Note: Descriptions are shown in the official language in which they were submitted.
1 341 422
RETINOIC ACID RECEPTOR COMPOSITION AND METHOD
ACKNOWLEDGMENT
This invention was made with government
support under a grant from the National Institutes
of Health (Grant No. GM 26444).
FIELD OF THE INVENTION
The present. invention relates generally to
ligand-responsive regulatory proteins and genes
encoding them. More particularly, the present
invention relates to retinoid related regulatory
proteins and genes encoding them, modification of
these and other regulatory proteins and genes by
recombinant DNA and other genetic engineering
techniques, plus uses of the retinoid related
regulatory proteins and genes, both unmodified and
modified.
In addition the invention relates to a
novel method for identifying functional ligands for
ligand-responsive proteins. This method is
especially useful for identifying functional
ligand(s) for newly discovered receptor proteins.
The method is exemplified in part by showing that a
vitamin A related morphogen, retinoic acid, is a
functional ligand for a newly discovered retinoid
receptor protein.
BACKGROUND OF THE INVENTION
A central problem in eukaryotic molecular
biology continues to be elucidation of molecules and
mechanisms that mediate specific gene regulation in
response to exogenous inducers such as hormones or
growth factors. Although much remains to be learned
about the specifics of such mechanisms, it is known
that exogenous inducers such as hormones modulate
v
~ 341 422
gene transcription by acting in concert with
intracellular components, including intracellular
receptors and discrete DNA known as hormone response
elements or HRE's.
More specifically, it is known that
hormones like the glucocorticoid and thyroid
hormones enter cells by facilitated diffusion. It
is also known that the hormones then bind to
specific receptor proteins, thereby creating a
hormone/receptor complex. The binding of hormone to
the receptor is believed to initiate 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 in
the art by a variety of names, including hormone
response elements or HRE's, modulate expression
(transcription of RNA) of nearby target gene
promoters.
A major obstacle to further understanding
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.
In an effort to overcome this lack of
availability of, receptor proteins, co-pending
Canadian application 550,151 which has been
assigned to the Salk Institute for Biological
/ t
' ~ 341 422
3
Studies, assignee of the present application,
discloses cloned genes for a variety of receptor
proteins, including glucocorticoid-, thyroid-,
mineralocorticoid- and new steroid-related
receptors. This application further discloses
detailed biochemical characterization of these
molecules which shows that the receptor proteins
contain discrete DNA- and ligand-binding domains.
l~ (Portions of this application have been published;
for portions relating to cloning of the
glucocorticoid receptor and characterization of this
molecule into discrete domains, see Hollenberg, er al.
(1985) and Giguere, eral., (1986); for other related
work regarding receptors, see Hollenberg, er al. ,
(1987), Green, eral., (1986), Green and Chambon,
( 1987 ) , Kumar, ec al. , ( 1987 ) , Miesfeld, er al. , ( 1987 )
and Evans (1988)).
Further with regard to biochemical
2d characterization of the receptors, sequence analysis
of the human glucocorticoid receptor gene revealed
homology with the product of the v-erb-A oncogene of
avian erythroblastosis virus (AEV) (see Weinberger, et
al., (1985)). This group and others subsequently
demonstrated the cellular homolog of v-erb-A to be
the beta thyroid hormone receptor (see Weinberger et al. ,
(1986) and Sap, et al. , (1986) ) .
The discovery that the DNA-binding domain
of the steroid and thyroid hormone receptors is
3n highly conserved raised the question of whether this
segment might be diagnostic for related ligand
inducible transcription factors. It also raised the
question of whether the DNA sequences encoding these
domains might be used as hybridization probes to
scan the genome for related, but novel, ligand-
responsive receptors. Utilizing this approach, our
group at the Salk Institute have identified several
BI
r ~~ '
1 X41 422
4
new gene products. As is shown in application 550,151,
one is the human aldosterone receptor (hit, ATCC No.
67201) (see Arriza, et al. , (1987) for the published
version of this portion the applicati.on).~ a
second is a novel thyroid hormone receptor expressed
at high levels in the rat central nervous system
(rTR alpha, ATCC No. 67281) (see Thompson, et al. ,
(1987) for the published version of this portion of
the application),
This disclosure describes the isolation and
characterization of a cloned full-length cDNA
encoding a novel retinoid receptor protein with
homology to the DNA-binding and ligand-binding
domains of the steroid and thyroid hormone
receptors. In addition the construction and
characterization of chimeric receptors made by
"swapping" functional domains between the
glucocorticoid, the mineralocorticoid, the thyroid,
the estrogen-related, and the retinoic acid
receptors is described. 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 a retinoic acid receptor DNA-
binding domain (i.e., is obtained from wild-type
retinoic acid receptor or is a mutant that contains
the functional elements of retinoic acid DNA-binding
domain), then the chimera will have DNA-binding
properties characteristic of a retinoic acid
receptor. The same is true of the ligand-binding
domain. If the ligand-binding domain in the
chimeric receptor binds to thyroid hormone, then the
chimera will have ligand-binding properties
characteristic of a thyroid hormone receptor.
1 X41 422
This disclosure also describes a new method
for identifying functional ligands for ligand-
responsive receptor proteins. The method is
5 illustrated by showing (1) that the retinoid,
retinoic acid and its metabolic precurser, retinol,
are functional ligands far the newly discovered
receptor protein, and (2) that the DNA- and ligand-
binding domains determine the functional
characteristics of the chimeric receptors.
BRIEF DESCRIPTION OF THE DRAWINGS
The follow.i.ng is a brief description of the
drawings. More detailed descriptions are faund in
the
section of the specification labeled, "Detailed
Description of the Drawings"'.
FIGURE 1 (A and B) is a drawing which shows
the DNA nucleotide sequence and the primary protein
sequence of phRARa. Fig. 1A shows the composite
structure of phRARa aligned with a line diagram of
some restriction endonuclease cleavage sites. Figs.
1B-1, 1B-2 and 1B-3 show the complete nucleotide
sequence of phRARa and its primary amino acid
sequence.
FIGURE 2 (A and B) is composed of a drawing
and a blot. Fig. 2A is a drawing which illustrates
construction of the chimeric receptor hRGR. Fig. 28
is a blot which illustrates induction of CAT
activity by retinoic acid.
FIGURE 3 (A and B) is composed of two
graphs. Fig. 3A is a graph illustrating dose-
response to retinoids. Fig. 3B is a bar graph
illustrating retinoic acid binding to cytosol
extracts of transfected COS-~. cells.
FIGURE 4 (A and B) shows a Southern blot
analysis of human genomic DNA. Fig. ~4A shows
digested human placenta DNA hybridized under
1 34~ 422
stringent conditions; Fig. 4B shows the same DNA
hybridized under non-stringent conditions.
FIGURE 5 shows a Northern blot analysis of
retinoic acid receptor mRNA in rat and human
tissues.
FIGURE 6 is a schematic drawing which shows
a comparison of hGR, hRR and hT~R~
FIGURE 7 is a schematic diagram of a
generalized steroid\thyroid\retinoic acid receptor
gene.
FIGURE 8 is a schematic drawing that shows
amino acid comparison of members of the steroid
hormone receptor superfamily.
FIGURE 9 is a schematic drawing that shows
the structure and activity of chimeric
thyroid/glucocorticoid receptors.
DEFINITIONS
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:
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.
As used herein, species are identified as
follows: h, human; r, rat; m, mouse; c, chicken; and
d, Drosophilia.
As used herein, "steroid hormone
superfamily of receptors" refers to the class of
related receptors comprised of glucocorticoid,
mineralocorticoid, progesterone, estrogen,
estrogen-related, vitamin Ds, thyroid, v-erb-A,
7 1 341 422
retinoic acid and E75 (Drosophrlia) receptors. See
Evans (1988) and the references cited therein.
As used herein, RR and RAR both mean
retinoic acid receptor. The acronyms, hRR and hRAR,
mean human retinoic acid receptor. The DNA referred
to as phRARa codes for human retinoic acid receptor
alpha. hRARa is encoded by deposited phRARl which
has been accorded ATCC No. 40392. The DNA referred
to as hRAR~ encodes human retinoic acid receptor beta.
See Brand et al. , ( 1988 ) .
As used herein, GR means glucocorticoid
receptor. The DNA referred to as hGR codes for
human glucocorticoid receptor GR. hGR is encoded by
deposited pRShGR which has been accorded ATCC No.
67200.
As used herein, MR means mineralocorticoid
receptor. The DNA referred to as hMR codes for
human mineralocorticoid receptor MR. hMR is encoded
by deposited pRShMR which has been accorded ATCC No.
67201.
