U.S. patent application number 10/236745 was filed with the patent office on 2003-05-01 for allosteric control of nuclear hormone receptors.
This patent application is currently assigned to The Salk Institute for Biological Studies. Invention is credited to Evans, Ronald M., Forman, Barry M., Umesono, Kazuhiko.
Application Number | 20030083469 10/236745 |
Document ID | / |
Family ID | 23467196 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030083469 |
Kind Code |
A1 |
Evans, Ronald M. ; et
al. |
May 1, 2003 |
Allosteric control of nuclear hormone receptors
Abstract
Heterodimerization is a common paradigm among eucaryotic
transcription factors, though it remains unclear how individual
monomers contribute to the overall transcriptional activities of
the complex. The 9-cis retinoic acid receptor (RXR) serves as a
common heterodimerization partner for several nuclear receptors
including the thyroid hormone (T.sub.3R), retinoic acid (RAR) and
vitamin D receptors. A strategy has been devised to examine the
transcriptional properties of each receptor individually or when
tethered to a heterodimeric partner. It has been found that the
intrinsic activity of RXR is masked in RXR-T.sub.3R and RXR-RAR
heterodimers. In contrast, a novel RXR-Nurrl heterodimer described
herein is highly responsive to RXR ligands, suggesting that
different partners exert unique allosteric control over the RXR
response. These findings establish a novel 9-cis retinoic acid
response pathway and resolve the paradox as to how T.sub.3R, RAR
and VDR contribute to distinct physiologic pathways while sharing a
common RXR subunit.
Inventors: |
Evans, Ronald M.; (La Jolla,
CA) ; Forman, Barry M.; (La Jolla, CA) ;
Umesono, Kazuhiko; (Nara, JP) |
Correspondence
Address: |
FOLEY & LARDNER
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
The Salk Institute for Biological
Studies
|
Family ID: |
23467196 |
Appl. No.: |
10/236745 |
Filed: |
September 6, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10236745 |
Sep 6, 2002 |
|
|
|
08877966 |
Jun 18, 1997 |
|
|
|
6458926 |
|
|
|
|
08877966 |
Jun 18, 1997 |
|
|
|
08372217 |
Jan 13, 1995 |
|
|
|
Current U.S.
Class: |
530/358 ;
435/7.21 |
Current CPC
Class: |
C07K 14/70567 20130101;
C07K 2319/00 20130101; G01N 33/6875 20130101; G01N 33/74
20130101 |
Class at
Publication: |
530/358 ; 514/12;
435/7.21 |
International
Class: |
A61K 038/17; G01N
033/567; C07K 014/705 |
Claims
That which is claimed is:
1. A method to suppress the constitutive activity of Nurrl, said
method comprising contacting Nurrl with at least the ligand binding
domain of RXR.
2. A method according to claim 1 wherein the ligand binding domain
of RXR is selected from RXR.alpha., RXR.beta. or RXR.gamma..
3. A method to render NurrI-containing cells inducibly responsive
to RXR selective ligands, said method comprising contacting said
cells with at least the ligand binding domain of RXR.
4. A method according to claim 3 wherein the ligand binding domain
of RXR is selected from RXR.alpha., RXR.beta. or RXR.gamma..
5. A method to render RXR-containing cells responsive to RXR
selective ligands, said method comprising contacting said cells
with a silent partner therefor.
6. A method according to claim 5, wherein said silent partner is an
isoform of Nurrl.
7. A method for the identification of nuclear receptor(s) which
participate as silent partner(s) in the formation of a heterodimer
with RXR, said method comprising introducing into a cell: at least
the ligand binding domain of a putative silent partner for RXR, a
chimeric construct containing a GAL4 DNA binding domain and at
least the ligand binding domain of RXR, and a reporter construct,
wherein said reporter construct comprises: (a) a promoter that is
operable in said cell, (b) a GAL4 response element, and (c) DNA
encoding a reporter protein, wherein said reporter protein-encoding
DNA is operatively linked to said promoter for transcription of
said DNA, and wherein said GAL4 response element is operatively
linked to said promoter for activation thereof, and thereafter
monitoring expression of reporter upon exposure of the
above-described cell to RXR selective ligand(s).
8. A method for the identification of nuclear receptor(s) which
participate as silent partner(s) in the formation of heterodimer(s)
with RXR, said method comprising introducing into a cell: a
putative silent partner for RXR, at least the ligand binding domain
of RXR, and a reporter construct, wherein said reporter construct
comprises: (a) a promoter that is operable in said cell, (b) a
response element for said putative silent partner, and (c) DNA
encoding a reporter protein, wherein said reporter protein-encoding
DNA is operatively linked to said promoter for transcription of
said DNA, and wherein said response element for said putative
silent partner is operatively linked to said promoter for
activation thereof, and thereafter monitoring expression of
reporter upon exposure of the above-described cell to RXR selective
ligand(s).
9. A method according to claim 8 wherein the response element for
the putative silent partner has the sequence AAAGGTCA.
10. A method for identifying ligands selective for heterodimers
comprising RXR and a silent partner therefor, said method
comprising comparing the level of expression of reporter when cells
containing a reporter construct, RXR and silent partner therefor
are exposed to test compound, relative to the level of expression
of reporter when cells containing a reporter construct, RXR and a
member of the steroid/thyroid superfamily which is not a silent
partner therefor are exposed to test compound, and selecting those
compounds which activate only the combination of RXR and silent
partner therefor.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to intracellular receptors,
and methods for the modulation thereof. In a particular aspect, the
present invention relates to novel heterodimeric complexes. In
another aspect, the present invention relates to methods for
modulating processes mediated by retinoid X receptor and/or orphan
receptor Nurrl.
BACKGROUND OF THE INVENTION
[0002] Heterodimerization is a common theme in eucaryotic
regulatory biology. Indeed, a number of transcription factor
families have been defined by their characteristic dimerization
interface. These include the leucine zipper (e.g. fos, jun, CREB,
C/EBP; see, for example, Lamb and McKnight, in Trends Biochem. Sci.
16:417-422 (1991)), helix-loop-helix (e.g. myc, max, MyoD, E12,
E47; see, for example, Amati and Land, in Curr. Opin. Genet. Dev.
4:102-108 (1994)), rel (NF.kappa.B, dorsal; see, for example, Blank
et al., in Trends Biochem. Sci. 17:135-140 (1992)), ankyrin (GABP;
see, for example, Brown and McKnight, in Genes Dev. 6:2502-2512
(1992)), and the nuclear receptor superfamilies (see, for example,
Evans, in Science 240:889-895 (1988), and Forman and Samuels, Mol.
Endocrinol. 4:1293-1301 (1990)). Detailed analyses of these
proteins have shown that heterodimerization produces novel
complexes that bind DNA with higher affinity or altered specificity
relative to the individual members of the heterodimer (see, for
example, Glass, in Endocr. Rev. 15:391-407 (1994)). Indeed, little
is known about the contributions of each monomer toward the
transcriptional properties of the complex.
