys Ala Ile Tyr Glu Lys Phe Ile Gln Thr
Asp Ala Pro Lys 135 140 145 gag gtt aac ctt gat ttt cac aca aaa gaa
gtc att aca aac agc atc 654 Glu Val Asn Leu Asp Phe His Thr Lys Glu
Val Ile Thr Asn Ser Ile 150 155 160 165 act caa cct acc ctc cac agt
ttt gat gct gca caa agc aga gtg tat 702 Thr Gln Pro Thr Leu His Ser
Phe Asp Ala Ala Gln Ser Arg Val Tyr 170 175 180 cag ctc atg gaa caa
gac agt tat aca cgt ttt ctg aaa tct gac atc 750 Gln Leu Met Glu Gln
Asp Ser Tyr Thr Arg Phe Leu Lys Ser Asp Ile 185 190 195 tat tta gac
ttg atg gaa gga aga cct cag aga cca aca aat ctt agg 798 Tyr Leu Asp
Leu Met Glu Gly Arg Pro Gln Arg Pro Thr Asn Leu Arg 200 205 210 aga
cga tca cgc tca ttt acc tgc aat gaa ttc caa gat gta caa tca 846 Arg
Arg Ser Arg Ser Phe Thr Cys Asn Glu Phe Gln Asp Val Gln Ser 215 220
225 gat gtt gcc att tgg tta taa agaaaattga ttttgctcat ttttatgaca
897 Asp Val Ala Ile Trp Leu 230 235 aacttataca tctgcttcta
acatatcgca tgtttatgtt aagatttggt cccatccttt 957 aaactgaaat
atgtcatgtg aaattatttt aaaaatgtaa aaacaaaact ttctgctaac 1017
aaaatacata cagtatctgc cagtatattc tgtaaaacct tctatttgat gtcattccat
1077 ttataatcag aaaaaaaact tatttcttaa tcaaaaggca gtacaaaaaa
agtaataatg 1137 ttttataaga ttgtagagtt aagtaaaagt taagcttttg
caaagttgtc aaaagttcaa 1197 acaaaagtct agttgggatt ttttaccaaa
gcagcataat atgtgttata taaacataat 1257 aatactcaga tatccaaatg
ttcagatagc atttttcata atgaatgttc tctttttttt 1317 ggtaatagtg
tagaagtgat ctggttctta caatgggaga tgaagaacat ttattattgg 1377
gttactacta accctgtccc aagaatagta atatcacctc tagttataag ccagcaacag
1437 gaacttttgt gaagacacat tcatctctac agaacttcag attaaatata
atctagatta 1497 atgactgaga ataagatcca catttgaact cattcctaag
tgaacatgga cgtacccagt 1557 tatacaaagt acttctgttg gtcacagaaa
catgaccaga ttttgcatat ctccaggtag 1617 ggaactaagt agactacctt
atcaccggct aagaaaactt gctactaaac tattaggcca 1677 tcaatggctt
gaataaaaac cagagaaggt ttttcccagg acgtctcatg tttggccctt 1737
tagaattggg gtagaaatca gaaatgagat gaggggaaga agcaaggagt ctaaggccct
1797 agcgatttgg gcatctgcca cattggttca tattcagaaa gtgttatctc
attgattata 1857 ttcttgttaa gcaaatctcc ttaagtaatt attattcaaa
taagattata ctcatacatc 1917 tatatgtcac tgttttaaag agatatttaa
tttttaatgt gtgttacatg gtctgtaaat 1977 atttgtattt aaaaatgcca
tgcattaggc tttggaaatt taatgttagt tgaaatgtaa 2037 aatgtgaaaa
ctttagatca tttgtagtaa taaatatttt taacttcatt catacagtta 2097
agtttatctg acaataaaag ctctgactga atgttgatta tccttcctat tatgtaataa
2157 ggaataaaca ttttcttctt ttagagtaaa aaaaaaaaaa aaaaaaaaaa
gggcggccgc 2217 2 235 PRT Homo sapiens 2 Met Glu Thr Thr Leu Leu
