Heterodimer complex of RXR and NURR1 or NGFI-B

Details Heterodimer complex of RXR and NURR1 or NGFI-B Heterodimer complex of RXR and NURR1 or NGFI-B

1997-06-18

2002-10-01

Eyler, Yvonne (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Reexamination Certificate

active

06458926

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to intracellular receptors, and method for the modulation thereof. In a particular aspect, th 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 Nurr1.
BACKGROUND OF THE INVENTION
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 (NFkB, 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.
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
3
R), the vitamin D
3
receptor (VDR) and the fatty acid/peroxisome proliferator activate 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).
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
n
-AGGTCA core-site.
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) an 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
n
(see, for example, Wilson et a ., in
Science
256:107-110 (1992) or Lee et al., in
Science
260:1117-1121 (1993)).
In direct repeats (DR, head-to-tail arrangement), the X
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
3
R 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.
7:1423-143 (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/Nurr1 orphan receptors provide well characterized example of this paradigm (Wilson et al., in
Mol. Cell Biol.
13:5794-5804 (1993)).
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 (&tgr;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).
The transactivation domain, &tgr;2, consists of approximately 20 amino acids with the potential to form an amphipathic &agr;-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 T2 domain displays constitutive transcriptional activity. However, in the natural context of the LBD, transcriptional activity requires the addition of ligand.
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
3
R. This suppression occurs at the level of ligand binding and transcriptional activation. Furthermore, RXR responsiveness has not been observed with other partners, including VDR.
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
In accordance with the present invention, it has been discovered that RXR can interact productively with Nurr1, 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 Nurr1 is suppressed, and the resulting complex becomes responsive to RXR-selective ligands (e.g., 9-cis retinoic acid). The unique ability of the Nurr1-RXR heterodimer complex to transduce RXR signals establishes a novel response pathway.
The result 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.

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