[0003] Nuclear hormone receptors are characterized by a central DNA
binding domain (DBD, see FIG. 1), which targets the receptor to
specific DNA sequences, known as hormone response elements (HREs) .
The retinoic acid receptor (RAR), the thyroid hormone receptor
(T.sub.3R) , the vitamin D.sub.3 receptor (VDR) and the fatty
acid/peroxisome proliferator activated receptor (PPAR)
preferentially bind to DNA as heterodimers with a common partner,
the retinoid X (or 9-cis retinoic acid) receptor (RXR; see, for
example, Yu et al., in Cell 67:1251-1266 (1991); Bugge et al., in
EMBO J. 11:1409-18 (1992); Kliewer et al., in Nature 355:446-449
(1992); Leid et al, in Cell 68:377-395 (1992); Marks et al., in
EMBO J. 11:1419-1435 (1992); Zhang et al., in Nature 355:441-446
(1992); and Issemann et al., in Biochimie. 75:251-256 (1993).
[0004] Naturally occurring HREs are composed of direct repeats
(i.e., DRs; see Umesono et al., in Cell 65:1255-1266 (1991),
inverted repeats (i.e., IRs; see Umesono et al., in Nature
336:262-265 (1988), and Williams et al. in J. Biol. Chem.
266:19636-19644 (1991)), and/or everted repeats (ERs; see Baniahmad
et al., in Cell 61:505-514 (1990); Farsetti et al., in J. Biol.
Chem. 267:15784-15788 (1992); Raisher et al., in J. Biol. Chem.
267:20264-20269 (1992); or Tini et al., in Genes Dev. 7:295-307
(1993)) of a degenerate X.sub.n-AGGTCA core-site.
[0005] The DNA binding domain (DBD) contains two helical regions,
one of which serves as a recognition helix that makes base-specific
contacts within the major groove of the core-site (see, for
example, Luisi et al., in Nature 352:497-505 (1991) and Schwabe et
al., in Cell 75:567-578 (1993)). A third helix has been identified
in some receptors which makes additional minor groove contacts in
the 5' portion of the core-binding site, X.sub.n (see, for example,
Wilson et al., in Science 256:107-110 (1992) or Lee et al., in
Science 260:1117-1121 (1993)).
[0006] In direct repeats (DR, head-to-tail arrangement) the X.sub.n
sequence also serves as a gap which separates the two core-binding
sites. Spacers of 1, 3, 4 and 5 nucleotides serve as preferred
response elements for heterodimers of RXR with PPAR, VDR, T.sub.3R
and RAR, respectively (see, for example, Naar et al., in Cell
65:1267-1279 (1991); Umesono et al., 1991, supra; Kliewer et al.,
in Nature 358:771-774 (1992); and Issemann et al., supra). The
optimal gap length for each heterodimer is determined by
protein-protein contacts which appropriately position the DBDs of
RXR and its partner (see, for example, Kurokawa et al., in Genes
Dev. 7:1423-1435 (1993); Perlmann et al., in Genes Dev. 7:1411-1422
(1993); Towers et al., in Proc. Natl. Acad. Sci. USA 90:6310-6314
(1993); and Zechel et al., in EMBO J. 13:1414-1424 (1994)). In
contrast to this mode of DNA binding, a growing number of
receptor-like proteins have been identified which bind as a monomer
to a single core-site. The NGFI-b/Nurrl orphan receptors provide
well characterized examples of this paradigm (Wilson et al., in
Mol. Cell Biol. 13:5794-5804 (1993)).
[0007] Once bound to an HRE, each receptor responds to its signal
through the C-terminal ligand binding domain (LBD), which binds its
cognate hormone with high affinity and specificity (see, for
example, Evans, 1988, supra; or Forman and Samuels, 1990, supra).
The LBD is a complex entity containing several embedded subdomains.
These include a C-terminal transactivation function (.tau.2), a
series of heptad repeats which serve as a dimerization interface
and a poorly-delineated transcriptional suppression domain (see
FIG. 1, and Forman and Samuels, 1990, supra).
[0008] The transactivation domain, .tau.2, consists of
approximately 20 amino acids with the potential to form an
amphipathic .alpha.-helix (see Zenke et al., in Cell 61:1035-1049
(1990); Danielian et al., in EMBO J. 11:1025-1033 (1992); Nagpal et
al., in EMBO J. 12:2349-2360 (1993); and Durand et al., in EMBO J.
13:5370-5382 (1994)). When linked to a heterologous DNA binding
domain, the isolated .tau.2 domain displays constitutive
transcriptional activity. However, in the natural context of the
LBD, transcriptional activity requires the addition of ligand.
[0009] The above-described evidence indicates that the LBD
functions as a modular unit whose transcriptional activities are
controlled by ligand. Accordingly, it should be possible for both
members of a receptor heterodimer to be simultaneously activated by
specific ligands therefor. However, in spite of this possibility,
it has been discovered that the ligand-induced transcriptional
activities of various receptor subtypes vary as a function of the
partner with which a subtype participates in the formation of a
heterodimer. For example, the ligand-induced transcriptional
activities of RXR are suppressed when complexed with RAR and
T.sub.3R. This suppression occurs at the level of ligand binding
and transcriptional activation. Furthermore, RXR responsiveness has
not been observed with other partners, including VDR.
[0010] Accordingly, the identification of receptor subtypes which
participate in the formation of RXR-containing heterodimers, yet
retain the ability to be activated by RXR-selective ligands, would
be highly desirable. The present invention identifies such receptor
subtypes and provides methodology for identifying additional
receptor species having such properties.
BRIEF DESCRIPTION OF THE INVENTION
[0011] In accordance with the present invention, it has been
discovered that RXR can interact productively with Nurrl, a member
of the nuclear receptor superfamily that (in the absence of
heterodimerizing partner therefor) is capable of binding DNA as a
monomer (see, for example, Law et al., in Mol. Endocrinol. 6
:2129-2135 (1992); and Scearce et al., in J. Biol. Chem.
268:8855-8861 (1993)). As a result of this interaction, the
constitutive activity of Nurrl is suppressed, and the resulting
complex becomes responsive to RXR-selective ligands (e.g., 9-cis
retinoic acid). The unique ability of the Nurrl-RXR heterodimer
complex to transduce RXR signals establishes a novel response
pathway.
[0012] The results described herein suggest that heterodimer
formation imparts allosteric changes upon the ligand binding domain
(LBD) of nuclear receptors. These allosteric changes confer
transcriptional activities onto the heterodimer that are distinct
from those of the component monomers. This arrangement permits a
limited number of regulatory proteins to generate a diverse set of
transcriptional responses to multiple hormonal signals.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 schematically represents the functional domains of
nuclear hormone receptors. "DNA" represents the DNA binding domain.
"LIGAND" reprsents the large C-terminal ligand binding domain.
Dimerization and transactivation (.tau.2) functions are embedded
within this region, as illustrated.
