U.S. patent application number 09/797727 was filed with the patent office on 2002-06-20 for gamma retinoic acid receptor.
This patent application is currently assigned to THE SALK INSTITUTE FOR BIOLOGICAL STUDIES. Invention is credited to Evans, Ronald Mark, Imawari, Michio, Ishikawa, Takashi, Takaku, Fumimaro, Umesono, Kazuhiko.
Application Number | 20020077457 09/797727 |
Document ID | / |
Family ID | 23459521 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020077457 |
Kind Code |
A1 |
Takaku, Fumimaro ; et
al. |
June 20, 2002 |
Gamma retinoic acid receptor
Abstract
A novel gamma retinoic acid receptor is disclosed. The novel
receptor is encoded for by cDNA carried on plasmid
pGEM-hRAR.gamma., which has been deposited with the American Type
Culture Collection for patent purposes. Chimeric receptor proteins
are also disclosed. The chimera contain at least one functional
domain from the new gamma retinoic acid receptor.
Inventors: |
Takaku, Fumimaro; (Tokyo,
JP) ; Ishikawa, Takashi; (Tokyo, JP) ;
Imawari, Michio; (Tokyo, JP) ; Evans, Ronald
Mark; (La Jolla, CA) ; Umesono, Kazuhiko; (La
Jolla, CA) |
Correspondence
Address: |
Stephen E. Reiter
FOLEY & LARDNER
23rd Floor
402 West Broadway
San Diego
CA
92101
US
|
Assignee: |
THE SALK INSTITUTE FOR BIOLOGICAL
STUDIES
|
Family ID: |
23459521 |
Appl. No.: |
09/797727 |
Filed: |
March 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09797727 |
Mar 1, 2001 |
|
|
|
08486325 |
Jun 7, 1995 |
|
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Current U.S.
Class: |
530/350 ; 506/14;
536/23.1 |
Current CPC
Class: |
C07K 14/70567 20130101;
Y10S 530/826 20130101; Y10S 530/846 20130101; C07K 2319/80
20130101; C07K 2319/715 20130101; C07K 14/72 20130101; A61K 38/00
20130101; C07K 14/721 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
530/350 ; 435/6;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C07K 005/00; C07K 007/00; C07K 016/00; C07K
017/00; A61K 038/00; C07K 001/00; C07K 014/00 |
Claims
What is claimed is:
1. A substantially pure double-stranded DNA wherein the sense
strand encodes the primary sequence of a protein which has
ligand-binding and transcription-activating properties
characteristic of gamma retinoic acid receptor protein.
2. A substantially pure DNA according to claim 1 wherein the sense
strand encodes the primary sequence of a protein having substantial
amino acid homology with the sequence of amino acids shown in FIG.
1.
3. A substantially pure DNA according to claim 1 wherein the sense
strand encodes the primary sequence of a protein which has
ligand-binding and transcription-activating properties
characteristic of human gamma retinoic acid receptor protein.
4. The plasmid pGEM-hRAR.gamma. (ATCC No. 40623).
5. A substantially pure DNA sequence having substantial sequence
homology with the DNA (hRAR.gamma.) of plasmid pGEM-hRAR.gamma.
(ATCC No. 40623).
6. A substantially pure DNA segment which has (a) one triplet
corresponding to a translation stop codon, which is in frame with a
triplet corresponding to a translation start codon and downstream
of said start codon triplet, and (b) as longest open reading frame,
a substantially pure segment with a sequence of triplets encoding
the primary sequence of gamma retinoic acid receptor protein.
7. A substantially pure DNA encoding human gamma retinoic acid
receptor.
8. A substantially pure DNA having substantial sequence homology
with any of the DNA's claimed in any of claims 1-7.
9. A mutant of any of the DNA's claimed in any of claims 1-8.
10. A substantially pure mRNA transcribed from any of the
substantially pure DNA sequences claimed in any of claims of
1-9.
11. A substantially pure protein wherein the amino acid sequence
comprising the DNA binding domain has at least about 98% homology
with the amino acid sequence shown in FIG. 1.
12. A substantially pure protein encoded by any of the
substantially pure nucleic acid sequences claimed in any of claims
1-10.
13. Cells transfected by any of the substantially pure DNA's
claimed in any of claims 1-9.