As used herein, TR means thyroid receptor.
TRalpha and TRheta refer to the alpha and beta forms of
the thyroid receptor. The DNA's referred to as
c-erb-A, herb-A 8 . 7 , peA101, rbeAl2 , and hFAB all code
for thyroid receptors. Plasmid pherb-A 8.7 encodes
hTRa; it has been deposited for patent purposes and
accorded ATCC No. 40374. Plasmid peA101 encodes
hTR~; it has been deposited for patent purposes and
accorded ATCC No. 67244. Plasmid rbeAl2 encodes
rTRa; it has been deposited for patent purposes and
accorded ATCC No. 67281. Plasmid phFA8 encodes a
partial clone of hTRa that has a deletion in the
"ligand-binding" region of the clone (i.e., the DNA
that codes for the carboxy terminal end of the
receptor protein). Plasmid phFAB has been accorded
ATCC No. 40372.
1 341 422
8
As used herein, ERR means estrogen-related
receptor. The acronyms, hERRl 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, eral, 1988). hERRi is encoded by deposited
plasmids pE4 and pHI~CA, which have been accorded ATCC
No. 67309 and 67310, respectively. (Neither pE4 or
pHKA are complete clones: hERR1 is constructed by
joining segments from both clones.) hERR2 is
encoded by deposited plasmid phH3 which has been
accorded ATCC No. 40373.
As used herein, VDR means vitamin D~
receptor.
As used herein, MTV means mammary tumor
virus; MMTV means mouse mammary tumor virus.
As used herein, RSV means Rous sarcoma
virus; SV means Simian virus.
As used herein, CAT means chloramphenicol
acetyltransferase.
As used herein, luciferase means firefly
luciferase. See, de Wet, I.R., Wood, K.V., DeLuca,
2.5 M. , Helinski, D.R. , and Subramani, S. , Mol. Celt. Biol. 7:
925-737 (1987).
As used herein, COS means monkey kidney
cells which express T antigen (Tag). See Gluzman,
Cell, 23:175 (1981). COS cells are receptor-
deficient cells that are useful in the functional
ligand identification assay of the present
invention.
As used herein, CV-1 means mouse kidney
cells from the cell line referred to as "CV-1".
CV-1 is the parental line of COS. Unlike COS cells,
which have been transformed to express SV40 T
antigen (Tag), CV-1 cells do not express T antigen.
9 1 341 422
CV-1 cells are receptor-deficient cells that are
also useful in the functional ligand identification
assay of the present invention.
As used herein, the generic terms of art,
"hormone response elements" or "HRE's",
"transcriptional control units", "hormone responsive
promoter/enhancer elements", "enhancer-like DNA
sequences" and "DNA sequences which mediate
transcriptional stimulation", all mean the same
thing, namely, short cis-acting sequences (about 20
by 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 try become
hormone responsive. These sequences, referred to
most frequently as hormone response elements or
HRE's, function in a position- and orientation-
independent fashion. Unlike other enhancers, the
activity of the HRE's is dependent upon the presence
or absence of ligand. (See Evans (1988) and the
references cited therein.) In the present
specification and claims, the phrase °'hormone
response element" is used in a generic sense to mean
and embody the functional characteristics implied by
all terms used in the art to describe these
sequences.
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 by 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.
'1 X41 422
As used herein, the acronym GRE means
glucocorticoid response element and THE means
thyroid receptor response element. GRE's are
5 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
10 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 TR and RR receptors
can activate TRE's, so a THE can be used with any
receptor that has a TR or RR-type DNA-binding
domain.
As used herein, ligand means an inducer,
such as a hormone or growth substance. Inside a
~5 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 TsRa and the T,~R~ bind thyroid
hormone such as Tg.
As used herein, the word "operative", in
the phrase "operative hormone response element
functionally linked to a ligand-responsive promoter
and an operative reporter gene", means that the
respective DNA sequences (represented by the terms
"hormone response element", "ligand-responsive
promoter" and "reporter gene") are operational, i.e.,
1341422
11
the hormone response element can bind with the DNA-
binding domain of receptor protein (either wild-type
or chimeric), the ligand-responsive promoter can
control transcription of the reporter gene (upon
appropriate activation by a HRE/receptor
protein/ligand complex) and the reporter gene is
capable of being expressed in the host cell. The
phrase "functionally linked" means that when the DNA
segments are joined, upon appropriate activation,
the reporter gene (e.g., CA7~ or luciferase) will be
expressed. This expression occurs as the result of
the fact that the "ligand responsive promoter"
(which is downstream from the hormone response
element, and "activated" when the HRE binds to an
appropriate ligand/receptor protein complex, and
which, in turn then "controls" transcription of the
reporter gene) was "turned on" or otherwise
activated as a result of the binding of a
2~ ligand/receptor protein complex to the hormone
response element.
As used herein, the phrase "DNA-binding
domain" of receptors refers to those portions of the
receptor proteins (such as glucocorticoid receptor,
thyroid receptor, mineralacorticoid receptor,
estrogen-related receptor and retinoic acid
receptor) that bind 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 Figure 8; also see
Giguere, et al. , ( 1986 ) ; Hollenberg, et a!. , ( 1987 ) ;
Green and Chambon (1987): and Miesfield, etal.,
(1987), Evans (1988).
12 1 341 422
The boundaries for the DNA-binding domains
for various steroid horm one superfamily receptors
are shown in Figure 8: t he boundaries are as
follows:
-hGR (pRShGR): nucleotide 1393 to 1590
amino acid 421 to 486
(See ATCC #67200)
-hTRb (peA101): nucleotide 604 to 807
amino acid 102 to 169
(See ATCC #67244)
-hTRa (pherb-A8 .7 ) : nucleotide 168 to 372
amino acid 50 to 117
(See ATCC #40374)
amino acid 291 to 358
(See ATCC #67281)
-ERR1 (pE4 & amino acid 176 to 241
pHKA): (See ATCC #67309 & #67310)
-ERR2 (phH3): amino acid 103 to 168
See ATCC #40373)
-hMR (pRShMR): nucleotide 2029 to 2226
amino acid 603 to 668
(See ATCC #67201)
-hRARa (phRARl): nucleotide 364 to 561
amino acid 88 to 153
(See ATCC #40392)
The DNA-binding dom ains of the steroid hormone
superfamily of receptors consist of an amino segment
varying between 66 to 68 amino acids in length.
This segment contains cysteine residues, one of
9
which is the first amino acid of the segment. This
first Cys residue begins a motif described as
Cys-XZ-Cys-Xl$_15-CYs-XZ-Cys,
where X is any amino acid
residue. The DNA-bindin g domain invariably ends
with the amino acids Gly -Met.
1341422
13
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.
As used herein, the phrase "ligand-binding
domain region" of receptors refers to those portions
of the receptor proteins that bind to ligands such
as growth substances or the hormones. These
boundaries of the ligand-binding domains for the
steroid receptor superfamily have been identified
and characterized. See Figure 8 and Evans (1988).
The ligand-binding domains fax the various
receptors are shown in Figure 8; some of those
domains are as follows:
-hGR (pRShGR): amino acid 528 to 777
(See ATCC #67200)
-hTRb (peA101): amino acid 232 to 456
(See ATCC #67244)
-hTRa (pherb-A8.7) : amino acid 183 to 410
(See ATCC #40374)
-rTR (rbeAl2) amino acid 421 to 639
(See ATCC #67281)
-ERR1 (pE4 & amino acid 295 to 521
pHKA): (See ATCC #67309)
-ERR2 (phH3) amino acid 212 to 433
(See ATCC #40373)
-hMR (pRShMR): amino acid 734 to 984
(See ATCC #67201)
-hRARa (phRARl): amino acid 198 to 462
(Sere ATCC #40392)
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 referred to in
Figure 8, the first common site can be introduced
immediately preceding the DNA-binding domain, the
1341422
second common site immediately following it. (For
example, in any of the steroid hormone superfamily
of receptors that are shown in Figure 8, a uniqu~a
NotI site can be introduced immediately preceding the
DNA-binding domain and a unique Xhvl 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.
As used herein, the nomenclature used to
identify the chimeric receptars is as follows: The
various functional domains (N-terminus, DNA-binding
and ligand-binding) are identified according to the
"parental" receptor from which they originated. For
example, domains from GR are "G" domains; TR domains
are "T" domains (unless otherwise further specified
as being "T$" or "Tb"' domains) p MR domains are "M"
domains; RAR domains are "R" domains ( unless
otherwise further specified as being "R," or "Rb"
domains), and ERR domains are "E" domains (unless
Qtherwise specified as being "E1" or "E2" domains).