[0014] FIG. 2 illustrates the differential modulation of RXR
response by T.sub.3R (shown in FIG. 2A) and RAR (shown in FIG.
2B).
[0015] FIG. 3 illustrates the differential modulation of RXR
transcriptional activity by the LBDs of T.sub.3R, RAR and VDR.
[0016] FIG. 4A illustrates the differential modulation of RXR
transcriptional activity by the LBD of T.sub.3R, wherein cells
treated according to FIG. 3 were additionally treated with T.sub.3
(i. e., T.sub.3R ligand) and LG69 (i. e., an RXR specific ligand) .
Normalized reporter activity was determined and plotted as
fold-activation relative to untreated cells.
[0017] FIG. 4B illustrates the differential modulation of RXR
transcriptional activity by the LBD of RAR, wherein cells treated
according to FIG. 3 were additionally treated with AM580 (i.e., an
RAR specific ligand) and LG69. Normalized reporter activity was
determined and plotted as fold-activation relative to untreated
cells.
[0018] FIG. 5 illustrates the ability of T.sub.3R and RAR to
suppress transcription of a constitutively active RXR derivative
(i.e., VP16-RXR).
[0019] FIG. 6 collectively illustrates that the ligand binding
acvitivy of RXR is altered by T.sub.3R and RAR.
[0020] FIG. 6A illustrates the binding of LG69 (an RXR specific
ligand), at-RA (all-trans retinoic acid, an RAR specific ligand)
and Am580 (an RAR specific ligand) to RXR and/or RAR.
[0021] FIG. 6B illustrates that the binding of LG69 to RXR is
reduced in RAR-RXR and T.sub.3R-RXR heterodimers.
[0022] FIG. 6C illustrates that competition of [.sup.3H] 9-cis RA
bound to RXR-RAR heterodimers requires RAR and RXR ligands.
[0023] FIG. 7 collectively demonstrates that a novel Nurrl-RXR
complex provides a signaling pathway for 9-cis retinoic acid.
[0024] Thus, FIG. 7A presents the results of transient transfection
analysis of GAL-Receptor LBD chimeras in the presence of the RXR
LBD.
[0025] FIG. 7B presents transient transfection analysis of
full-length Nurrl and/or RXR.
[0026] FIG. 7C presents a comparison of the responsivity of
Nurrl-RXR complex, or RXR alone, in the presence and absence of RXR
specific ligand in the presence of a Nurrl specific response
element (NBRE) or an RXR specific response element (CRBPII).
[0027] FIG. 7D demonstrates that the RXR LBD activates through
Nurrl but inhibits activation of other receptors.
[0028] FIG. 8 presents an allosteric control model of ligand
responsiveness.
DETAILED DESCRIPTION OF THE INVENTION
[0029] In accordance with the present invention, there is provided
a heterodimer complex comprising RXR and a silent partner
therefor.
[0030] As employed herein, the term "silent partner" refers to
members of the steroid/thyroid superfamily of receptors which are
capable of forming heterodimeric species with RXR, wherein the
silent partner of the heterodimer is not capable of binding ligand
(i.e. , only the RXR co-partner of the heterodimer is capable of
binding ligand).
[0031] As employed herein, the phrase "members of the
steroid/thyroid superfamily of receptors" (also known as "nuclear
receptors" or "intracellular receptors") refers to hormone binding
proteins that operate as ligand-dependent transcription factors,
including identified members of the steroid/thyroid superfamily of
receptors for which specific ligands have not yet been identified
(referred to hereinafter as "orphan receptors") . These hormone
binding proteins have the intrinsic ability to bind to specific DNA
sequences. Following binding, the transcriptional activity of
target gene (i.e. , a gene associated with the specific DNA
sequence) is modulated as a function of the ligand bound to the
receptor.
[0032] The DNA-binding domains of all of these nuclear receptors
are related, consisting of 66-68 amino acid residues, and
possessing about 20 invariant amino acid residues, including nine
cysteines.
[0033] A member of the superfamily can be identified as a protein
which contains the above-mentioned invariant amino acid residues,
which are part of the DNA-binding domain of such known steroid
receptors as the human glucocorticoid receptor (amino acids
421-486), the estrogen receptor (amino acids 185-250), the
mineralocorticoid receptor (amino acids 603-668), the human
retinoic acid receptor (amino acids 88-153). The highly conserved
amino acids of the DNA-binding domain of members of the superfamily
are as follows:
1 (SEQ ID No 1) Cys - X - X Cys - X - X - Asp* - X - Ala* - X -
Gly* - X - Tyr* - X - X - X - X - Cys - X - X - Cys - Lys* - X -
Phe - Phe - X - Arg* - X - X - X - X - X - X - X - X - X - (X - X
-) Cys - X - X - X - X - X - (X - X - X -) Cys - X - X - X - Lys -
X - X - Arg - X - X - Cys - X - X - Cys - Arg* - X - X - Lys* - Cys
- X - X - X - Gly* - Met;
[0034] wherein X designates non-conserved amino acids within the
DNA-binding domain; the amino acid residues denoted with an
asterisk are residues that are almost universally conserved, but
for which variations have been found in some identified hormone
receptors; and the residues enclosed in parenthesis are optional
residues (thus, the DNA-binding domain is a minimum of 66 amino
acids in length, but can contain several additional residues).
[0035] Examples of silent partners contemplated for use in the
practice of the present invention are various isoform(s) of Nurrl,
HNF4 [see, for example, Sladek et al., in Genes & Development
4: 2353-2365 (1990)], the COUP family of receptors [see, for
example, Miyajima et al., in Nucleic Acids Research 16: 11057-11074
(1988), Wang et al., in Nature 340: 163-166 (1989)], COUP-like
receptors and COUP homologs, such as those described by Mlodzik et
al., in Cell 60: 211-224 (1990) and Ladias et al., in Science 251:
561-565 (1991), the ultraspiracle receptor [see, for example, Oro
et al., in Nature 347: 298-301 (1990)], and the like.
[0036] RXR species contemplated for use in the practice of the
present invention are selected from RXR.alpha., RXR.beta.,
RXR.gamma., and the like.
[0037] In accordance with another embodiment of the present
invention, there is provided a method to suppress the constitutive
activity of Nurrl. Such method comprises contacting Nurrl with at
least the ligand binding domain of RXR.
[0038] In accordance with yet another embodiment of the present
invention, there is provided a method to render Nurrl-containing
cells inducibly responsive to RXR selective ligands. Such method
comprises contacting such cells with at least the ligand binding
domain of RXR.
[0039] In accordance with still another embodiment of the present
invention, there is provided a method to render RXR-containing
cells responsive to RXR selective ligands. Such method comprises
contacting said cells with a silent partner therefor.