Phe Phe Ser Gln Ile Asn Met Cys Glu Ser 1 5 10 15 Lys Glu Lys Thr
Phe Phe Lys Leu Ile His Gly Ser Gly Lys Glu Glu 20 25 30 Thr Ser
Lys Glu Ala Lys Ile Arg Ala Lys Glu Lys Arg Asn Arg Leu 35 40 45
Ser Leu Leu Val Gln Lys Pro Glu Phe His Glu Asp Thr Arg Ser Ser 50
55 60 Arg Ser Gly His Leu Ala Lys Glu Thr Arg Val Ser Pro Glu Glu
Ala 65 70 75 80 Val Lys Trp Gly Glu Ser Phe Asp Lys Leu Leu Ser His
Arg Asp Gly 85 90 95 Leu Glu Ala Phe Thr Arg Phe Leu Lys Thr Glu
Phe Ser Glu Glu Asn 100 105 110 Ile Glu Phe Trp Ile Ala Cys Glu Asp
Phe Lys Lys Ser Lys Gly Pro 115 120 125 Gln Gln Ile His Leu Lys Ala
Lys Ala Ile Tyr Glu Lys Phe Ile Gln 130 135 140 Thr Asp Ala Pro Lys
Glu Val Asn Leu Asp Phe His Thr Lys Glu Val 145 150 155 160 Ile Thr
Asn Ser Ile Thr Gln Pro Thr Leu His Ser Phe Asp Ala Ala 165 170 175
Gln Ser Arg Val Tyr Gln Leu Met Glu Gln Asp Ser Tyr Thr Arg Phe 180
185 190 Leu Lys Ser Asp Ile Tyr Leu Asp Leu Met Glu Gly Arg Pro Gln
Arg 195 200 205 Pro Thr Asn Leu Arg Arg Arg Ser Arg Ser Phe Thr Cys
Asn Glu Phe 210 215 220 Gln Asp Val Gln Ser Asp Val Ala Ile Trp Leu
225 230 235 3 1164 DNA Mus sp. CDS (134)..(841) 3 tttttgtaag
aaaaatctga ggaaagattc gggatagcgc tttattcagg atgttttcct 60
atgaaatagc attcatctgt gggagagaga aggactaagg aaatctgaca tctgttggtc
120 actgggacag aat atg gat atg tca ctg gtt ttc ttc tct caa tta aat
169 Met Asp Met Ser Leu Val Phe Phe Ser Gln Leu Asn 1 5 10 atg tgt
gaa tca aaa gag aaa act ttt ttc aaa cta atg cat ggg tca 217 Met Cys
Glu Ser Lys Glu Lys Thr Phe Phe Lys Leu Met His Gly Ser 15 20 25
ggg aaa gaa gaa aca agc atc gag gcc aaa atc aga gcg aaa gaa aaa 265
Gly Lys Glu Glu Thr Ser Ile Glu Ala Lys Ile Arg Ala Lys Glu Lys 30
35 40 agg aat aga cta agt ctt ctc cta cag agg cct gac ttc cat gga
gag 313 Arg Asn Arg Leu Ser Leu Leu Leu Gln Arg Pro Asp Phe His Gly
Glu 45 50 55 60 act caa gcc agt aga tct gcc ctc ttg gcc aaa gaa aca
aga gtc tct 361 Thr Gln Ala Ser Arg Ser Ala Leu Leu Ala Lys Glu Thr
Arg Val Ser 65 70 75 cct gaa gaa gca gtg aaa tgg gct gaa tca ttt
gac aaa ttg ctc tct 409 Pro Glu Glu Ala Val Lys Trp Ala Glu Ser Phe
Asp Lys Leu Leu Ser 80 85 90 cat aga gat gga gtg gat gct ttt acc
aga ttt ctt aaa act gaa ttc 457 His Arg Asp Gly Val Asp Ala Phe Thr
Arg Phe Leu Lys Thr Glu Phe 95 100 105 agt gag gag aac att gaa ttt
tgg gtc gcc tgt gaa gac ttc aag aaa 505 Ser Glu Glu Asn Ile Glu Phe
Trp Val Ala Cys Glu Asp Phe Lys Lys 110 115 120 tgc aag gaa cct caa
caa atc atc cta aaa gca aag gca atc tat gag 553 Cys Lys Glu Pro Gln