According to this notation, unless otherwise
specified, "T" is used generically to mean either
the T$Ra or the TsR~ receptors: "E" means either
hERRl or hERR2; and "R" means either the RARa or the
RARE receptors. Wild-type receptors do not contain
any exchanged domains, and so according to this
notation system would be identified as G-G-G (or
GGG ) , Ta-Ta-Ts ( or TsTaTa ) , Tb-Tb-Tb ( or TbTbTb ) , M-M-I''!
3 5 ( or I~iM) . ~-~-R. ( or R"R,Ra ) . Rb-Rb-Re ( or RbRbRb ) .
E1-E1-E1 or EZ-EZ-EZ, where the first domain listed is
the N-terminus domain, the middle domain is the
1 341 422
DNA-binding domain, and the last domain is the
ligand-binding domain. Any chimeric receptor will
have functional domains from at least two wild-type
or parental sources. For example, the chimeric
receptor GGRa would have N-termimus and DNA-binding
domains from glucocorticoid receptor and the
ligand-binding domain from the alpha retinoic acid
receptor; GT Rb would have the N-terminus from
s
glucocorticoid, the DNA-binding domain from thyroid
receptor alpha and the ligand-binding domain from
retinoic acid receptor bera.
As used herein,. hGR~X, hTR~~X, and hRRNx refer
to hGR, hTR~ and hRR receptors that have been
engineered to contain the unique sites for NorI and
XhoI flanking the baundaries for the DNA-binding
domains in these receptors. These mutant receptors
exemplify construction o~ hybrid receptors that are
comprised of all possible combinations of amino
termini, DNA-binding domains, and ligand-binding
domains from hGR, hMR, hERRl, hERR2, hTRa, hTR~,
rTRa, hRARa, and hRAR~.
As used herein, Southern blot analysis refers
to a procedure for transferring denatured DNA from
an agarose gel to a nitrocellulose filter where it
can be hybridized with a complementary nucleic acid.
- As used herein, Northern blot analysis refers
to a technique for transferring RNA from an agarose
gel to a nitrocellulose filter on which it can be
hybridized to complementary DNA.
As used herein, "mutant" DNA of the invention
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 new nucleotides into wild-type
sequences, deletion of nucleotides from wild-type
sequences, substitution of nucleotides in the wild-
'~ 341 422
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 to include new
nucleotides, deletion of nucleotides, substitution
of nucleotides, etc.
Use of the term "substantial sequence homology"
in the present specification arid claims means it is
intended that DNA, RNA, or amino acid sequences
which have slight and non-consequential sequence
variations from the actual sequences disclosed and
claimed herein 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.
As used herein, the term "recomb:inantly
produced" means made using genetic engineering
. techniques, not merely purified from nature.
The amino acids which comprise the various
amino acid sequences appearing herein may be
identified according to the following three-letter
or one-letter abbre~riations:
35
~ X41 4~~
m
Three-Letter One-Letter
Amino Abbreviation Abbreviation
Acid
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
The nucleotides which compris e the various
nucleotide appearing her ein have their
sequences
usual designations A, G, T, C or
single-letter (
U) used routinely the art.
in
As used herein, by means base pairs and kb
means .
kilobase
pairs
In the present specification and claims,
the
Greek (a), beta (~),etc. are
letters
alpha
sometimes o as a, b, .
referred etc
t
DEPOSITS
Plasmids pRShGR (hGR), pRShMR (hMR), peA101
(hT~) and GMCAT, all of which are in E. coli HB101,
plus plasmids rebAl2 (rTRa), pE4 and phKA (which
together encode hERRl ) , phH3 ( hERR2 ) , pherb-A 8 . 7
(hTRa), phFA 8 (a partial clone of hTRa), and
1 341 422
m
plasmid phRARl have been deposited at the
American Type Culture Collection, Rockville,
Maryland, U.S.A. (ATCC) under the terms of the
Budapest Treaty on the International Recognition
of Deposits of Microorganisms for Purposes of
Patent Procedure and the Regulations promulgated
under this Treaty. Samples of the plasmids are
and will be available to industrial. property
offices and other persons legally entitled to
receive them under the terms of said Treaty and
Regulations and otherwise in compliance with the
patent laws and regulations of the United States
of America and all other nations or international
organizations in which this application, or an
application claiming priority of this
application, is filed or in which any patent
granted on any such application is granted.
The ATCC Deposit Numbers and Deposit Dates
for the deposits are as follows:
pRShGR (hGR) 67200 Sept. 9, 1986
pRShMR (hMR) 67201 Sept. 9, 1986
pE4 (hERRl*) 67309 Jan. 30, 1987
phHKA (hERRl*) 67310 Jan. 30, 1987
phH3 (hERR2) 40373 Sept. 29, 1987
GMCAT (reporter) 67282 Dec. 18, 1986
pherb-A 8.7 (hTRa) 40374 Sept. 29, 1987
phFA 8 (hTRa*) 40372 Sept. 29, 1987
peA101 (hTRb) 67244 Oct. 22, 1986
prbeAl2 (rTRa) 67281 Dec. 18, 1986
phRARa (hRARa) 40392 Nav. 20, 1987
(* means a partial clone)
(pE4 & phHKA together encode complete hERRl)
SUMMARY OF "THE INVENTION
In one aspect, the present invention
comprises a double-stranded DNA segment wherein
the plus or sense strand of the segment contains
1 341 422
19
a sequence of triplets coding for the primary
sequence of a protein which has ligand-binding
and DNA-binding (or transcription-activating)
properties characteristic of a retinoid receptor
protein referred to herein as human 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
retinoic acid receptor protein.
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 protein, and an RNA made by
transcription of this double-stranded DNA.
In another aspect, the invention comprises a
plasmid, phRARl, which contains DNA coding for a
retinoic acid receptor protein of the present
invention (RARa). This plasmid has been
deposited with the American Type Culture
Collection for patent purposes; it has been
accorded ATCC No. 40392.
In still another aspect, the invention
comprises a cell, preferably a mammalian cell,
transformed with a DNA coding for retinoic acid
receptor protein. According to this aspect of
the invention, the transforming DNA is capable of
being expressed in the cell, thereby increasing
the amount of retinoic acid receptor, encoded by
this DNA, in the cell.
Further the invention comprises novel
retinoic acid receptors made by expression of a
DNA coding for retinoic acid receptor or
translation of an mRNA transcribed from such a
retinoic acid receptor ceding DNA. According to
this aspect of the invention, the retinoic acid
receptors will be protein products of
as ' X41 422
"unmodified" retinoic acid coding DNA's and
mRNA's, or will be modified or genetically
engineered 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" retinoic acid receptor
proteins. Preferably these retinoic acid
receptors, whether "unmodified" or "engineered",
will have at least about 5% of the retinoic acid
binding activity and/or at least about 5% of the
DNA-binding or transcription-activating activity
of the corresponding naturally occurring retinoic
acid receptor.
Further the invention comprises chimeric
receptors made by exchanging the functional
domains of one 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. The chimeric receptors of the
invention include double-stranded DNA's that code
for 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 transformed with
chimeric receptors encoding DNA's of the
invention.
According to the chimeric receptor aspect of
the invention, to effect the chimeric DNA
fusions, two restriction endonuclease sites are
introduced into each receptor cDNA at comparable
1 341 ~~2
21
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 No~I 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
1.0 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. This
1.5 aspect of the invention is illustrated in the
section of the specification labeled "Detailed
Description of the invention'°.)
In the present specification and claims, the
chimeric receptors (referred to also as chimera
or hybrids) are named by letters referring to the
origin of the various domains. Domains from hGR
are referred to as "G" domains, domains from hTR
are "T" domains, domains from hERR are "E" and
domains from hRR are "R" domains. For example,
~5 the chimeric receptor "RGR" has the amino and
carboxyl termini of hRR and the DNA-binding
domain of hGR: the chimeric receptor "TGG" has
the amino terminus from hTR, and the DNA-binding
and carboxyl terminus from hGR. (In the diagram
shown in Figure 7, the amino terminus of the
receptor is referred to domain A/B and the
carboxyl terminus is referred to as domain E.)
According to the notation used in the
specification and claims, unless. otherwise
specified, "T" is used generically to mean either
the TsRa or the TsR~g receptor: "E" means either
22 1 3 4 1 4 2 2
hERRl or hERR2: and "R" means either the RARa or
the RARE receptor.