14. Chimeric receptors having an N-termimus domain, a DNA-binding
domain, and a ligand-binding domain wherein at least one of the
domains is selected from the group consisting of the RAR.gamma.
N-terminus domain, the RAR.gamma. DNA-binding domain, and the
RAR.gamma. ligand-binding domain, and at least one of the remaining
domains is selected from the group consisting of N-terminus,
DNA-binding and ligand-binding domains from the glucocorticoid
receptor, the mineralocorticoid receptor, the progesterone
receptor, the estrogen receptor, the steroid-related receptors
(ERR1 and ERR2), the vitamin D.sub.3 receptor, the thyroid
receptors, the V-erb-A receptor, the E75 (Drosophilia) receptor,
and the retinoic acid receptors alpha and beta.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to ligand-responsive
regulatory proteins and genes encoding them. More particularly, the
present invention relates to a new retinoic acid receptor protein
and the gene that encodes it, modification of the new retinoic acid
receptor protein and gene by recombinant DNA and other genetic
engineering techniques, plus uses of the new retinoic acid receptor
protein and gene, both unmodified and modified.
BACKGROUND OF THE INVENTION
[0002] It is known that hormones like the glucocorticoid and
thyroid hormones enter cells by facilitated diffusion. It is also
known that hormones then bind to specific receptor proteins,
thereby creating a hormone/receptor complex. The binding of hormone
to the receptor initiates an alosteric alteration of the receptor
protein. As a result of this alteration, it is believed that the
hormone/receptor complex is capable of binding with high affinity
to certain specific sites on the chromatin DNA. Such sites, which
are referred to as hormone response elements or HRE's, modulate
expression of nearby target gene promoters.
[0003] A major obstacle to further understanding of the specifics
of gene regulation by exogenous inducers such as hormones has been
the lack of availability of receptor proteins in sufficient
quantity and purity to allow such proteins to be adequately
analyzed and characterized. This same lack of availability has
thwarted the use of receptors in diagnostic assays to determine the
presence of exogenous inducers (e.g., the hormones) in various body
fluids and tissues, as well as their use as "prototypes" for
engineering chimeric receptor protein analogs.
[0004] In an effort to overcome this lack of availability of
receptor proteins, scientific investigators are working to discover
the genes that encode such proteins. To date several such genes
have been disclosed and characterized. The cloned genes include
those encoding the following receptors: glucocorticoid,
mineralocorticoid, progesterone, estrogen, the two steroid-related
receptors (known in the art as ERR1 and ERR2), vitamin D.sub.3,
thyroid, V-erb-A, E75 (Drosophilia and two retinoid receptor
proteins, retinoic acid receptor alpha (RAR.alpha.) and retinoic
acid receptor beta (RAR.beta.). (See Giguere, et al. (1987)
regarding RAR.alpha., and Petkovich, et al. (1987) and Brand, et
al. (1988) regarding RAR.beta..).
[0005] This disclosure describes the isolation and characterization
of a cDNA encoding a third functional retinoid receptor protein
that is referred to herein as the gamma retinoic acid receptor
(RAR.gamma.). Like RARs alpha and beta, the new gamma retinoic acid
receptor has homology with the DNA-binding and ligand-binding
domains of the steroid and thyroid hormone receptors.
[0006] The retinoic acid receptor genes belong to the superfamily
of genes known as the steroid hormone receptor family. All genes in
this family can be divided into discrete regions or domains that
are sometimes referred to as regions A/B, C, D, E, and F. See FIG.
2; also see Robertson, (1987) and Evans, (1988). The C region
encodes the DNA-binding domain, the E region encodes the
ligand-binding domain and the F region encodes the carboxy-terminus
domain. The D region is believed to function as a "hinge". The
function of the A/B (or N-terminus) region is not entirely clear;
it may be involved with enhancement and repression of receptor
transcription activity. See for example, Hollenberg, et al. (1988)
and Oro, et al. (1988).