Gln Ile Ile Leu Lys Ala Lys Ala Ile Tyr Glu 125 130 135 140 aaa ttc
att cag aat gat gcc ccc aaa gag gtt aac att gat ttt cat 601 Lys Phe
Ile Gln Asn Asp Ala Pro Lys Glu Val Asn Ile Asp Phe His 145 150 155
act aaa gaa gta att gct aag agc atc gcc cag ccc act ctc cac agt 649
Thr Lys Glu Val Ile Ala Lys Ser Ile Ala Gln Pro Thr Leu His Ser 160
165 170 ttt gat acg gca caa agc aga gtg tac cag ctc atg gaa cat gac
agt 697 Phe Asp Thr Ala Gln Ser Arg Val Tyr Gln Leu Met Glu His Asp
Ser 175 180 185 tat aaa cgc ttt ttg aaa tct gag acc tac tta cat ttg
ata gaa gga 745 Tyr Lys Arg Phe Leu Lys Ser Glu Thr Tyr Leu His Leu
Ile Glu Gly 190 195 200 aga cct cag aga cca aca aac ctt agg aga cga
tca cga tca ttt act 793 Arg Pro Gln Arg Pro Thr Asn Leu Arg Arg Arg
Ser Arg Ser Phe Thr 205 210 215 220 tac aat gat ttc caa gat gta aag
tca gat gtt gcc att tgg tta tga 841 Tyr Asn Asp Phe Gln Asp Val Lys
Ser Asp Val Ala Ile Trp Leu 225 230 235 gtaaaagtca tttgtcttct
tttgatagtg tatgtgtata tctaaaatat atactaatac 901 taatgtgtac
ttctaaaata tagcttgtgt ataagaagag atgatttcat ttttaaaata 961
caccatgcaa atacatatta aatgtaagaa ctttttatat tatactaaaa taattcatca
1021 tctatcttcc gaaatatttt atgaaaatct atctgatatt ctattctaat
aaaattcttt 1081 atttctacaa taacagtcag taagaagaag ctttgaagcc
gaattccagc acactggcgg 1141 ccggtactag tggatccgag ctc 1164 4 235 PRT
Mus sp. 4 Met Asp Met Ser Leu Val Phe Phe Ser Gln Leu Asn Met Cys
Glu Ser 1 5 10 15 Lys Glu Lys Thr Phe Phe Lys Leu Met His Gly Ser
Gly Lys Glu Glu 20 25 30 Thr Ser Ile Glu Ala Lys Ile Arg Ala Lys
Glu Lys Arg Asn Arg Leu 35 40 45 Ser Leu Leu Leu Gln Arg Pro Asp
Phe His Gly Glu Thr Gln Ala Ser 50 55 60 Arg Ser Ala Leu Leu Ala
Lys Glu Thr Arg Val Ser Pro Glu Glu Ala 65 70 75 80 Val Lys Trp Ala
Glu Ser Phe Asp Lys Leu Leu Ser His Arg Asp Gly 85 90 95 Val Asp
Ala Phe Thr Arg Phe Leu Lys Thr Glu Phe Ser Glu Glu Asn 100 105 110
Ile Glu Phe Trp Val Ala Cys Glu Asp Phe Lys Lys Cys Lys Glu Pro 115
120 125 Gln Gln Ile Ile Leu Lys Ala Lys Ala Ile Tyr Glu Lys Phe Ile
Gln 130 135 140 Asn Asp Ala Pro Lys Glu Val Asn Ile Asp Phe His Thr
Lys Glu Val 145 150 155 160 Ile Ala Lys Ser Ile Ala Gln Pro Thr Leu
His Ser Phe Asp Thr Ala 165 170 175 Gln Ser Arg Val Tyr Gln Leu Met
Glu His Asp Ser Tyr Lys Arg Phe 180 185 190 Leu Lys Ser Glu Thr Tyr
Leu His Leu Ile Glu Gly Arg Pro Gln Arg 195 200 205 Pro Thr Asn Leu
Arg Arg Arg Ser Arg Ser Phe Thr Tyr Asn Asp Phe 210 215 220 Gln Asp
Val Lys Ser Asp Val Ala Ile Trp Leu 225 230 235
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References