Chimeric receptors of the invention include
chimera having (1) an N-terminus domain selected
from the group of wild-type receptors consisting
of hGR, hMR, hERRl, hERR2, rTRa, hT~a, hT~i, hRARa
and hRAR~9, (2) a DNA-binding domain selected from
the group of wild-type receptors consisting of
hGR , hMR , hERRl , hERR2 , rTRa , hT3a ,
hT~, hRARa and hRAR~3, and (3) a ligand-binding
domain selected from the group of wild-type
receptors consisting of hGR, hMR, hERRl, hERR2,
rTRa, hTga, hT~, hRARa and hRAR~, wherein any one
chimeric receptors will have N-terminus, DNA-
binding, and ligand-binding domains that
originate from at least two different "wild-type
receptor" sources.
Preferred chimeric receptor DNA's of the
2p invention include GRR, GRG, GGR, RGG, RGR, RRG,
GTT, GTG, GGT, TGG, TGT, TTG, TTR, TRT, TRR, RTT,
RTR, RRT, GTT, GTG, GGT, TGG, TGT, arid TTG
receptor DNA's, plus the chimeric hybrid receptor
proteins made by expression of a chimeric DNA of
the invention translation of an mRNA transcribed
from such a chimeric: receptor coding DNA.
Preferably these chimeric receptors will have
activity that exceeds exogenous background
binding or transcriptional activation activity
levels in any given cell, or will have at least
about 5% of the DNA-binding or transcription-
activating activity of the corresponding
naturally occurring receptor DNA-binding domain,
and/or about 5% of the li.gand-binding activity of
the corresponding naturally occurring ligand-
binding domain.
23 1 3 4 1 4 2 2
The invention also comprises a method for
identifying functional ligand(s) for receptor
proteins. According to the method, DNA sequences
(referred to herein as the sample sequences) can
be isolated which code for receptor proteins and
which have at least an operative portion of a
ligand-binding domain and a DNA-binding domain.
(As those skilled in the art will appreciate, not
all of the DNA sequences in the ligand-binding
domains are necessary in order for the domains to
be functional. The operative sequences, i.e.,
those that must be present if the domain is to
bind ligand, can be identified by deletion
studies on any given domain.) Once the sample
DNA sequences are isolated, a chimeric gene can
be created by substituting the DNA-binding domain
region in the sample DNA sequence with a DNA-
binding domain region taken from a DNA sequence
coding for another receptor protein, ~.g.,
glucocorticoid receptor protein, thyroid receptor
protein, mineralocorticaid receptor protein or
retinoic acid receptor protein. Next a suitable
receptor-deficient host cell is transfected with:
(1) the chimeric receptor gene, which is
preferably carried on an expression plasmid, and
(2) a reporter gene, such as the C,~?" gene or the
firefly luciferase gene, which is also preferably
carried on plasmid, and which is refered to in
application 550,151 as a reporter plasmid. In any
case, the reporter gene is functionally linked to
an operative hormone response element (HRE)
(either wild-type or engineered) wherein the
hormone zesponse element is capable of being
activated by the DNA-binding domain used to make
the chimeric receptor gene. (For example, if the
chimeric receptor gene contains the DNA-binding
",~:r~..
24
domain region from glucocorticoid receptor coding
DNA, then the HRE should be a wild-type, an
engineered, or a synthetic GRE, i.e., one that can
be activated by the operative portion of the
DNA-binding region of a glucocorticoid receptor
protein. If a thyroid receptor DNA-binding
domain region is used, then the wild-type or
engineered HRE should be responsive to a thyroid
(or retinoic acid) receptor protein, etc.) Next
the transfected host. cell is challenged with a
battery of candidate ligands which can
potentially bind with the ligand-binding domain
region of the chimeric protein coded for by the
chimeric gene. To determine which of these
ligands can functionally complex with the
chimeric receptor protein, induction of the
reporter gene is monitored by monitoring changes
in the protein levels of the protein coded for by
the reporter gene. (For example, if luciferase
is the reporter gene, the production of
luciferase is indicative of receptor-regulated
gene transcription.) Finally, when a ligand(s)
is found that can induce transcription of the
reporter gene, it is concluded that this
ligand(s) can bind to the receptor protein coded
for by the initial sample DNA sequence. This
conclusion can be further verified by testing the
binding properties of the receptor protein, coded
for by the initial sample DNA sequences, vis-a-vis
the ligand(s) that induce expression of the
reporter gene.
As those skilled in the art will appreciate,
if a cell already captains (a) a chimeric DNA
sequence (C) comprised of (1) operative portions
of a DNA-binding domain of a first receptor
sequence (i.e., a first sequence) linked to (2)
1 341 422
operative portions of a ligand-binding domain of
a second receptor sequence ( i.e. , a second
sequence), and (b) a reporter nucleic acid
5 sequence functionally linked to an operative
hormone response element wherein the operative
portions of the DNA-binding domain of the first
receptor sequence can functionally bind to and
activate the hormone response element that is
1U functionally linked to the reporter sequence,
then the method for identifying a functional
ligand for a receptor protein will be comprised
of challenging the cell with at least one
candidate ligand and then monitoring induction of
15 the reporter sequence by means of changes in the
amount of expression product of the reporter
sequence.
The new functional ligand identification
assay makes it possible to screen a large number
2U of potential ligands or any given receptors,
regardless of whether the receptor is a wild-type
receptor or a chimeric one.
The functional ligand identification method
is illustrated herein by showing (1) that the
25 retinoid, retinaic acid and its metabolic
precursor, retinol, are functional ligands for
the receptor protein coded for by phRARl DNA, and
(2) that the DNA- and ligand-binding domains
determine the functional characteristics of the
3U chimeric receptors.
The new functional assay, as well as the new
retinoic acid receptor and the new chimeric
receptors, are described more fully below.
~ 3~1 X22
26
DESCRIPTION OF THE INVENTION
The Retinoic Acid Receptor
In a continuing effort to explore the
steroid hormone receptor superfamily, advantage
was taken of the fortuitous identification of a
novel genomic sequence with striking homology to
the DNA-binding domain of the steroid hormone
receptors (see Dej can et al. , 1986 ) . This sequence
spans the integration site of a hepatitis B virus
(HBV) from a human hepatocellular carcinoma.
To pursue the hypothesis that this gene
might code for a previously unknown receptor, an
oligonucleotide derived from this sequence was
labeled and used to probe a number of human cDNA
libraries. Five positive clones were initially
isolated from a testis cDNA library. The insert
from one of these clones (lhTlR} was used to
isolate additional cDNA clones from a agtl0
kidney cDNA library. A restriction map of the
largest clone (phRARl) is shown in Figure 1A.
Nucleotide sequence analysis reveals a long open
reading frame of 462 amino acids beginning with a
presumptive initiator methionine codon
corresponding to nucleotides 103-105 as shown in
Fig. 1B-1. The sequence surrounding this ATG
agrees with the consensus described by Kozak
(1987) for a translation initiation site.
Upstream of the ATG is an in-frame terminator
Providing support for the initiator methionine.
Another methionine found 30 codons downstream
fails to conform to the consensus and is an
unlikely initiator. Following the terminator
codon at position 1489-1491 is a 3'-untranslated
region with a consensus polyadenylation signal
(AATAAA) found 20 nucleotides upstream of a
polyadenylated tract (see Proud foot, er al. , 1976) .
1 341 422
27
A polypeptide of relative molecular mass
50,772 d (51 Kd) is encoded within the
translational open reading frame. The size of
the protein encoded by the insert of phRARl was
verified by in vitro translation of RNA ( see Krieg, er
al., (1984)) derived from this insert and found to
correspond to the predicted size of 54 Kd (data
not shown), Amino acid sequence of this protein
has been compared to the glucocorticoid and
thyroid hormone receptors. The highest degree of
similarity is found in a cysteine-rich sequence
of 66 amino acids beginning at residue 88. Our
group has previously demonstrated that this
region of the hGR represents the DNA-binding
domain for this receptor. See Giguere, et al. ,
( 1987 ) and Hollenberg, er al. , ( 1987 ) . In
addition, mutagenesis and expression studies have
provided direct evidence for its role in
transcriptional activation of genes harboring
glucocorticoid response elements (GREs). See
Giguere, e1 al. , ( 1987 ) and Hal lenberg, er al. ,
( 1987 ) .
Domain Switching and Transcriptional Activation
Since the ligand for the gene product of
phRARl was unknown, it was desirable to develop a
quick and sensitive assay to reveal its identity.
