Heterodimeric nuclear receptors proteins, genes encoding...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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C435S069100, C435S069400, C435S320100, C536S023100, C536S023510, C530S350000

Reexamination Certificate

active

06635429

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of nuclear receptors. Specifically, the present invention is based in part on the isolation of DNA encoding RX receptors and on the novel observation that two different types of nuclear receptors, retinoic acid receptors (RAR) and thyroid receptors (TR) dimerize with RX receptor (RXR) to form a heterodimer. The heterodimer is capable of binding to retinoic acid response elements (RARE), thyroid receptor response elements (TRE), or RX response elements (RXRE) at physiological conditions. Based on this observation, the present invention provides methods of identifying agents capable of binding the disclosed heterodimers, as well as identifying DNA sequences capable of being bound by the heterodimers. In addition, the present invention describes a method to identify mammalian-specific enzymes involved in RA metabolism, novel heteromeric partners of RXR and co-factors involved in the activation function of retinoic acid receptors.
BACKGROUND OF THE INVENTION
Retinoids are metabolites of vitamin A (retinol) which are thought to be important signaling molecules during vertebrate development and for controlling the differentiation state of several adult tissues (for reviews see Brockes,
Neuron
2:1285-1294 (1989) and Brockes,
Nature
345:766-768 (1990); Sherman,
Retinoids and Cell Differentiation,
Sherman, M. I. (ed). CRC Press (1986); Summerbell et al.,
Trends in Neurosci.
13:142-147 (1990)). Two families of nuclear retinoid receptors have been characterized. Retinoic acid receptors, which include RAR-&agr;, RAR-&bgr; and RAR-&ggr; (for reviews see Ruberte et al.,
Development
111:45-60 (1991b) and Chambon et al.,
Seminars in Dev. Biol.
2:153-159 (1991a)), have a high affinity for all-trans retinoic acid (RA) and belong to the same class of nuclear receptors as thyroid hormone (TRs), vitamin D3 (VDR) and ecdysone (EcR) receptors (see Koelle et al.,
Cell
67:59-77 (1991)). Members of the RXR family, RXR-&agr; (Mangelsdorf et al.,
Nature
345:224-229 (1990) herein incorporated by reference), RXR-&bgr; (Hamada et al.,
Proc. Natl. Acad. Sci. USA
86:8289-8293 (1989) herein incorporated by reference) and RXR-&ggr; respond to much higher concentrations of RA, and the natural ligand for RXRs appears to be a new stereoisomer of RA. RXRs belong to a different class of nuclear receptors which includes the Drosophila ultraspiracle (usp) gene product (Oro et al.,
Nature
347:298-301 (1990)).
Synthetic and natural DNA response elements (REs) have been characterized for TRs (Glass et al.,
Nature
329:738-741 (1987); Umesono et al.,
Cell
65:1255-1266 (1991) and see refs therein), RARs (Vasios et al.,
Proc. Natl. Acad. Sci. USA
86:9099-9103 (1989) and Vasios et al.,
EMBO J.
10:1149-1158 (1991); de Thé et al.,
Nature
343:177-180 (1990); Leroy et al.,
Proc. Natl. Acad. Sci. USA
88:10138-10142 (1991a) and refs therein), and RXRs (Mangelsdorf et al.,
Cell
66:555-561 (1991)). All of these REs consist of the repetition of a core motif, PuG
T
G
TCA (Pu=purine) (or a related sequence), in different configurations with respect to both the orientation (direct or inverse repetition) and the spacing of the two motifs. The recognition of REs by a given receptor appears to be dependent on the actual sequence, orientation and spacing of the repeated motifs. Systematic studies of the influence of the spacing between directly repeated motifs have shown that RARs have a preference for 5 bp spaced motifs (Umesono et al.,
Cell
65:1255-1266 (1991)), whereas TRs and RXRs preferentially recognize motifs separated by 4 bp (Umesono et al.,
Cell
65:1255-1266 (1991)) and 1 bp (Mangelsdorf et al.,
Cell
66:555-561 (1991)), respectively. The presence of repeated motifs in these REs, and the demonstration that the glucocorticoid and oestrogen receptors bind as dimers to palindromic REs made up of similar motifs (Schwabe et al.,