[0007] The present specification also discloses chimeric receptors
made by "swapping" functional domains between the new gamma
retinoic acid receptor and the glucocorticoid, the
mineralocorticoid, the progesterone, the estrogen, the
estrogen-related (ERR1 and ERR2), the vitamin D.sub.3 receptor, the
thyroid receptors, the V-erb-A receptor, the E75 (Drosophilia)
receptor and the alpha and beta retinoic acid receptors. These
chimeric receptors have hybrid functional characteristics based on
the "origin" of the "parental" DNA-binding and ligand-binding
domains incorporated within the chimeras. For example, if the
DNA-binding domain in the chimeric receptor is the gamma retinoic
acid receptor DNA-binding domain (i.e., is obtained from wild-type
gamma retinoic acid receptor or is a mutant that contains the
functional elements of the gamma retinoic acid DNA-binding domain),
then the chimera will have DNA-binding properties characteristic of
the gamma retinoic acid receptor. The same is true of the
ligand-binding domain.
DESCRIPTION OF THE DRAWINGS
[0008] The drawings comprise three figures of which:
[0009] FIG. 1 is a drawing which shows the DNA nucleotide sequence
and the primary protein sequence of hRAR.gamma. encoded by the
EcoRI fragment harbored in PGEM-hRAR.gamma..
[0010] FIG. 2 is a drawing that shows the amino acid comparison
among the three human RARs (alpha, beta and gamma).
[0011] FIGS. 3(A and B) is composed of two blots.
[0012] FIG. 3A shows induction of CAT activity and thus retinoic
acid-dependent transactivation by the protein encoded by the cDNA
insert of pGEM-hRAR.gamma..
[0013] FIG. 3B shows that hRAR.gamma. recognizes ERE and TRE, but
not GRE.
DEFINITIONS
[0014] In the present specification and claims, reference will be
made to phrases and terms of art which are expressly defined for
use herein as follows:
[0015] As used herein, the generic term "retinoids" means a group
of compounds which includes retinoic acid, vitamin A (retinol) and
a series of natural and synthetic derivatives that can exert
profound effects on development and differentiation in a wide
variety of systems.
[0016] As used herein, the human species is identified with a lower
case "h".
[0017] As used herein, "steroid hormone superfamily of receptors"
refers to the class of related receptors comprised of
glucocorticoid, mineralocorticoid, progesterone, estrogen,
estrogen-related (ERR1 and ERR2), vitamin D.sub.3, thyroid,
v-erb-A, E75 (Drosophilia) and the retinoic acid receptors. See
Evans (1988) and the references cited therein.
[0018] As used herein, RAR means retinoic acid receptor. The
acronym hRAR means human retinoic acid receptor. HRAR.alpha. refers
to human retinoic acid receptor alpha. See Giguere, et al., (1987).
HRAR.beta. refers to human retinoic acid receptor beta. See Brand,
et al., (1988). hRAR.gamma. refers to human retinoic acid receptor
gamma.
[0019] As used herein, GR means glucocorticoid receptor. hGR means
human glucocorticoid receptor.
[0020] As used herein, MR means mineralocorticoid receptor. hMR
means human mineralocorticoid receptor.
[0021] As used herein, T.sub.3R means thyroid hormone receptor
triiodthyronine. T.sub.3R.alpha. and T.sub.3R.beta. refer to the
alpha and beta forms of the thyroid receptor.
[0022] As used herein, ER means estrogen receptor.
[0023] As used herein, ERR means estrogen-related receptor. The
acronyms, hERR1 and hERR2 refer to human estrogen-related receptors
1 and 2. These receptors are more related to steroid receptors than
to the thyroid receptors, yet they do not bind any of the major
classes of known steroid hormones (Giguere, et al., 1988).
[0024] As used herein, VDR means vitamin D.sub.3 receptor.
[0025] As used herein, PR means progesterone receptor.
[0026] As used herein, CAT means chloramphenicol
acetyltransferase.
[0027] As used herein, CV-1 means mouse kidney cells from the cell
line referred to as "CV-1". CV-1 cells are receptor-deficient cells
that are useful in functional ligand identification assays.
[0028] As used herein, hormone response elements or HRE's mean
short cis-acting DNA sequences (about 20 bp in size) that are
required for hormonal (or ligand) activation of transcription. The
attachment of these elements to an otherwise hormone-nonresponsive
gene causes that gene to become hormone responsive. These sequences
function in a position- and orientation-independent fashion. Unlike
other transcriptional regulators, the activity of the HRE's is
dependent upon the presence or absence of ligand. See Evans (1988)
and the references cited therein.