Previous studies have demonstrated that the DNA-
binding domain of the human glucocorticoid and
estrogen receptors can be interchanged to yield a
functional hybrid receptor. This chimera
recognizes the glucocorticoid respansive element
of the MMTV-LTR but stimulates transcription in
an estrogen-dependent fashion (see Green, er al. ,
(1987)). This led us to wonder is if a general
domain-swapping strategy could be exploited to
identify the ligand-binding properties of a novel
28 1 341 422
hormone receptor. To test this approach we first
substituted the DNA-binding domain of the phRARl
gene product with the well described DNA-binding
domain from the hGR (Fig. 2A). (This chimeric
construction, when expressed in suitable host
cells, produces a hybrid receptor protein whose
ligand-binding domain region must bind with a
functional exogenous ligand before the
ligand/receptor complex can bind to a GRE,
thereby activating a glucocorticoid inducible
promoter.)
To assay for the presence of a functional
ligand the chimeric receptor gene was transfected
into suitable host cells along with a suitable
GRE linked reporter gene. CV-1 cells were used
for the assay along with a MMTV-CAT reporter
gene. (MMTV-CAT is carried on reporter plasmid,
GM-CAT, which has been deposited with the
American Type Culture Collection for patent
purposes; see the section of this specification
labeled, "Deposits", As those skilled in the art
will appreciate, reporter plasmids suitable for
assaying hybrid thyroid receptor prateins, i.e.,
hybrid proteins having the DNA-binding domain of
a thyroid receptor protein, can be constructed by
substituting the GRE on plasmid GM-CAT with a
thyroid hormone responsive transcription element.
For example, the growth hormone promoter can be
functionally linked to the bacterial CAT gene.
Since the growth hormone promoter contains a
thyroid responsive transcription element, such a
reporter plasmid can be used to assay hybrid
thyroid receptor proteins. See the subheading:
"Construction of Reporter and Expression
Plasmids" in this specification. (Since
mineralocorticoid receptors can activate GRE's, a
1 ~~+~ x+22
29
reporter plasmid such as GM-CAT can be used to
assay hybrid mineralacorticoid receptor
proteins.)
Returning to the functional ligand
identification assay, the transfected cells were
then systematically challenged with a battery of
candidate ligands and .induction monitored by
changes in CAT activity.
Because of their hormonal-like activities,
the retinoids, including retinol (Vitamin A) and
retinoic acid, were evaluated as potential
inducers. Remarkably, retinoic acid elicited a
dramatic increase in C.4T activity of the hybrid
receptor (Fig. 28). No effect upon CAT activity
was observed using the parent vector, pRShRRNx~
or the wild type gene product from phRARl, herein
referred to as human retinoic acid receptor
(hRARa). As expected, the hybrid receptor is not
induced by glucocorticoids, and the hGR is not
induced by retinoic acid.
As shown in Figure ~A, retinoic acid
exhibits an ED5° value of 6 x 101° M on CAT
activity induced by the hybrid receptor, which is
consistent with EDb~, values observed for retinoic
acid in a variety of biological assays (see Sporn
and Roberts, 1984). Retinal functions as a weak
agonist with an ED6° value greater than 100 ziNi.
Retinyl acetate and retinyl palmitate function as
even weaker inducers. A number of natural and
synthetic ligands including testosterone,
dihydrotestosterone, estrogen, dexamethasone,
cortisol, aldosterone, progesterone, Ts, T,~,
Vitamin D$ and 25-OH-cholesterol failed to induce
CAT activity.
1 341 422
To corroborate the identity of the phRAR1
gene product as the retinoic acid receptor, the
binding properties of the expressed product were
5 evaluated following transfection of COS-1 cells.
As shown in Figure 3B, transfected cells reveal
increased capacity to specifically bind $H-
retinoic acid. This increase occurs over an
endogenous background that is a likely
10 consequence of the presence of cellular retinoid
binding proteins as well as a significant non-
specific binding. Consistent with the activation
studies, the binding is fully competed by
retinoic acid but only partially by retinol.
15 Thyroid hormones, dexamethasone and vitamin D~ did
not compete the binding of retinoic acid.
A Gene Family
To determine if the new retinoic acid gene
was unique and to identify potentially related
20 genes, human DNA was examined by Southern blot
analysis. Hybridization of restriction
endonuclease-digested human DNA with a labeled
DNA fragment derived from the coding region of
the hRR gene produced three bands in every
25 digestion consistent with a single hybridizing
genetic locus (Fig. 4A). This hybridization
pattern is unrelated to the restriction
endonuclease map described by Dejean etal. (1986)
for the HBV pre-integration site. However, when
30 the hybridization conditions were relaxed, six
additional bands were observed in the products of
each enzyme digestion (Fig. 4B). These
observations suggested that there were at least
one additional locus, and possibly more, in the
human genome related to the retinoic acid
receptor. The RARE has now been found. See
Brand, et al, (1988) .
1 341 422
31
Expression of the hRR Gene
Since retinoic acid is known to exert
effects on a large number of different cell
types, we examined the expression of the hRR
gene. Total cytoplasmic RNAs isolated from a
variety of rat and human tissues were size
fractionated and transferred to a nitrocellulose
filter. Hybridization with a 600-by restriction
fragment from phRAR1 reveals a major RNA species
of 3,200 nucleotides with highest levels in the
hippocampus, adrenals, cerebellum, hypothalamus
and testis (Fig. 5). Longer exposure shows that
most tissues contain a small amount of the 3.2 kb
l~ transcript while it is undetectable in some
tissues such as liver.
Retinoic Acid Receptor Data Summary
The data disclosed herein identify the gene
product of phRAR1 as a human retinoic acid
receptor based on three criteria. First, the
overall structural homology of the hRR to steroid
and thyroid hormone receptors (Fig. 6~ suggests
that it is likely to be a ligand-responsive
- regulatory protein. Second, an expressed
chimeric receptor, consisting of the DNA-binding
domain of the hGR and the presumptive ligand-
binding domain of the hRR acts as a
transcriptional regulator of a glucocorticoid-
inducible reporter gene only in the presence of
retinoic acid. This induction occurs at
physiological levels. Third, expression of the
candidate hRR in transfected cells selectively
increases the capacity of those cells to bind
retinoic acid.
~ 341 422
32
Development and ~ncoaenesis
The retinoids comprise a group of compounds
including retinoic acid, retinol (vitamin A) and
a series of natural and synthetic derivatives
that together exert profound effects on
development and differentiation in a wide variety
of systems. See Sparn & Roberts, (1980 ; Mandel lx
Cohen, (1985); Wolback & Howe, (1925)p Lotan
(1980); and Fuchs & Green, (1980). A~.though
early studies focused on the effects of retinoids
on epithelial growth and differentiation, their
actions have been shown to be more widespread
than previously suspected. Many recent studies
demonstrate the effects of these molecules on a
variety of cultured cell lines including
neuroblastomas (see Hausler, er al. , ( 1983 ) ) ,
melanomas (see Lotan, er al. , ( 1983 ) ) and
fibroblasts (see Shroder er al., (1982) ) . In the
human promyelocytic leukemia cells (HL-60),
retinoic acid is a potent inducer of granulocyte
differentiation (see Breitman, er al. , (1980) ) . In
F9 teracarcinoma stem cells, retinoic acid will
induce the differentiation of parietal endoderm,
characteristic of a late mouse blastocyst (see
Strickland & Mahdavi, (1978) Jetten eral.,
(1979); and Wang eral., (1985)). Retinoic acid
has been shown to exert equally potent effects in
development. For example, in the developing
chick limb bud, retinoic acid is able to
substitute for the action of the polarizing
region in establishing the anterior-posterior
axis (see Tickle & Eichele, (1985)). By
controlling the exposure to retinoic acid, it is
possible to generate novel patterns of limb
structures. Although retino:ic acid is primarily
considered a morphogen, Northern blot analysis
9 341 422
33
suggests a re-evaluation of its function in the
adult. In humans, retinol deficiency has been
linked to an alarming increase in a variety of
cancers (see Moon & Itri, (1984)). Retinoids have
also been shown to inhibit tumor progression in
animals and block the action of tumor promoters in
vitro. In this context, the hRR may be considered
as a negative regulator of oncogenesis.