[0040] In accordance with a further embodiment of the present
invention, there is provided a method for the identification of
nuclear receptor(s) which participate as silent partner(s) in the
formation of a heterodimer with RXR. Such method comprises
[0041] introducing into a cell:
[0042] at least the ligand binding domain of a putative silent
partner for RXR,
[0043] a chimeric construct containing a GAL4 DNA binding domain
and at least the ligand binding domain of RXR, and
[0044] a reporter construct, wherein said reporter construct
comprises:
[0045] (a) a promoter that is operable in said cell,
[0046] (b) a GAL4 response element (or a response element for the
putative silent partner, when substantially full length putative
receptor is employed), and
[0047] (c) DNA encoding a reporter protein,
[0048] wherein said reporter protein-encoding DNA is operatively
linked to said promoter for transcription of said DNA, and
[0049] wherein said GAL4 response element is operatively linked to
said promoter for activation thereof, and thereafter
[0050] monitoring expression of reporter upon exposure of the
above-described cell to RXR selective ligand(s).
[0051] In accordance with a still further embodiment of the present
invention, there is provided a method for identifying ligands
selective for heterodimers comprising RXR and a silent partner
therefor. Such method comprises
[0052] comparing the level of expression of reporter when cells
containing a reporter construct, RXR and silent partner therefor
are exposed to test compound, relative to the level of expression
of reporter when cells containing a reporter construct, RXR and a
member of the steroid/thyroid superfamily which is not a silent
partner therefor are exposed to test compound, and
[0053] selecting those compounds which activate only the
combination of RXR and silent partner therefor.
[0054] The LBD of nuclear hormone receptors is a complex
multifunctional unit containing subdomains for dimerization,
transcriptional suppression and hormone-induced transactivation
(Forman and Samuels, 1990, supra). The dimerization domain incudes
a series of heptad repeats flanked by sequences required for ligand
binding. Thus, the dimerization domain is embedded within the
larger LBD. This structural arrangement raises the possibility that
dimerization may serve as an allosteric modulator of ligand binding
and transactivation. This possibility has been investigated with
the following observations.
[0055] First, dimerization within the LBD is utilized to confer
transcriptional suppression upon certain heterodimeric complexes.
This is exemplified by unliganded T.sub.3R and RAR, which confer
transcriptional suppression upon RXR. Similarly, in accordance with
the present invention, it is demonstrated that RXR can suppress
constitutive activation by Nurrl.
[0056] Second, the intrinsic ligand binding capacity of the LBD can
be modulated by dimerization. This is illustrated by the ability of
unliganded RAR to abrogate the ligand binding activity of RXR. It
has also been found that T.sub.3R induces a similar suppression,
but the presence of ligand therefor, i.e., T.sub.3, is required for
the complete effect. Thus, RXR is seen to serve as a silent partner
when participating in the T.sub.3R and RAR pathways.
[0057] However, not all heterodimeric interactions restrict
ligand-responsiveness. Indeed, in accordance with the present
invention, it is demonstrated that RXR actively confers
ligand-responsiveness upon the Nurrl-RXR heterodimer complex.
Similarly, it has previously been shown that the Drosophila
ecdysone receptor (EcR) acquires ligand binding activity after
heterodimerization with USP (Drosophila homolog of RXR; see Yao et
al., in Nature 366:476-479 (1993)). Thus, differential interactions
among receptor LBDs can either restrict, redirect or lead to an
acquisition of new ligand binding phenotypes.
[0058] In accordance with the results described herein, a
structural model is proposed (see FIG. 8) to account for the
observations. In FIG. 8, RXR (dark shading) and its partner
receptor (e.g., T.sub.3R, RAR or Nurrl (designated "R" in the
figure, shown in light shading) initially exist as monomers in
solution. RXR in monomeric form is capable of binding ligand.
RXR-receptor heterodimers then form, driven by the dimerization
interface that is embedded within the ligand binding domain (LBD) .
Subsequent to dimerization, binding of ligand (e.g., 9-cis RA) to
RXR is modestly reduced by T.sub.3R and dramatically reduced by
RAR. Addition of ligand for T.sub.3R (e.g., T.sub.3) results in a
further reduction in 9-cis RA binding, while certain retinoids
(shown as "RA" in the figure) such as Am580 (an RAR specific
ligand) may restore 9-cis RA binding to RXR-RAR. It is of
particular note that the Nurrl-RXR heterodimer maintains the
ability to bind 9-cis RA.
[0059] The above-described structural model relies on the
observation that a major dimerization interface is embedded within
the larger LBD. It is proposed that upon dimerization, the
structure of the RXR ligand binding/dimerization domain is altered.
Each RXR partner gives rise to unique conformational changes that
either maintain or abrogate RXR ligand binding activity. Binding of
ligand by the partner receptor induces a conformational change that
can be propagated through the dimerization interface onto the LBD
of RXR. This model allows one to explain how the dimerization
partner and its specific ligand exert allosteric control over the
RXR ligand response.
[0060] In the above-described model, the RXR monomer (or homodimer)
is capable of binding ligand with high affinity. When RXR interacts
with one of its non-permissive partners (i.e., T.sub.3R or RAR),
its ability to bind ligand is diminished. On the other hand,
dimerization of USP/RXR with EcR promotes high affinity binding of
ecdysone to EcR. It is believed that these effects are a direct
consequence of the localization of a major dimerization interface
within the LBD (see FIGS. 1 and 8). The above-described model
predicts that this structural arrangement serves to functionally
link dimerization and ligand binding activities. This would then
provide a mechanism by which dimerization could exert allosteric
control over the ligand response.
[0061] In addition to dimerization, ligand binding by one receptor
may also result in allosteric modification of its partner.
Specifically, binding of ligand to the RXR partner can either
restore (as in the case of RAR) or further decrease (as in the case
of T.sub.3R) the ligand binding potential of RXR (see FIG. 6). It
is already known that upon ligand binding the cognate receptor
undergoes a conformation change (see, for example, Toney et al., in
Biochemistry 32:2-6 (1993)). The results provided herein support
the suggestion that ligand-induced conformation changes in the LBD
of one heterodimer partner will be propagated through the
dimerization interface onto the LBD of the partner. Thus, the model
presented above can explain how a dimerization partner and its
specific ligand can exert allosteric control over the RXR ligand
response. Similarly, the above-described model can account for the
ability of ligand to either promote EcR-USP, (Yao et al., 1993,
supra) or destabilize VDR-RXR and T.sub.3R-T.sub.3R dimers (see,
for example, Andersson et al., in Nucleic Acids Res. 20:4803-4810
(1992); Ribiero et al., in Mol. Endocrinol. 6:1142-1152 (1992); Yen
et al. , in J. Biol. Chem. 267:3565-3568 (1992); MacDonald et al,
in Mol. Cell Biol . 13:5907-5917 (1993); and Cheskis and Freedman,
in Mol. Cell Biol. 14:3329-3338 (1994)).
[0062] The restriction of RXR activity within certain heterodimers
indicates that 9-cis RA responsiveness is not an obligatory
consequence of heterodimerization with RXR. This allows RXR to
function as both a receptor and as a heterodimerization partner,
without requiring all target genes to be 9-cis RA responsive. This
explains the paradox as to how RXR serves as a common subunit for
receptors which display independent physiologic effects (e.g.