[0029] As used herein, synthetic HRE's refer to HRE's that have
been synthesized in vitro using automated nucleotide synthesis
machines. Since the HRE's are only about 20 bp in size, they are
easily synthesized in this manner. If wild-type, engineered or
synthetic HREs are linked to hormone-nonresponsive promoters, these
promoters become hormone responsive. See Evans (1988) and the
references cited therein.
[0030] As used herein, the acronym GRE means glucocorticoid
response element and TRE means thyroid receptor response element.
(TRE.sub.p is a TRE that has been engineered to maximize the
palindrominicity of this response element.) GRE's are hormone
response elements that confer glucocorticoid responsiveness via
interaction with the GR. See Payvar, et al., Cell 35:381 (1983) and
Schiedereit, et al., Nature 304:749 (1983). GRE's can be used with
any wild-type or chimeric receptor whose DNA-binding domain can
functionally bind (i.e., activate) with the GRE. For example, since
GR, MR and PR receptors can all activate GRE's, a GRE can be used
with any wild-type or chimeric receptor that has a GR, MR or
PR-type DNA-binding domain. TRE's are similar to GRE's except that
they confer thyroid hormone responsiveness via interaction with TR.
TRE's can be used with any wild-type or chimeric receptor whose
DNA-binding domain can functionally bind (i.e., activate) with the
TRE. Both thyroid and retinoic acid receptors can activate TRE's,
so a TRE can be used with any receptor that has a TR or RAR-type
DNA-binding domain.
[0031] As used herein, ligand means an inducer, such as a hormone
or growth substance. Inside a cell, the ligand binds to a receptor
protein, thereby creating a ligand/receptor complex, which in turn
can bind to an appropriate hormone response element. Single ligands
may have multiple receptors. For example, both the
T.sub.3R.sub..alpha. and the T.sub.3R.sub..beta. bind thyroid
hormone such as T.sub.3.
[0032] As used herein, the phrase "DNA-binding domain" refers to
that portion of the receptor protein (such as glucocorticoid,
mineralocorticoid, progesterone, estrogen, estrogen-related
receptors, vitamin D.sub.3, thyroid, v-erb-A, E75 (Drosophilia) and
the retinoic acid receptors) that binds to HRE sites on the
chromatin DNA. The boundaries for these DNA-binding domains have
been identified and characterized for the steroid hormone
superfamily. See Evans (1988) and the references cited therein.
[0033] The DNA-binding domains of the steroid hormone superfamily
of receptors consist of an amino segment varying between 66 to 68
amino acids in length. This segment contains 9 cysteine residues,
one of which is the first amino acid of the segment. This first Cys
residue begins a motif described as
Cys-X.sub.2-Cys-X.sub.13-15-Cys-X.sub.2-Cys, where X is any amino
acid residue. The DNA-binding domain invariably ends with the amino
acids Gly-Met.
[0034] For convenience in the cloning procedure, between 1 and 6
amino acids residues preceding and/or following the DNA-binding
domain can be switched along with the DNA-binding domain.
[0035] As used herein, the phrase "ligand-binding domain region"
refers to that portion of the receptor proteins that binds to
ligands such as growth substances or hormones. These boundaries of
the ligand-binding domains for the steroid receptor superfamily
have been identified and characterized. See Evans (1988) and the
references cited therein.
[0036] Common restriction endonuclease sites must be introduced
into receptor cDNA clones to allow exchange of functional domains
between receptors. In any of the various receptors in the steroid
receptor superfamily of genes, the first common site can be
introduced immediately preceding the DNA-binding domain, the second
common site immediately following it. (For example, in any of the
steroid hormone superfamily, a unique NotI site can be introduced
immediately preceding the region of the cDNA encoding the
DNA-binding domain and a unique XhoI site can be introduced
immediately following it. This divides the receptors into three
functional regions or "cassettes"; (1) an N-terminus cassette, (2)
a DNA-binding domain cassette, and (3) a ligand-binding domain
cassette. The three regions or cassettes from any one receptor can
be combined with cassettes from other receptors to create a variety
of chimeric receptors.
[0037] As used herein, "mutant" DNA refers to DNA which has been
genetically engineered to be different from the "wild-type" or
unmodified sequence. Such genetic engineering can include the
insertion of nucleotides into wild-type sequences, deletion of
nucleotides from wild-type sequences, substitution of nucleotides
in the wild-type sequences, or "swapping" of functional domains
from one receptor to another. Receptors that have been engineered
by "swapping" functional domains from one receptor to another are
also referred to as chimeric or hybrid receptors. Chimeric
receptors can be further engineered by insertion of nucleotides,
deletion of nucleotides, substitution of nucleotides, etc.