A Superfamily of RegulatorY Genes
Two surprising results have emerged from the
studies presented here. The first is the
discovery of a family of retinoic acid receptor-
related genes which predicts the existence of one
or more other proteins with closely related
properties (e.g., the RARE described by Brand er al,
(1988)). Physiological studies demonstrate that
both retinoic acid as well as retinol (vitamin A)
can exert potent effects on cellular
differentiation and that these effects are often
not linked. It thus seems likely that at least
one related gene product might be a specific
retinol receptor or a receptor for another member
of the retinoid family. The second surprising
observation from these results is the close
kinship of the retinoid receptor with the thyroid
hormone receptor. (As we show below, the
retinoic acid receptor can activate a thyroid
response element or TRE; see the section of the
specification labeled "Retinoic Acid and Thyroid
Hormone Induce Gene Expression Through a Common
Response Element".) This relationship is
surprising in part because of the structural
dissimilarity of the thyroid hormones and the
retinoids. Thyroid hormones being derived from
the condensation of two tyrosine molecules
whereas, the retinoids are derived from mevalonic
1 X41 422
34
acid. The observation that chemically distinct
molecules interact with receptors sharing common
structures most likely reflects a common mode of
action with which they elicit their particular
regulatory effects. Based on this analogy, we
can now propose that the interaction of retinoids
with their intracellular receptors induces a
cascade of regulatory events that results from
the activation of specific sets of genes by the
hormone/receptor complex. Although animals
employ diverse means to control their development
and physiology, the demonstration that the
retinoic acid receptor is part of the steroid
receptor superfamily suggests that mechanisms
controlling morphogenesis and homeostasis may be
more universal than previously suspected.
Construction and Characterization
of Chimeric Receptars
Construction of chimeric receptor genes is
discussed above in the sectians of the
specification labeled "Definitions", "Summary of
the Invention" and "Domain Switching and
Transcriptional Activation". In the sections
that follow, construction and characterization of
the chimeric receptors is illustrated by showing
construction and and characterization of GR/TR
hybrids.
Materials and Methods
Cell Culture and Transfection
CV-1 cells were used as the receptor-
deficient host cells that were transfected with
expression plasmids that carry the chimeric RR/TR
receptors, and reporter plasmids carrying the CAT
reporter gene. Conditions for growth and
transfection of CV-1 (African Green monkey
kidney) cells were as previously described
1 X41 422
(Giguere etal. (1986)), except that the calcium
phosphate precipitate was left on the cells for
4-8 hours, at which time the media was changed to
5 DMEM with 5% Ts free bovine serum (Scantibodies)
minus or plus 10-? M Ts (Sigma) . Cells were
harvested 36 hours after the addition of Ts, and
CAT assays were performed as described by Gorman
et al. ( 1982 ) . Typically, 5 pg reporter and 1 y~g
10 expression vector were cotransfected, along with
2.5 pg RSV-pgal as a control for transfection
efficiency. Acetylated and non-acetylated forms
of [14C]chloramphenicol were separated by thin
layer chromatography, excised, and quantitated by
15 liquid scintillation counting in Econofluor
(DuPont) with 5% DMSO. l9-galactosidase assays
were performed as described by Herbomel etal.
(1984). CAT activity is expressed as percent
conversion divided by p-galactosidase activity.
20 Construction of Reporter
and Expression Plasmids
Synthetic oligonucleotides corresponding to
-169 to -200 of the rat growth hormone gene was
inserted into a linker scanning mutant of MTV- CAT
25 that has a HiradIII site at position -190/-181
(Buetti and Kuhnel {1986)). Expression vectors
were constructed for the thyroid hormone
receptors by inserting the full-length cDNAs of
pheAl2 (hTRp, see Weinberger, et al. (1986) ) and
30 rbeAl2 ( rTRa, see Thampson, et al. ( 1987 ) ) between
the KpnI and BamHI sites of the pRS vector
(Giguere, et al. (1986) and (1987) ) .
Construction of Chimeric Receptors
The construction of hGRN~ has been described
35 (Giguere, et al. ( 1987 ) . To construct hTR~NX, the
cDNA insert of pheAl2 (hTRf, see, Weinberger, et al.
(1985) and (1986)) was subcloned between the KpnI
1 341 422
36
and BamHI sites of M13mp19 and mutagenized by the
method of Runkel (1985). The oliganucleotide
used to create the NotI site changed three amino
acids: Asp97 to Arg, Lys98 to Pro, Asp99 to Pro.
The oligonucleotide used to create the XhoI site
changed two amino acids: Thrl?1 to Leu, Asp172 to
Gly. The mutant receptor cDNA was then
transferred to the expression vector pRS
(Giguere, etal. (1986) and (1987)); hybrids were
constructed by exchanging KpnI-NotI, Kpr~I-XhoI, or
NotI-XhoI restriction fragments between RShGRNx and
RShTRpNX. RShGRNX has about 75% of wild-type
DNA-binding activity, and RShTRpNX has about 60%
1:5 of wild-type DNA-binding activity.
The Cis~Trans Functional Liqand
Identification A.__ssay
The cis/trans functional ligand identification
cotransfection assay was used to study chimeric
receptors constructed by swapping domains between
the glucocorticlod, the thyroid and the retinoic
acid receptors. (As those skilled in the art
will appreciate, the cis/trans cotransfection assay
can be used to study chimeric receptors made by
swapping functional between any of the wild-type
or genetically engineered receptors.) In the
cis/trans assay, preferably two plasmids are
transfected into a receptor deficient cell line.
The first plasmid is used to express the receptor
protein (whether wild-type, chimeric or
genetically engineered). The second plasmid is
used to monitor transcription from a ligand or
hormone responsive promoter. For the thyroid
hormone receptor assay, the expression plasmid
3~ consists of the Rous Sarcoma Virus long terminal
repeat (RSV-LTR) directing the expression of a
cDNA encoding a thyroid hormone receptor. For
1 341 422
37
the hGR, the reporter plasmid is the mouse
mammary tumor virus long terminal repeat (MTV-
LTR) fused to the bacterial chloramphenicol
acetyltransferase ( CAT) gene. To convert MTV-
CAT to a thyroid hormane responsive reporter, an
oligonucleotide containing a thyroid hormone
response element (TRE) was inserted at position
-191 of the MTV-LTR. This sequence, -169 to -200
of the rat growth hormone (rGH) gene,
specifically binds thyroid hormone receptors and
can confer Ts responsiveness to a heterologous
promoter (Glass eta~'. (1987)). Expression and
reporter plasmids were cotransfected into CV-1
cells and CAT activity was measured in the
absence and presence of Ts. The assays showed
that neither the alpha nor the beta thyroid hormone
receptor activates transcription from MTV- CAT, in
the absence ar presence of Ts. (Data not shown.)
However, the addition of a THE produces an MTV
promoter that is thyroid hormone responsive.
Induction of CAT activity is dependent on the
cotransfectian of a functional alpha or beta thyroid
hormone receptor and the addition of Ts. In the
presence of Ts, the alpha receptor (rTRa) induces
CAT activity approximately 15-fold, while the beta
receptor (hTR~) induces activity by about 5-fold.
The hybrid thyroid hormone/glucocorticoid
receptors were constructed to compare the
functional properties of the thyroid and
glucocorticoid hormone receptors. To facilitate
the construction of the chimeric hybrid
receptors, unique sites for the restriction
enzymes NotI and Xhol were inserted flanking the
DNA binding domains of hGR and hTRQ. These
mutant receptors, termed hGRNx and hTR~NX, can be
used to create hybrids with all possible
1 34~ 422
38
combinations of amino termini, DNA-binding
domains, and ligand-binding domains for these
receptors. (As those skilled in the art will
appreciate, comparable plasmids, such as pRARaNx
or pMRNx for example, can be used to create
chimeric receptors consisting of all possible
combinations of all functional domains from the
various receptors in the steroid hormone receptor
superfamily. The receptors and the locations of
the various functional domains are shown in
Figure 8.) The hybrid arid parental receptors
were assayed using both thyroid hormone and
glucocorticoid responsive promoters, in the
absence or presence of Tg or the synthetic
glucocorticoid dexamethasone.
The structures and activities of the hybrid
thyroid/glucocorticaid receptors are shown in
Figure 9. The receptors are divided into three
sections, and hybrids are named by letters
referring to the "origin" of the domain; for
example, "T-G-T" has the amino and carboxyl
termini of hTR~ (T-,-T) and the DNA binding
domain of the hGR (-G-). Hybrids with a putative
hTR~ DNA binding domain (TTG, GTT, GTG) activated
transcription from TRE- CAT, while hybrids with an
hGR DNA binding domain (GGT, TGG, TGT) activated
transcription from GRE- CAT. This demonstrates
that this region of hTRp is analogous to the hGR
DNA binding domain and is responsible for
promoter recognition. Hybrid receptors with an
hTRp carboxyl terminus were activated by Ts, while
those with an hGR carboxyl terminus were
activated by dexamethasone. This is consistent
with the identification of the carboxyl terminus
as the part of the receptor that is responsible
for hormone binding and activation specificity.
~ X41 422
39
Taken together, the functional properties of
these hybrids support the assignment of the DNA-
and l:igand-binding domains of hTR~.