T.sub.3R, RAR, VDR).
[0063] In contrast, the ability of RXR to transduce signals when
complexed with Nurri suggests an alternative pathway for 9-cis RA
signaling. Nurrl expression is induced by physiological stimuli
(see Davis and Lau, in Mol. Cell Biol. 14:3469-3483 (1994))
including membrane depolarization and liver regeneration (Scearce
et al., 1993, supra). Based on the results presented herein, it is
clear that RXR contributes to the regulation of these events.
[0064] Unlike previously described heterodimers, RXR functionally
interacts with Nurrl in the absence of RXR-specific DNA contacts
(see FIG. 7D) . Indeed, the ability to tether to a DNA bound
monomer is a distinguishing feature of the Nurrl-RXR heterodimer
complex. As a result, an RXR mutant that is deficient in DNA
binding activates through Nurrl while it inhibits other receptor
heterodimers (see FIG. 7D).
[0065] In accordance with the present invention, there are provided
methods for the modulation of Nurrl expression induced by
physiological stimulus of a subject. Such method comprises
administering to the subject an effective amount of a composition
comprising at least the ligand binding domain of RXR. Physiological
stimuli contemplated for treatment in accordance with the present
invention include any event which induces production of calcium
ions, cyclic AMP, ACTH, and the like.
[0066] The invention will now be described in greater detail by
reference to the following non-limiting examples.
Example 1
Cell Culture and Transfection
[0067] CV-1 cells were grown in Dulbecco's Modified Eagle's medium
supplemented with 10% resin-charcoal stripped (Samuels et al.,
Endocrinology 105:80-85 (1979)) fetal bovine serum, 50 U/ml
penicillin G and 50 .mu.g/ml streptomycin sulfate (DMEM-FBS) at
37.degree. C. in 5% CO.sub.2. One day prior to transfection, cells
were plated to 50-80% confluence using phenol-red free DMEM-FBS.
Cells were transfected by lipofection using
N-{2-(2,3)-dioleoyloxy)propyl-N,N,N-trimethyl ammonium methyl
sulfate} according to the manufacturer's instructions (DOTAP,
Boehringer Mannheim). After 2 hours, the liposomes were removed and
cells treated for 40 hours with phenol-red free DMEM-FBS alone or
with the following ligands: 100-300 nM T.sub.3
(L-triiodothyronine), 100 nM LG69 (4-{1- (3, 5, 5, 8,
8-pentamethyl-5, 6, 7, 8-tetrahydro-2-napthalenyl)-1--
propenyl}benzoic acid), 50-100 nM Am580 (4-(5, 6, 7,
8-tetrahydro-5, 5, 8, 8-tetramethyl-2-napthamido) benzoic acid) or
100 nM VD.sub.3 (1.alpha., 25-dihydroxyvitamin D.sub.3) . Cells
were harvested and assayed for luciferase and .beta.-galactosidase
activity. All points were performed in triplicate in each
experiment and varied by less than 10%. Each experiment was
repeated three or more times with similar results.
Example 2
Expression and Reporter Constructs
[0068] For luciferase assays, response elements with HindIII
overhangs were cloned into the HindIII site of the TK-LUC reporter
which contains the Herpes virus thymidine kinase promoter
(-105/+51). Response elements with the underlined consensus
hexanucleotide sequence were as follows:
2 UAS.sub.G .times. 4 (i.e., 4 copies of the following sequence):
SEQ ID NO:2 5'-CGA CGGAGTACTGTCCTCCGAGCT; IRO = TREp (i.e., 1 &
2 copies of the following sequence): SEQ ID NO:3 5'-TCAGGTCA
TGACCTGAG; DR4 .times. 2 SEQ ID NO:4 5'-A A A G G T C A C G A A A G
G T C A CCATCCCGGGA AAAGGTCACGAAAGGTCACC; DR5 SEQ ID NO:5
5'-CAGGTCA-CCAGGAGGTCAGAG; DR5 .times. 2 SEQ ID NO:6 5'-A A A G G T
C A C C G A A A G G T C A CCATCCCGG GAAAAGGTCACCGAAAGGTCACC; ER8
SEQ ID NO:7 5'-TGACCTTTCTCTCC AGGTCA; NERE .times. 3 (i.e., 3
copies of the following sequence): SEQ ID NO:8 5'-GAGTTTAAAAGGTCA
TGCTCAATTTTC; CRBPIT SEQ ID NO:9 5'-GTCACAGGTCACAGGTCACAGGT-
CACAGTTCA; MLV-DR4 .times. 2 (i.e., 2 copies of the following
sequence): SEQ ID NO:10 5'-AAGGTTCACGAGGTTCACGT.
[0069] All mammalian expression vectors were derived from pCMX
(Umesono et al., 1991, supra) which contains the CMV
promoter/enhancer followed by a bacteriophage T7 promoter for
transcription in vitro. pCMX expression vectors for T.sub.3R.sub.62
, hRAR.alpha. (Umesono et al., 1991, supra) and hRXR.alpha. (Yao et
al., 1993, supra) were used as previously described. CMX-Nurrl
(provided by Thomas Perlmann), an expression vector for full-length
mouse Nurrl, was cloned by inserting the BglII-XhoI fragment from
pBS34-1 (excised from .lambda.ZAP34) (see Law et al., 1992, suPra)
into pCMX. The VP16-RXR fusion contains the 78 amino acid
transactivation domain of Herpes VP16 from pVP16Cl (Novagen) fused
N-terminal to the full-length hRXR.alpha..
[0070] GAL4 fusions were made by fusing the following receptor
ligand binding domains to the C-terminal end of the yeast GAL4 DNA
binding domain (amino acids 1-147) from pSG424 (see Sadowski and
Ptashne, in Nucleic Acids Res. 17:7539 (1989)): human RXR.alpha.
LBD (Glu 203 - Thr 462); mouse Nurrl (Cys 318 - Phe 598); human
T.sub.3R.beta. (Leu 173 - Asp 456); human RAR.alpha. (Glu 156 - Pro
462); and human VDR (Glu 92 - Ser 427). The LBD expression
constructs contain the SV40 TAg nuclear localization signal
(APKKKRKVG; SEQ ID NO:11) fused upstream of the human
T.sub.3R.beta. LBD (Leu 173 - Asp 456), HRAR.alpha. LBD (Glu 156 -
PRO 462) or the human RXR.alpha. LBD (Glu 203 -Thr 462).
CMX-.beta.gal contains the E. coli .beta.-galactosidase coding
sequences derived from pCH110 (Pharmacia) cloned into PCMX.
[0071] In the left panel of FIG. 5, CV-1 cells were transfected
with the following plasmids: IRO TK-LUC (300 ng/10.sup.5 cells),
CMX-.beta.gal (500 ng/10.sup.5 cells) alone (-) or with
CMX-VP16-RXR.alpha. (100 ng/10.sup.5 cells) and/or CMX-hRAR.alpha.