[0038] Use of the term "substantial sequence homology" in the
present specification and claims refers to DNA, RNA, or amino acid
sequences that have slight and non-consequential sequence
variations from the actual sequences disclosed and claimed herein
and means that these sequences are within the scope of the appended
claims. In this regard, the "slight and non-consequential" sequence
variations mean that the homologous sequences will function in
substantially the same manner to produce substantially the same
compositions as the nucleic acid and amino acid compositions
disclosed and claimed herein.
[0039] As used herein, the term "recombinantly produced" means made
using genetic engineering techniques, not merely purified from
nature.
[0040] The amino acids which comprise the various amino acid
sequences appearing herein may be identified according to the
following three-letter or one-letter abbreviations:
1 Three-Letter One-Letter Amino Acid Abbreviation Abbreviation L -
Alanine Ala A L - Arginine Arg R L - Asparagine Asn N L - Aspartic
Acid Asp D L - Cysteine Cys C L - Glutamine Gln Q L - Glutamic Acid
Glu E L - Histidine His H L - Isoleucine Ile I L - Leucine Leu L L
- Lysine Lys K L - Methionine Met M L - Phenylalanine Phe F L -
Proline Pro P L - Serine Ser S L - Threonine Thr T L - Tryptophan
Trp W L - Tyrosine Tyr Y L - Valine Val V
[0041] The nucleotides which comprise the various nucleotide
sequences appearing herein have their usual single-letter
designations (A, G, T, C or U) used routinely in the art.
[0042] As used herein, bp means base pairs and kb means kilobase
pairs.
DEPOSITS
[0043] Plasmid pGEM-hRAR.gamma. was deposited Jun. 22, 1989 at the
American Type Culture Collection, Rockville, Md., U.S.A. (ATCC) for
patent purposes. It has been accorded ATCC No. 40623.
DESCRIPTION OF THE INVENTION
[0044] In one aspect, the present invention comprises a
double-stranded DNA segment wherein the plus or sense strand
encodes the primary sequence of a protein that has ligand-binding
and DNA-binding properties characteristic of a retinoid receptor
protein referred to herein as human gamma retinoic acid receptor
protein. According to this aspect of the invention, the
double-stranded DNA segment is one which is capable of being
expressed into human gamma retinoic acid receptor protein.
[0045] In another aspect, the invention comprises a single-stranded
DNA, which is the sense strand of a double-stranded DNA coding for
retinoic acid receptor gamma protein.
[0046] In another aspect, the invention comprises an mRNA made by
transcription of the double-stranded DNA of the invention.
[0047] In another aspect, the invention comprises a plasmid,
pGEM-hRAR.gamma., which contains DNA encoding the human gamma
retinoic acid receptor protein of the present invention
(hRAR.gamma.). This plasmid has been deposited with the American
Type Culture Collection for patent purposes; it has been accorded
ATCC No. 40623.
[0048] In still another aspect, the invention comprises a cell,
preferably a mammalian cell, transfected with a DNA coding for
retinoic acid receptor gamma protein. According to this aspect of
the invention, the transfected DNA is capable of being expressed in
the cell, thereby increasing the amount of gamma retinoic acid
receptor encoded by this DNA in the cell.
[0049] Further the invention comprises novel retinoic acid
receptors made by expression of DNA coding for gamma retinoic acid
receptor or translation of an mRNA transcribed from such gamma
retinoic acid receptor encoding DNA. According to this aspect of
the invention, the gamma retinoic acid receptors will be protein
products of "unmodified" gamma retinoic acid encoding DNA's and
mRNA's, or will be modified or genetically engineered gamma
retinoic acid receptor protein products which, as a result of
engineered mutations in the receptor DNA sequences, will have one
or more differences in amino acid sequence from the corresponding
naturally occurring "wild-type" gamma retinoic acid receptor
proteins. Preferably these gamma retinoic acid receptors, whether
"unmodified" or "engineered", will have at least about 5% (over
background) of the retinoic acid binding activity and/or at least
about 5% (over background) of the DNA-binding or
transcription-activating activity of the corresponding naturally
occurring gamma retinoic acid receptor.