Trends Biochem. Sci.
116:291-296 (1991); Luisi et al.,
Nature
352:497-505 (1991); and references therein) have suggested that RARs, TRs and RXRs also bind as dimers to REs. This possibility has been directly supported by in vitro binding evidence in the case of TRs and RARs (Glass et al.,
Cell
59:697-708 (1989); Glass et al.,
Cell
63:729-738 (1990); Lazar et al.,
Mol. Cell. Biol.
11:5005-5015 (1991); Forman et al.,
Gene
105:9-15 (1991)). However, it has also been reported that the in vitro binding of RAR (Glass et al.,
Cell
63:729-738 (1990)) and TR (Murray et al.,
Mol. Endocrinol.
3:1434-1442 (1989); Burnside et al.,
J. Biol. Chem.
265:2500-2504 (1990)) to REs can be greatly stimulated by the addition of, as yet, uncharacterized factor(s) present in nuclear extracts of a variety of cells. Furthermore, evidence has been presented indicating that these factors may form heterodimers with RAR (Glass et al.,
Cell
63:729-738 (1990)) and TR (Lazar et al.,
Mol. Cell. Biol.
11:5005-5015 (1991); Näär et al.,
Cell
65:1267-1279 (1991)), which bind with greater affinity to REs than the isolated receptors.
In the course of purification of RARs overexpressed in a variety of host-vector systems, these receptors lose the capability to bind the RA response element (RARE) of the RAR-&bgr;2 promoter (&bgr;-RARE) (de Thé et al.,
Nature
343:177-180 (1990); Sucov et al.,
Proc. Natl. Acad. Sci. USA
87:5392-5398 (1990); Mendelsohn et al.,
Development
113:723-734 (1991)), which could be recovered by addition of HeLa cell nuclear extracts, irrespective of the source of over expressed RARs.
Retinoids have been used in the treatment of actinally aged skin (Ellis et al.,
Pharmacol. Skin.
3:249-253 (1989)), various types of dermatoses (Gollnick,
Dermatological
175(1): 182-195 (1987)), disorders of keratinization (Happle et al.,
Dermatological
175(1):107-124 (1987)), rheumatoid arthritis (Brinckerhoff et al., 1985
Retinoids, Differentiation and Disease,
Pitman, London (Ciba Foundation Symposium 113) p. 191-211), basal cell carcinoma (Peck,
Dernatological
175(1):138-144 (1987)), and systemic sclerosis (Maurice et al.,
Pharmacol. Skin.
3:235-239 (1989)). In addition, retinoids have been demonstrated to possess immunostimulating activity (Dennert, 1985
Retinoids, Differentiation and Disease,
Pitman, London (Ciba Foundation Symposium 113) p. 117-131), inhibit epidermal terminal differentiation (Lichti et al., 1985
Retinoids, Differentiation and Disease,
Pitman, London (Ciba Foundation Symposium 113) p. 77-89), modulate carcinogenesis in the urinary bladder (Hicks et al., 1985
Retinoids, Differentiation and Disease,
Pitman, London (Ciba Foundation Symposium 113) p. 168-190), regulate differentiation in embryonal carcinoma cells (Sherman et al., 1985
Retinoids, Differentiation and Disease,
Pitman, London (Ciba Foundation Symposium 113) p. 42-60), regulate differentiation in tracheal epithelial cells (Jetten et al., 1985
Retinoids, Differentiation and Disease,
Pitman, London (Ciba Foundation Symposium 113) p. 61-76), inhibit neoplastic transformation (Bertram et al., 1985
Retinoids, Differentiation and Disease,
Pitman, London (Ciba Foundation Symposium 113) p. 29-41), possess anti-inflammatory activity (Ney et al.,
Dermatological
175(1):93-99 (1987)), modulate melanoma growth (Amos et al.,
Pharmacol. Skin.
3:29-36 (1989)), and may play an important role in cholesterol metabolism (Rottman et al.,
Mol. Cell. Biol.
11:3814-3820 (1991)).
It is unknown what the molecular basis is for the various effects retinoids are able to regulate. One possibility is that the various effects regulated by retinoids are caused by the interactions of the retinoid ligand with a tissue specific RAR receptor. Alternatively, the various receptors may bind to different RE motifs with differing affinities.
Using the observations disclosed in the present invention, it is now possible to examine the interactions of retinoids, or derivatives thereof, with specific RAR/RXR, and TR/RXR heterodimeric combinations. Additionally, each of the heterodimeric combinations can be examined for it's affinity for diff

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