(50 ng/10.sup.5 cells) as indicated. No ligand treatment was
employed. Luciferase activity was normalized to the
.beta.-galactosidase internal control. In each experiment, the
normalized activity obtained in the presence of VP-RXR, T.sub.3R or
RAR is plotted as activity relative to the reporter alone, which
was defined to have a relative activity of 1.
Example 3
Ligand Binding Assays
[0072] Bacterially expressed proteins were used for ligand binding
assays. GST-hRXR.alpha. (see Mangelsdorf et al., in Cell 66:
555-561 (1991)), chicken T.sub.3R.alpha.1 (see Forman et al., in
Mol. Endocrinol. 6:429-442 (1992)) and human RAR.alpha. (Forman et
al., 1992, supra) were expressed and purified to near homogeneity
as previously described. GST-RXR (150 ng) or a GST control (150 ng)
were incubated with or without approximately 500 ng of T.sub.3R or
RAR in the presence of 50 nM [.sup.3H]-ligands (LG69, 56 Ci/mmol;
at-RA, 49 Ci/mmol; 9-cis RA, 29 Ci/mmol) , 3 ng/.mu.l poly dI-dC,
50 fmol/.mu.l of the indicated oligonucleotide, 10 .mu.l of 50%
(v/v) epoxy-linked glutathione-sepharose (Sigma) in ligand binding
buffer (25 mM Tris, pH 7.8, 0.5% CHAPS, 100 mM KCl, 8% Glycerol, 1
mM DTT).
[0073] Where indicated (see, for example, FIG. 6) unlabeled ligands
were added as follows: LG69, 2 .mu.M; Am580, 2 .mu.M; T.sub.3, 1
.mu.M. The reaction was mixed for 30 minutes at 25.degree. C. and
then chilled to 4.degree. C. for 10 minutes. The
glutathione-sepharose beads were washed three times in ligand
binding buffer and the amount of [.sup.3H] bound was determined in
a liquid scintillation counter. Background binding was determined
with the GST control and represented 3-5% of the total binding seen
with GST-RXR.
Example 4
RXR Responsiveness is Diminished in T.sub.3R-RXR
and RAR-RXR Heterodimers
[0074] Since T.sub.3R and RAR function as heterodimers with RXR,
RXR responsiveness was examined in the context of RXR-T.sub.3R and
RXR-RAR heterodimers. Attention is directed to FIG. 2, wherein
transient transfection analysis of T.sup.3R-RXR and RAR-RXR
heterodimers is described. Reporter constructs employed contain the
HRE indicated in the figure, cloned upstream of the TK-LUC
reporter. In the left panel of the figure, CV-1 cells were
transfected with the following plasmids: HRE.times.2 - TK-LUC (300
ng/10.sup.5 cells), CMX-hT.sub.3R.beta. (20 ng/10.sup.5 cells),
CMX-hRXR.alpha. (20 ng/10.sup.5 cells) and the internal control
CMX-.beta.gal (500 ng/10.sup.5 cells) . Cells were treated without
ligand or with 100 nM T.sub.3, 100 nM LG69 or 100 nM T.sub.3+100 nM
LG69.
[0075] In the right panel of FIG. 2, cells were transfected with
HRE.times.1 TK-LUC (300 ng/10.sup.5 cells), CMX-hRAR.alpha. (50
ng/10.sup.5 cells) CMX-hRXR.alpha. (50 ng/10.sup.5 cells) and
CMX-.beta.gal (500 ng/10.sup.5 cells) . Cells were treated without
ligand or with 50 nM Am580, 100 nM LG69 or 50 nM Am580+100 nM LG69.
Normalized luciferase activity was determined and plotted as
fold-activation relative to untreated cells.
[0076] Although cells transfected with both T.sub.3R.beta. and
RXR.alpha. expression vectors were responsive to T.sub.3, they were
surprisingly not responsive to the RXR specific ligand LG69 (see
FIG. 2; Boehm et al., in J. Med. Chem. 37:408-414 (1994) ) .
Treatment of these cells with both T.sub.3 and LG69 did not result
in further stimulation of the T.sub.3 response, rather the response
to T.sub.3 was somewhat reduced. Similarly, cells simultaneously
transfected with RAR.alpha. and RXR.alpha. expression vectors
responded to the RAR-specific ligand Am580, but remained
unresponsive to LG69. In contrast, treatment with Am580+LG69
resulted in increased transcriptional activity over that seen with
AM580 alone.
Example 5
Suppression of RXR Activity is Mediated by the LBD
[0077] Since RXR homodimers are activated RXR agonists, the results
presented above suggest that RXR activity is suppressed in
unliganded RXR-T.sub.3R and RXR-RAR heterodimers. It is suspected
that heterodimerization within the LBD (see FIG. 1) could induce an
allosteric change in the RXR LBD that blocks its ability to bind
ligand and/or transactivate. To test this hypothesis, a system was
developed to examine the responsiveness of RXR-containing
heterodimers in a manner that relies solely on interactions between
the LBDs.
[0078] Thus, a chimeric protein was constructed containing the
yeast GAL4 DBD linked to the RXR LBD (GAL-RXR). The ability of this
RXR-chimera to respond to LG69 was initially examined in the
presence of truncated receptors containing the LBDs of T.sub.3R or
RAR. Thus, transient transfection analysis of GAL-RXR LBD was
carried out in the presence of T.sub.3R, RAR or VDR LBDs. Reporter
constructs contained 4 copies of the UAS.sub.G cloned upstream of
the TK-LUC reporter. CV-1 cells were tranfected with UAS.sub.G
.times.4 TK-LUC (300 ng/10.sup.5 cells), CMX-GAL-RXR (100
ng/10.sup.5 cells), CMX-.beta.gal (500 ng/10.sup.5 cells) alone or
with either CMX-T.sub.3R LBD, CMX-RAR LBD or CMX-VDR LBD (100
ng/10.sup.5 cells). Following transfection, cells were treated
without ligand or with 100 nM LG69, 100 nM T.sub.3, 50 nM Am580 or
100 nM VD.sub.3. Normalized luciferase activity was determined and
plotted as reporter activity (see FIG. 3).
[0079] Although GAL-RXR activated the UAS.sub.G reporter in
response to LG69, the absolute levels of induced and uninduced
activity were dramatically suppressed by both T.sub.3R and RAR LBDs
(see FIG. 3) In contrast, the VDR LBD failed to suppress RXR
responsiveness. These results indicate that suppression of RXR by
unliganded T.sub.3R and RAR is mediated solely by interactions
between the LBDs of these receptors.
[0080] These results are consistent with previous experiments which
have shown that receptor LBDs remain tethered to the GAL-RXR LBD in
cells (see, for example, Nagpal et al., 1993, supra). Thus, it was
next sought to determine whether the tethered LBDs can activate
transcription in response to their specific ligands. As seen in
FIG. 3, the T.sub.3R, RAR and VDR LBDs conferred ligand-dependent
activation upon GAL-RXR, but not GAL4 alone. Thus, receptor LBDs
tethered to RXR provide all the functions required for
ligand-dependent transcriptional activation in the absence of
direct DNA contact.