[0050] Further the invention comprises chimeric receptors made by
exchanging the functional domains of the gamma retinoic acid
receptor with functional domains of another type. The chimeric
DNA's thus produced encode chimeric receptor proteins that have
functional characteristics based on the "origin" of their
respective DNA- and ligand-binding domains.
[0051] The chimeric receptors of the invention include
double-stranded DNA's that encode the chimeric receptors, as well
as single-stranded DNA's which are the sense strands of the
double-stranded DNA's, and mRNA's made by transcription of the
double-stranded DNA's. The invention also comprises cells, both
eukaryotic and prokaryotic, that are transfected with chimeric
receptors encoding DNA of the invention.
[0052] According to the preferred method for making the chimeric
receptor genes and proteins of the present invention, to effect the
chimeric DNA fusions, two restriction endonuclease sites are
preferably introduced into each receptor DNA at comparable
locations in or near the DNA-binding domains in order to divide the
receptor DNA's into three functional domains or regions. (For
example, a unique NotI site can be introduced immediately preceding
the DNA-binding domain and a unique XhoI site can be introduced
immediately following it. This divides the receptors into three
functional regions or "cassettes"; (1) an N-terminus cassette, (2)
a DNA-binding domain cassette, and (3) a ligand-binding domain
cassette. The three regions or cassettes from the RAR.gamma.
receptor can be combined with cassettes from other receptors from
the steroid superfamily to create a variety of chimeric
receptors.
EXPERIMENTAL ASPECTS OF THE INVENTION
Isolation of the Gamma Retinoic Acid Receptor
[0053] An oligonucleotide from RAR.alpha. was labeled and used to
probe a human cDNA library constructed from human tumor liver cell
mRNA. Nucleotide sequence analysis of one of the clones thus
isolated revealed a long open reading frame of 454 amino acids
beginning with a presumptive initiator methionine codon at position
200 as shown in FIG. 1.
RAR Amino Acid Sequence Comparison
[0054] The amino acid sequence of the newly discovered RAR.gamma.
was compared with the amino acid sequences from RAR.alpha. and
hRAR.beta.. The results of this comparison are shown in FIG. 2. As
the drawing in the figure illustrates, remarkable identity in the
amino acid sequence exists in the DNA-binding domains and in the
ligand-binding domains.
Ligand Assay
[0055] To assay for the ligand for the putative new retinoic acid
gamma receptor protein, the NcoI-EcoRI fragment of pGEM-hRAR.gamma.
was recloned in the pRS eukaryotic expression vector giving
pRShRAR.gamma.. The plasmid was introduced into monkey kidney CV-1
cells via calcium-phosphate transfection together with a reporter
plasmid .DELTA.MTV-TRE.sub.p-CAT. As a control, pRSerbA.sup.-1
(encodes no protein, stands as a negative control), pRShRAR.alpha.,
and pRShRAR.beta. were also examined. The transfected cells were
incubated in the presence or absence of 100 nM retinoic acid for 36
hours, and the induced CAT activities were analyzed by
chromatography. The results indicate that a protein encoded by the
NcoI-EcoRI insert transactivates through the .DELTA.MTV-TRE.sub.p
promoter in a retinoic acid dependent fashion, providing evidence
that it is a functional new retinoic acid receptor. See FIG.
3A.
Response Element Specificity of hRAR.gamma.
[0056] To assay for the hormone response elements activated by the
putative new gamma retinoic acid receptor protein, the NcoI-EcoRI
fragment of pGEM-hRAR.gamma. was recloned in the pRS eukaryotic
expression vector giving pRShRAR.gamma.. The plasmid was introduced
into monkey kidney CV-1 cells via calcium-phosphate transfection
together with one of the following reporter plasmids:
.DELTA.MTV-GRE-CAT, .DELTA.MTV-ERE-CAT, or .DELTA.MTV-TRE-CAT, with
.DELTA.MTV-CAT as the control. As above, the transfected cells were
incubated in the presence or absence of 100 nM retinoic acid for 36
hours, and the induced CAT activities were analyzed by
chromatography. As FIG. 3B illustrates, hRAR.gamma. recognizes ERE
and TRE, but not GRE, which is consistent with the other two known
human retinoic acid receptors.