Retinoic Acid and Thyroid Hormone Induce Gene
Expression Through a Common Responsive Element
identification of a functional retinoic acid
responsive element (RARE) is crucial to our
understanding of the mechanisms by which retinoic
acid receptors activate gene expression and
regulate cell differentiation. One impediment to
such a study is the absence of any identified
gene whose transcription is directly dependent on
the retinoic acid receptor-hormone complex. An
alternative approach to localize a RARE is to
systematically challenge the inducibility of
known hormonally responsive promoters with
retinoic acid receptor produced from cloned cDNA.
(As discussed above under the heading "The
Cis/Trans Assay" , in this system, transcriptional
activation from a promoter containing a HRE is
dependent on expression of functional receptor
from cotransfected expression plasmids in
receptorless cells such as CV-1.) Because the
DNA-binding domains of the retinoic acid and
thyroid hormone receptors are highly related (62%
identical in their amino acid sequences, see
Figure 6), the possibility that the retinoic acid
receptor could activate gene expression through a
THE was investigated.
TRE's are known: see, for example, Glass, ec
al, (1987) for a discussion of a cis-acting element
in the rat growth hormone 5' flanking genomic
sequence that is necessary far thyroid hormone
(3,5,3'-triiodo-L-thyronine, T~) regul tion.
~ 3~~ 4zz
To test if a THE could effectively function
as a RARE, a novel Ts responsive promoter was
constructed by replacing the glucocorticoid
responsive elements present in the mouse Mammary
Tumour Virus-Long Terminal Repeat (MTV-LTR) with
an oligonucleotide encoding the natural TREGx»
This promoter was then fused to the bacterial
chloramphenicol acetyl transferase (CAT) gene to
10 generate the reporter plasmid MTV-TRE~x-CAT.
After transient transfection into CV-~. cells, the
inducibility of the promoter was determined by
measuring CAT activity, When CV-1 cells are
cotransfectd with the expression vector
15 containing a human thyroid hormone receptor beta
(pRShTsRp) and the reporter plasmid OMTV-
TRE~H-CAT, induction in CAT activity is observed
in the presence of TS. In contrast,
cotransfection of an expression vector encoding
20 the human glucocorticoid receptor (pRShGRa) and
the same reporter plasmid did not stimulate CAT
activity from this promoter in response to the
synthetic glucocorticoid dexamethasone. These
results clearly demonstrate that the induction of
25 CAT activity by RARa is conferred by the THE
because the wild-type MTV-LTR construct was not
responsive. (Data not shown.) These results also
show that the hRARa can specifically induce gene
expression from a promoter containing a TRE.
30 RAR, and GR Chimeric Receptors
As discussed above, the modular structure of
steroid hormone receptors makes it ;possible to
exchange functional domains from ane receptar to
another to create functional chimeric receptors.
35 This strategy was used to create hGR/hRARa
chimera that had the RAR DNA-binding domain and
the GR ligand-binding domain» When CV-1 cells
1 341 422
41
were cotransfected with the expression plasmid
encoding hGRG and the reporter OMTV-TREGH-CAT,
dexamethasone specifically elicited CAT activity.
(Data not shown.) This experiment provided
direct evidence that the DNA-binding damain of
the hRARa determined the specificity of target
gene activation.
DETAILED DESCRIPTION OF THE DRAWINGS
FIGURE 1. DNA and primary amino acid
sequence of phRARl. A, Schematic representation
and restriction enzyme map of the phRARl clone.
The stippled box represents the predicted open
reading frame. B, (shown as B-1, B-2 and B-3)
The complete nucleotide sequence of phRARl is
shown with the amino acid sequence given above
the long open reading frame. An upstream in-
frame stop codon at nucleotides 85-87 and
polyadenylation signal are underlined.
Z0 Figure 1 Methods. A 63-mer oligonucleotide
corresponding to nucleotides 408-477 of the
genomic sequence published by Dejean er al. (1986)
was used as a hybridization probe to screen a
human testis agtl0 library. The hybridization
25 mixture contained 35% formamide, 1X Denhardt's,
5X SSPE, 0.1% sodium dodecyl sulfate (SDS), 100
~g ml~l denaturated salmon sperm DNA and lOg c.p.m.
ml-1 of SZP-labeled oligonucleotide. Duplicate
nitrocellulose filters were hybridized at 42°C for
16 h, washed three times for 20 min each in 2X
SSC, 0.1% SDS (1XSSC = 150 mM NaCl, 15 mM sodium
citrate) at 55°C and autoradiographed at -70°C
with an intensifying screen. Clone lhTlR
obtained from this screening was partially
characterized and then used as a hybridizing
probe to screen a human kidney WgtlO cDNA library
(see Bell, et al. , (1986) ) . For this screening, the
1 341 422
42
washing conditions were modified to 1XSSC with
0.1% SDS at 68°C. Several cDNA clones were
isolated and the longest clone, phRARl, was
digested with a number of restriction enzymes and
the resulting fragments were subcloned in both
orientations into the M13 sequencing vectors mpl8
and mpl9 and sequenced by the dideoxy procedure
(see Sanger, er al. , ( 1977 ) ) . DNA sequences were
compiled and analyzed by the programs of Devereux
et al . ( 1984 ) and Staden ( 1982 ) .
FIGURE 2. A, Construction of the chimeric
receptor hRGR. The domain-structure of the
various constructions are shown schematically,
the numbers correspond to the amino acid
positions of each domain. The DNA-binding
domains are represented by "DNA" and the ligand-
binding domains by their respective inducers.
The Notl and Xhol sites created by site-directed
mutagenesis to permit the exchange of the DNA-
binding domains between receptors are indicated.
B, Induction of CAT activity by retinoic acid.
The expression vectors were cotransfected into
CV-1 cells with the reporter plasmid MTVCAT and
cultured for 2 days in absence or presence of 100
nM dexamethasone (DEX) or retinoic acid (RA).
The receptor inserted into the expression vectors
are: pRShGR, human glueocorticoid receptor;
pRShRR, human retinaic acid receptor; pRShRRnX,
mutated human retinoic acid receptor with Nori and
Xhol sites: pRShRGR, chimeric receptor composed of
the human retinoic acid receptor which DNA-
binding domain has been replaced by the human
glucocorticoid receptor DNA-binding dr~main.
Figure 2 Methods. A, Restriction enzyme
fragments of the cDNA inserts of phRARl and hGR
(see Hollenberg, et al. , (1985) were subcloned into
~ 3'~~ 422
43
the Kpnl and BamHl sites of the mpl9 vector and
mutagenized according to the method of Kunkel
(1985). The oligonucleotides used for the
creation of the Nor1 site within hGR and hRR were
28 and 31 nucleotides respectively, while the
oligonucleotides used for the creation of the Xhol
site within hGR and hRR were 24 and 23
nucleotides. The creation of the Norl site
resulted in the mutation of Pro,,~s to an Arg
residue in hGRNX, and in the mutation of I1e84 and
Tyr85 to Pro residues in hRRNX. The introduction
of the Xhol site did not alter the hGRNX amino
acid sequence but resulted in the mutation of
LyslSS to a Leu residue in hRRNX. The mutant
receptors were then transferred to the expression
vector pRS (see Giguere, er al. , (198f) , and the
Nor1/Xhol restriction fragment of pRShGR~X
containing the hGR DNA-binding domain was
introduced into pRShRnx between the Notl and Xhol
sites to create pRShRGR. B, Cell transfection
and CAT assay. The recombinant DNA constructs (5
~g each) were introduced into CV-1 cells by
calcium phosphate coprecipitation (see Wigler, er
al., (1979)). The cells were then cultured for
two days in serum free media supplemented with
Nutridoma (Boehringer Mannheim) in presence or
absence of inducers. CV-1 cells were then
prepared for CAT assays as described by Gorman, er
al. (1982) and the assays performed for 3 h using
25 ug of protein extract. All experiments with
retinol were conducted in subdued light.
FIGURE 3. A, Dose-response to retinoids.
CV-1 cells cotransfected with pRShRGR and pMTVCAT
were treated with increasing concentrations of
retinoids or a single 1 ~M dose (*) of
testosterone, dihydrotestosterone, estrogen,
44 1 3 ~ 1 4 2 2
cortisol, aldosterone, progesterone, triiodo-
thyronine (TS), thyroxine (T~), dihydroxy-vitamin
Ds (VDS) and 25-OH-cholesterol. The levels of CAT
activity were plotted as percentages of the
maximal response observed in this experiment. B,
Retinoic acid binding to cytosol extracts of
transfected COS-1 cells. Bars represent bound
sH-retinoic acid determined in absence (black
bars) or presence (stippled bars) of a 1000-fold
excess of various competitors. The values
represent the mean of quadruplicate
determinations. Competitors are retinoic acid
(RA), retinol (R), T" dexamethasone (DEX) and
vitamin D$ (VDs) .