[0081] The experiment described with respect to FIG. 3 was also
performed with the combination of RXR-specific ligand (e.g., LG69)
and T.sub.3R or RAR specific ligand (see FIG. 4, which illustrates
the differential modulation of RXR transcriptional activity by the
LBD of T.sub.3R. Thus, cells treated according to the procedure
described above with respect to FIG. 3 were additionally treated
with 100 nM T.sub.3+100 nM LG69 (see FIG. 4A) or 50 nM AM580+100 nM
LG69 (see FIG. 4B). Normalized luciferase activity was determined
and plotted as fold-activation relative to untreated cells.
[0082] In order to compare the effects of T.sub.3R and RAR LBDs on
LG69 inducibility of GAL-RXR, these data were re-plotted as
fold-induction. Comparison of FIGS. 2 and 4 indicate that the
effects of ligand-occupied T.sub.3R and RAR are qualitatively
similar, regardless of whether the full-length receptors or their
LBDs are used. Note that the T.sub.3R LBD led to a coordinate
reduction in both basal and LG69-induced activities of GAL-RXR,
hence the fold response to LG69 was only modestly inhibited from
69-fold (see FIG. 4A, GAL-RXR alone) to 57-fold by the T.sub.3R LBD
(FIG. 4B, GAL-RXR+T.sub.3R LBD) . Addition of T.sub.3 resulted in
strong activation of T.sub.3R and the combination of T.sub.3+LG69
resulted in slightly less activity than with T.sub.3 alone. In
contrast to T.sub.3R, unliganded RAR LBD strongly suppressed the
fold-responsiveness of GAL-RXR to LG69. Treatment with Am580+LG69
resulted in increased transcriptional activity over that seen with
AM580 alone suggesting that RXR responsiveness to LG69 may be
restored by addition of the PAR agonist Am580 (FIG. 4B).
Example 6
RAR and T.sub.3R Differentially Suppress
the Ligand Binding Activity of RXR
[0083] In addition to decreasing basal and activated transcription,
RAR also blocks the ability of RXR to respond to its ligand. Thus,
the possibility that RXR is incapable of binding ligand when
tethered to RAR was examined. A bacterially expressed
glutathione-S-transferase-RXRa fusion protein (GST-RXR) was
incubated with recombinant T.sub.3R or RAR in the presence of
radiolabeled RXR ligands. The amount of ligand bound to RXR or
RXR-containing heterodimers was quantitated using
glutathione-sepharose as an affinity probe. In the left panel of
FIG. 6A, purifed GST-hRXR.alpha. was incubated with 50 nM [.sup.3H]
LG69 (56 Ci/mmol) and the optimized PAR reponse element 5'-GCAAA
AGGTCA AAAAG AGGTCA TGC-3'; SEQ ID NO:12; Kurokawa et al., Genes
Dev. 7:1423-1435 (1993)) alone or with 2 .mu.M LG69, 2.mu.M Am580.
In the right panel of FIG. 6A, purified GST-hRXR.alpha. and the RAR
response element were incubated with 25 nM [.sup.3H] at-RA (49
Ci/mmol) without or with 500 ng of hRAR.alpha.. The amount of
specifically bound [.sup.3H] label was then determined employing
standard techniques as previously described.
[0084] As expected, binding of [.sup.3H] LG69 to GST-RXR was
specifically completed by unlabeled LG69, but not by the
RAR-specific ligand Am580 (see FIG. 6A, right panel); specific
binding of [.sup.3H] all-trans RA (at-RA) was observed when GST-RXR
was mixed with excess PAR (see FIG. 6A, right panel) . A
quantitation of the amount of specifically bound [.sup.3H] LG69,
[.sup.3H] at-RA or (.sup.125I] T.sub.3 indicates that GST-RXR could
be saturated with approximately equimolar amounts of RAR or
T.sub.3R, respectively. Electrophoretic mobility shift experiments
indicate that ligands do not alter the binding activity of
T.sub.3R-RXR or RAR-RXR heterodimers.
[0085] Next, the ligand binding activity of RXR was examined in the
presence of RAR-T.sub.3R. Thus, purified GST-hRXR.alpha. and 50 nM
[.sup.3H] LG69 (56 Ci/mmol) were incubated alone or with 500 ng of
hRAR.alpha. or chicken T.sub.3R.alpha.l and the optimized RAR
response element or the optimized T.sub.3R response element
5'-GCAAA AGGTCA AATA AGGTCA CGT-3'; SEQ ID NO:13; Kurokawa et al.,
supra), respectively. Where indicated, unlabeled T.sub.3 was added
to a concentration of 1 .mu.M. Specifically bound [.sup.3H] LG69
was determined.
[0086] Surprisingly, addition of RAR resulted in a dramatic
(<85%) decrease in the amount of [.sup.3H] LG69 bound to GST-RXR
(see FIG. 6B), indicating that the ligand binding potential of RXR
is reduced in the RXR-RAR heterodimer. These findings account for
the ability of unoccupied RAR to suppress the ligand inducibility
of RXR (see FIG. 4B).
[0087] Similar experiments were performed on the RXR-T.sub.3R
heterodimer. In contrast to PAR, unliganded T.sub.3R led to a
modest reduction in [.sup.3H] LG69 binding. However, ligand binding
was strongly diminished upon addition of T.sub.3 (FIG. 6B) . These
findings are consistent with the observation that unoccupied
T.sub.3R results in a modest suppression of RXR inducibility,
whereas no induction is elicited when T.sub.3R is occupied by
T.sub.3 (FIG. 4B)
[0088] The transfection experiments summarized in FIGS. 2 and 4B
indicate that RAR-RXR heterodimers exhibit RXR responsiveness only
in the presence of an RAR ligand, suggesting that RXR binding
activity may be restored by RAR ligands. To test this hypothesis,
the observation that 9-cis RA binds with high affinity to both RAR
and RXR (Allegretto et al., 1993; Allenby et al., 1993) was applied
as follows. Thus, GST-RXR/RAR heterodimers were allowed to form in
the presence of [.sup.3H] 9-cis RA. Reactions were performed as
described above with reference to FIG. 6A, using both
GST-hRXR.alpha. and hRAR.alpha. with 50 nM [.sup.3H] 9-cis RA (29
Ci/mmol) . Specifically bound [.sup.3H] 9-cis RA was determined in
the absence or presence of 2 .mu.M LG69 and/or 2 .mu.M Am580. In
all experiments, maximal binding was in the range of 200-300 fmol
of [.sup.3H] ligand.
[0089] Although Am580 fully competed with [.sup.3H] at-RA for
binding to GST-RXR/RAR heterodimers (FIG. 6A, right panel), Am580
resulted in only a partial decrease in [.sup.3H] 9-cis RA binding
(see FIG. 6C) . Nearly complete competition was observed by
addition of both Am580 and the RXR-specific ligand LG69 (see FIG.