Gamma Retinoic Acid Receptor Data Summary
[0057] The data disclosed herein identify the protein product
encoded by the cDNA insert in pGEM-hRAR.gamma. as human gamma
retinoic acid receptor based on three criteria. First, the overall
structural homology that the pGEM-hRAR.gamma. gene product has with
hRAR.alpha. and hRAR.beta. suggests that it is a retinoic acid
receptor. Second, the RAR.gamma. receptor protein acts as a
transcriptional regulator of a TRE- or an ERE-inducible reporter
gene in the presence of retinoic acid. Third, the hRAR.gamma.
recognizes ERE and TRE, but not GRE.
REFERENCES
[0058] The present specification refers to the following
publications, each of which is expressly incorporated by reference
herein.
[0059] 1. Brand, N., Petkovich, M., Krust, A., and Chambon, P.,
"Identification of a Second Human Retinoic Acid Receptor", Nature
332, 850-853 (1988).
[0060] 2. Evans, R., "The Steroid and Thyroid Hormone Receptor
Superfamily", Science 24-0, 889-895 (1988).
[0061] 3. Giguere, V., Ong, E. S., Segui, P., and Evans, R. M.,
"Identification of a Receptor for the Morphogen Retinoic Acid",
Nature 330, 624-629 (1987).
[0062] 4. Giguere, V., Yang, N., Segui, P., and Evans, R. M.,
Nature 331, 91-(1988).
[0063] 5. Hollenberg, S. and Evans, R. M., "Multiple and Coperative
Trans-Activation Domains of the Human Glucocorticoid Receptor",
Cell, 55, 899-906 (1988).
[0064] 6. Kozak, M., Nucleic Acid Res. 16, 8125-8148 (1987).
[0065] 7. Oro, A. E., Hollenberg, S., and Evans, R. M., Cell, 55,
1109-1114 (1988).
[0066] 8. Petkovich, M., Brand, N. J., Krust, A., and Chambon, P.,
"A Human Retinoic Acid Receptor Which Belongs to the Family of
Nuclear Receptors", Nature 330, 444-450 (1987).
[0067] 9. Robertson, M., "Towards a Biochemistry of Morphogenesis",
Nature 330, 420-421 (1987).
SPECIFICATION SUMMARY
[0068] From the foregoing description, one of ordinary skill in the
art can understand that the present invention provides
substantially pure DNA which encodes the retinoid receptor protein
referred to as the gamma retinoic acid receptor protein. The
invention also provides a plasmid containing the gamma retinoic
acid receptor DNA. This plasmid, pGEM-hRAR.gamma. has been
deposited with the American Type culture Collection for patent
purposes.
[0069] The invention is also comprised of gamma retinoic acid
receptor proteins, including modified functional forms thereof,
expressed from the DNA (or mRNA) of the invention.
[0070] The present invention also includes chimeric hybrid
receptors made by exchanging (1) the N-terminal domains, (2) the
DNA-binding domains, and (3) the ligand-binding domains from hGR,
hMR, PR, hERR1, hERR2, T.sub.3R.sub..alpha., T.sub.3R.sub..beta.,
D.sub.3, V-erb-A, E75 and the alpha and beta RAR receptors with the
domains of the new RAR.gamma. receptor. The chimeric receptors so
constructed have DNA-binding domain and ligand-binding domain
characteristics of the DNA-binding domain and ligand-binding
domains of the respective "parental" receptors from which they
originated.
[0071] The hRAR.gamma. DNA of the invention can be used to make the
gamma retinoic acid receptor proteins, and functional modified
forms thereof, in quantities that were not previously possible. The
same is true of the chimeric receptors. With the quantities of
gamma receptor protein available as a result of the present
invention, the receptor proteins can be used to screen for gamma
retinoic acid receptor-agonists or gamma retinoic acid
receptor-antagonists. Availability of the gamma receptor proteins
also means that they can be used in diagnostic assays to determine
levels of retinoic acid present in various tissues and body fluids.
Alternatively, the receptor proteins can be used to assay for
levels of mRNA.
[0072] Without departing from the spirit and scope of this
invention, one or ordinary skill can make various changes and
modifications to the invention to adapt it to various usages and
conditions. As such, these changes and modifications are properly,
equitable, and intended to be, within the full range of equivalence
of the following claims.
* * * * *