Figure 3 Methods. A, CV-1 cell
cotransfections and GAT assays were performed as
described in Figure 2. Retinoic acid was
dissolved in a minimum volume of dimethyl
sulfoxide and diluted in ethanol. All other
products were diluted in ethanol and control
cultures received 0.1% solvent (v/v) in media.
Dose-response curves of retinoid treatment were
performed in triplicate. B, Subconfluent COS-1
cells were transfected with 10 ~g/dish of a
control plasmid (pRS) or pRShRR by the DEAE-
Dextran method (see Deans, et ar. , ( 1984 ) ) . Cells
were maintained for 2 days in DMEM with 5%
charcoal-treated fetal calf serum, then harvested
in THE (40 mM tris-HCl pH 7.5, 150 mM NaCl, 1mM
EDTA) and lysed by bounce homogenization in
hypotonic buffer (50 mM tris-HCl pH 7.4, 0.1 mM
EDTA, 5 mM dithiothreitol, 10 mM NaMoO,~, 10%
glycerol, 0.5 mM phenylmethylsulfonyl fluoride)
and centrifuged at 100,000 X g for 30 min to
yield the cytosol fractian. Incubatians were
performed in hypotonic buffer with 150 ~g of
~ ~4~ ~z2
protein from the cytosolic fraction and 2 X 10'$ M
SH-retinoic acid (NEN, 52.5 Gi/mmole) in a total
volume of 200 ~1. Specific binding was measured
by the addition of 2 X 10'6 M of competitors.
Reactions were carried out at 4°C for 16 h. Bound
sH-retinoic acid was quantitated using DE-81
filters. Reactions were placed on filters for 1
min and then rinsed with 5 ml of washing buffer
10 (50 mM tris-HC1 pH 7.4, 0.1 mM EDTA, 0.1 % Triton*
X-100). Filters were dried and counted by liquid
scintillation spectrophotometry.
FIGURE 4. Southern blot analysis of human
genomic DNA. A, Human placenta DNA was digested
15 with the indicated restriction enzymes. After
separation of the digested DNA in a 0.8% agarose
gel (10 ~g/lane) and transfer to nitrocellulose
filters (see Southern, (1975}, the blots were
hybridized with an EcoRl X Pv~II fragment from
phRARl ('600 bp) encompassing the DNA-binding
domain of the hRR under high stringency
conditions (50% formamide, 5X SSPE, 1X
Denhardt's, 0.1% SDS, 100 beg m1'1 salmon sperm
DNA}. The filter was washed in O.iX SSC, 0.1%
25 SDS at 65°C. Lambda HrrrdIII DNA markers (size in
Kb) are aligned to left of the autoradiogram. B,
Analysis of human placenta DNA using the same
probe as in A under non-stringent conditions. A
parallel blot containing identical samples was
30 hybridized as in A, except that 35% formamide was
used. The filter was washed in 2XSSC, 0.1% SDS
at 55°C.
FIGURE 5. Northern blot analysis of
retinoic acid receptor mRNA in rat and human
35 tissues.
* trade mark
~...;. ;
1 X41 422
46
Figure 5 Methods. Total RNA was isolated
from various tissues using guanidine thyocyanate
(see Chirgwin, et al. , (1980) , separated on 1%
agarose- formaldehyde gel, transferred to
nitrocellulose, and hybridized under stringent
conditions using the probe described in Fig. 4.
Twenty ~g of total RNA was used in all lanes.
Migration of ribosomal RNA's (28S and 18S) are
indicated for size markers. The nitrocellulose
filter was autoradiographed at -70°C with an
intensifying screen for 1 week.
FIGURE 6. Schematic amino acid comparisons
of the hGR, hRR and hT$R~ structures. Amino acid
1.5 sequences have been aligned schematically with
the percentage amino acid identity for each
region of homology in the intervals between
dotted lines.
FIGURE 7 is a schematic diagram of a
generalized steroid\thyroid\retinoic acid
receptor gene, showing the divisian of the gene
into regions A/8, C, D, and E. The functian of
the A/B region is just beginning to be
elucidated; the C region encodes the DNA-binding
domain; the D region is believed to be a hinge
region: and the E region encodes the ligand-
binding domain.
FIGURE 8 is a schematic drawing that shows
amino acid comparison of members of the steroid
hormone receptor superfamily. Primary amino acid
sequences have been aligned on the basis of
regions of maximum amino acid similarity, with
the percentage amino acid identity indicated for
each region in relation to the hGR (Miller etal.,
(1985). Domains shown are: a domain at the NHZ-
terminal end that is required for "maximum
activity". the 66- to 68-amino acid DNA-binding
.. 1 34~ ~z2
47
domain core ("DNA"): and the 250-amino acid
ligand-binding (or hormone-binding domain)
("Hormone"). The amino acid position of each
domain boundary is shown. Amino acid numbers for
all receptors represent the human forms with the
exception of v-erb-A and E75 (Segraves, 1988).
Functional assignments have been determined by
characterization of the glucocorticoid and
estrogen receptors. Designations are as follows:
GR, glucocorticoid receptor; MR mineralocorticoid
receptor: PR, progesterone receptor: ER, estrogen
receptor: ERR1 or ERR2, estrogen-related 1 or 2;
VDR, vitamin D$ receptar: and TsR~ and TsRa,
thyroid hormone receptors. The (+) or (-)
indicates whether a part~.cular property has been
demonstrated for the products of cloned receptor
cDNA or with purified receptor. HRE, hormane
response element. This relates to whether the
binding site has been identified structurally and
whether its enhancement properties have been
demonstrated by gene transfer studies. For PR,
DNA-binding properties have been shown only with
the native purified receptor. "Hormone binding
in vitro" indicates whether this property has
been demonstrated by translation in a rabbit
reticulocyte lysate system (Hollenberg era!,
1985). "Hormone binding in vivo" refers to
expression of the cloned receptor in transfected
cells. "Chromosome" indicates the human
chromosome location. Species are as follows: h,
human; r, rat; m, mouse: c, chicken; and d,
Drosophilia.
FIGURE 9. Structure and activity of
chimeric thyroid/glucocorticoid receptors.
1 34~ 422
48
Figure 9 Methods. To construct hybrid
receptors, unique NotI and XhoI sites were
inserted flanking the DNA binding domains of the
hGR and hTR~. Hybrids were created by exchanging
the appropriate 'segments of the receptor cDNA's.
"DNA" indicates the DNA binding domain; "Ts/Ts"
and "cortisol" indicate the l,igand binding
domains of hTR~ and hGR respectively. The
numbers above the boxes indicate amino acid
residues. Hybrids are named by letters referring
to the origin of the domain: for example, "TGT"
has the amino and carboxyl termini of hTR~ and
the DNA binding domain of the hGR. All receptors
1~~ were assayed on TRE-M CAT arid GRE-M CAT in the
absence and presence of T$ and the synthetic
glucocorticoid dexamethasone ("dex"). All of the
combinations shown gave activation above
background.
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SPECIFICATION SUMMARY
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
10 to as retinoic acid receptor protein. The invention
also provides a plasmid containing retinoic acid
receptor DNA. This plasmid, phRARl, has been
deposited with the American Type culture Collection
for patent purposes.
15 The invention is also comprised of retinoic
acid receptor proteins, including modified
functional forms thereof, expressed from the DNA (or
mRNA) of the invention.
In addition to novel retinoic acid receptor
20 DNA, RNA and protein compositions, the present
invention 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, hERRl, hERR2, T3Ra, TSR~, RARa, and
25 RARE receptors with one another. The chimeric
receptors so constructed have DNA-binding domain and
ligand-binding domain characteristics similar to the
DNA-binding domain and ligand-binding domain
characteristics of the respective "parental"
30 receptors from which they originated.
Finally, the present invention involves a
bioassay for determining the functional ligands for
receptor proteins, both wild-type and chimeric.
The phRARi DNA of the invention can be used
35 to make the retinoic acid receptor proteins, and
functional modified forms thereof, in quantities
that were not previously possible. The same is true
1 34~ 422
56
of the chimeric receptors. With the quantities of
receptor protein available as a result of the
present invention, detailed studies can be made of
both the ligand/receptor complexes and the
ligand/receptor/HRE complexes. In addition, an
adequate supply of the retinoic acid receptor
proteins means that they can now be used to screen
compounds for retinoic acid receptor-agonists or
retinoic acid receptor-antagonist activity.
Availability of the receptor proteins also means
that they can be used in diagnostic assays to
determine the levels of retinoic acid present in
various tissues and body fluids.
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.