6C), suggesting that RXR can bind ligand, provided the RAR LBD is
occupied. These findings are consistent with the restoration of RXR
responsiveness in RAR-occupied heterodimers (FIG. 4B).
Example 7
Identification of a Novel RXR-permissive Heterodimer
[0090] Since RXR serves as a silent partner in the T.sub.3R and RAR
pathways, it was next investigated whether RXR could serve as an
active component in other complexes. To search for such complexes,
the LBD of a number of nuclear receptors were fused to the GAL4
DBD, and tested to determine whether the RXR LBD could confer LG69
responsiveness upon these GAL-LBD chimeras. Thus, CV-1 cells were
transfected with UAS.sub.g.times.4 TK-LUC (300 ng/10.sup.5 cells),
CMX-.beta.gal (500 ng/10.sup.5 cells) and the indicated
CMX-GAL-receptor LBD construct (100 ng/10.sup.5 cells) with or
without CMX-RXR LBD (100 ng/10.sup.5 cells). Following
transfection, cells were treated without ligand or with 100 nM
LG69. Normalized luciferase activity was determined and plotted as
fold-activation relative to untreated cells.
[0091] As expected, LG69 responsiveness was not seen when the RXR
LBD was expressed alone, or with GAL-T.sub.3R and GAL-RAR (see FIG.
7A). Similarly, LG69 inducibility was not observed with chimeras
containing the LBDs of VDR (see FIG. 7A) or several other members
of the nuclear receptor superfamily. Unexpectedly, strong
responsiveness to LB69 was observed when the RXR-LBD was
co-expressed with a GAL-Nurrl chimera (see FIG. 7A). These results
suggest that the LBDs of Nurrl and RXR form a novel heterodimer
complex which promotes potent RXR responsiveness.
[0092] Nurrl (also known as RNR-1, NOT, HZF-3), the .beta. isoform
of NGFI-b (also known as nur77, N10, NAK-1, TR3), is reported to be
a constitutively active orphan receptor that binds as a
high-affinity monomer to an AA-AGGTCA core-site (NBRE) (see, for
example, Law et al., 1992, supra; Wilson et al., 1992, supra;
Scearce et al., 1993, supra; and Wilson et al., 1993, supra). This
prompted further investigation as to whether full-length Nurrl and
RXR could interact productively on the NBRE.
[0093] Thus, CV-1 cells were tranfected with NBRE.times.3 TK-LUC
(300 ng/10.sup.5 cells) , CMX-.beta.gal (500 ng/10.sup.5 cells),
alone or with CMX-Nurrl (100 ng/10.sup.5 cells) and CMX-hRXR.alpha.
(100 ng/10.sup.5 cells) as indicated in FIG. 7B. Following
transfection, cells were treated with or without 100 nM LG69.
Normalized luciferase activity was determined and plotted as
reporter activity.
[0094] Consistent with published results (see, for example, Scearce
et al., 1993, supra), Nurrl constitutively activates the NBRE
reporter (see FIG. 7B), but was not responsive to LG69 (FIG. 7B).
RXR, which does not bind to the NBRE, did not activate this
reporter. However, when Nurrl and RXR are co-expressed, the
constitutive activity of Nurri is suppressed, and the complex
becomes strongly responsive to LG69 (FIG. 7B) . Similar results
were obtained with RXR.alpha., RXR.beta. and RXR.gamma..
[0095] The ability of the Nurrl-RXR heterodimer complex to
transduce RXR signals suggested the desirability of comparing the
activity of this complex with that of RXR on an established RXR
response element (CRBPII, cellular retinol binding protein II; see
Mangelsdorf et al., 1991, supra). Using sub-optimal amounts of
RXR-expression vector, the CRBPII reporter was compared with a
3-copy NBRE reporter as follows. Cells were transfected as
described with respect to FIG. 7B, but with a 5-fold lower amount
of CMX-hRXR.alpha. (20 ng/10.sup.5 cells). CRBPII TK-LUC (300
ng/10.sup.5 cells) was used where indicated.
[0096] Since RXR was limiting in this assay, only minimal
activation of the CRBPII reporter was observed (see FIG. 7C) . In
contrast, Nurrl-RXR displayed a potent response to LG69, despite
the fact that the NBRE reporter contains 1 less core-binding site
than CRBPII (see FIG. 7C). Thus, Nurrl-RXR can efficiently
transduce RXR signals. However, unlike other heterodimers, the
Nurrl-RXR complex is strongly responsive to LG69 and 9-cis RA,
suggesting that this complex establishes a novel signaling pathway
for 9-cis RA.
Example 8
Nurrl Does not Require the RXR DBD for Coupling
[0097] The Nurrl-RXR complex is unique in several ways. First, the
Nurrl DBD recognizes its response element in the absence of RXR
(see, for example, Wilson et al., 1992, supra; Scearce et al.,
1993, supra; and Wilson et al., 1993, supra) . Second, the
monovalent NBRE serves as a response element for a multimeric
Nurrl-RXR complex (see FIG. 7B). These observations raise the
possibility that RXR associates with NBRE-bound Nurrl in the
absence of RXR-specific DNA contacts. Such behavior would be in
sharp contrast with T.sub.3R, RAR and VDR, which rely on
RXR-specific contacts to recognize hormone response elements.
Indeed, RXR mutants lacking the DBD associate with wild-type RAR;
however, these complexes do not bind DNA or activate transcription
(see Minucci et al., in Mol. Cell Biol. 14:360-372 (1994)).
[0098] This prompted an investigation of the question of whether
the RXR DBD is required for activation through the Nurrl pathway.
Thus, CV-1 cells were transfected with TK-LUC reporters (300
ng/10.sup.5 cells), CMX-.beta.gal (500 ng/10.sup.5 cells) and the
indicated CMX-receptor construct (20 ng/10.sup.5 cells; see FIG.
7D) with or without CMX-RXR-LBD (100 ng/10.sup.5 cells) . The
following receptor, reporter, ligand combinations were used: Nurrl,
NBRE.times.3, 100 nM LG69; hT.sub.3R.beta.), MLV.times.2, 100 nM
T.sub.3; hRAR.alpha., DR5.times.2, 100 nM Am580; hVDR,
SPP1.times.3, 100 nM VD.sub.3. Normalized luciferase activity was
determined and plotted as percent of maximal fold-activation where
100% is defined as the fold activation by T.sub.3R, RAR, VDR, the
RXR LBD, or Nurrl+RXR LBD. The actual fold-activation values are
shown above each bar in the figure.
[0099] As shown in FIG. 7D, the RXR LBD is sufficient to confer
strong LG69 responsiveness upon Nurrl. In contrast, the RXR LBD
acts as a dominant-negative inhibitor of wild-type VDR, T.sub.3R
and RAR (FIG. 7D). These findings indicate that the RXR DBD is not
required for ligand-dependent activation of Nurrl-RXR, a property
that further distinguishes this novel complex from previously
described RXR-containing complexes.
[0100] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
* * * * *