Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-11-13
2004-03-30
Pak, Michael (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200
Reexamination Certificate
active
06713270
ABSTRACT:
This invention relates to the field of receptors belonging to the superfamily of nuclear hormone receptors, in particular to steroid receptors. The invention relates to DNA encoding a novel steroid receptor, the preparation of said receptor, the receptor protein, and the uses thereof.
Steroid hormone receptors belong to a superfamily of nuclear hormone receptors involved in ligand-dependent transcriptional control of gene expression. In addition, this superfamily consists of receptors for non-steroid hormones such as vitamine D, thyroid hormones and retinoids (Giguère et al, Nature 330, 624-629, 1987; Evans, R. M., Science 240, 889-15 895,1988). Moreover, a range of nuclear receptor-like sequences have been identified which encode socalled ‘orphan’ receptors: these receptors are structurally related to and therefore classified as nuclear receptors, although no putative ligands have been identified yet (B. W. O'Malley, Endocrinology 125, 1119-1170, 1989; D. J. Mangelsdorf and R. M. Evans, Cell, 83, 841-850, 1995).
The superfamily of nuclear hormone receptors share a modular structure in which six distinct structural and functional domains, A to F, are displayed (Evans, Science 240, 889-895, 1988). A nuclear hormone receptor is characterized by a variabel N-terminal region (domain A/B), followed by a centrally located, highly conserved DNA-binding domain (hereinafter referred to as DBD; domain C), a variable hinge region (domain D), a conserved ligand-binding domain (herein after referred to as LBD; domain E) and a variable C-terminal region (domain F).
The N-terminal region, which is highly variable in size and sequence, is poorly conserved among the different members of the superfamily. This part of the receptor is involved in the modulation of transcription activation (Bocquel et al, Nucl. Acid Res., 17, 2581-2595, 1989; Tora et al, Cell 59, 477-487, 1989).
The DBD consists of approximately 66 to 70 amino acids and is responsible for DNA-binding activity: it targets the receptor to specific DNA sequences called hormone responsive elements (hereinafter referred to as HRE) within the transcription control unit of specific target genes on the chromatin (Martinez and Wahli, In ‘Nuclear Hormone Receptors’, Acad. Press, 125-153, 1991).
The LBD is located in the C-terminal part of the receptor and is primarily responsible for ligand binding activity in this way, the LBD is essential for recognition and binding of the hormone ligand and, in addition possesses a transcription activation function, thereby determining the specificity and selectivity of the hormone response of the receptor. Although moderately conserved in structure, the LBD's are known to vary considerably in homology between the individual members of the nuclear hormone receptor superfamily (Evans, Science 240, 889-895, 1988; P. J. Fuller, FASEB J., 5, 3092-3099, 1991; Mangelsdorf et al, Cell, Vol. 83, 835-839, 1995).
Functions present in the N-terminal region, LBD and DBD operate independently from each other and it has been shown that these domains can be exchanged between nuclear receptors (Green et al, Nature, Vol. 325, 75-78, 1987). This results in chimeric nuclear receptors, such as described for instance in WO-A-8905355.
When a hormone ligand for a nuclear receptor enters the cell by diffusion and is recognized by the LBD, it will bind to the specific receptor protein, thereby initiating an allosteric alteration of the receptor protein. As a result of this alteration the ligand/receptor complex switches to a transcriptionally active state and as such is able to bind through the presence of the DBD with high affinity to the corresponding HRE on the chromatin DNA (Martinez and Wahli, ‘Nuclear Hormone Receptors’, 125-153, Acad. Press, 1991). In this way the ligand/receptor complex modulates expression of the specific target genes. The diversity achieved by this family of receptors results from their ability to respond to different ligands.
The steroid hormone receptors are a distinct class of the nuclear receptor superfamily, characterized in that the ligands are steroid hormones. The receptors for glucocorticoids (GR), mineralcorticoids (MR), progestins (PR), androgens (AR) and estrogens (ER) are classical steroid receptors. Furthermore, the steroid receptors have the unique ability upon activation to bind to palindromic DNA sequences, the so-called HRE's, as homodimers. The GR, MR, PR and AR recognize the same DNA sequence, while the ER recognizes a different DNA sequence. (Beato et al, Cell, Vol. 83, 851-857, 1995). After binding to DNA, the steroid receptor is thought to interact with components of the basal transcriptional machinery and with sequence-specific transcription factors, thus modulating the expression of specific target genes.
Several HRE's have been identified, which are responsive to the hormone/receptor complex. These HRE's are situated in the transcriptional control units of the various target genes such as mammalian growth hormone genes (responsive to glucocorticoid, estrogen, testosterone), mammalian prolactin genes and progesterone receptor genes (responsive to Estrogen), avian ovalbumin genes (responsive to progesterone), mammalian metallothionein gene (responsive to glucocorticoid) and mammalian hepatic &agr;
2&mgr;
-globulin gene (responsive to estrogen, testosterone, glucocorticoid).
The steroid hormone receptors have been known to be involved in embryonic development, adult homeostasis as well as organ physiology. Various diseases and abnormalities have been ascribed to a disturbance in the steroid hormone pathway. Since the steroid receptors exercise their influence as hormone-activated transcriptional modulators, it can be anticipated that mutations and defects in these receptors, as well as overstimulation or blocking of these receptors might be the underlying reason for the altered pattern. A better knowledge of these receptors, their mechanism of action and of it the ligands which bind to said receptor might help to create a better insight in the underlying mechanism of the hormone signal transduction pathway, which eventually will lead to better treatment of the diseases and abnormalities linked to altered hormone/receptor functioning.
For this reason cDNA's of the steroid and several other nuclear receptors of several mammalians, including humans, have been isolated and the corresponding amino acid sequences have been deduced, such as for example the human steroid receptors PR, ER, GR, MR, and AR, the human non-steroid receptors for vitamine D, thyroid hormones, and retinoids such as retinol A and retinoic acid. In addition, cDNA's encoding well over 100 mammalian orphan receptors have been isolated, for which no putative ligands are known yet (Mangelsdorf et al, Cell, Vol.83, 835-839, 1995). However, there is still a great need for the elucidation of other nuclear receptors, in order to unravel the various roles these receptors play in normal physiology and pathology.
The present invention provides for such a novel nuclear receptor. More specific, the present invention provides for novel steroid receptors, having estrogen mediated activity. Said novel steroid receptors are novel estrogen receptors, which are able to bind and be activated by, for example, estradiol, estrone and estriol.
According to the present invention it has been found that a novel estrogen receptor is expressed as an 8 kb transcript in human thymus, spleen, peripheral blood lymphocytes (PBLs), ovary and testis. Furthermore, additional transcripts have been identified. Another transcript of approximately 10 kb was identified in ovary, thymus and spleen. In testis, an additional transcript of 1.3 kb was detected. These transcripts are probably generated by alternative splicing of the gene encoding the novel estrogen receptor according to the invention.
Cloning of the cDNA's encoding the novel estrogen receptors according to the invention revealed that several splicing variants of said receptor can be distinguished. At the protein level, these variants differ only at the C
Dijkema Rein
Mosselman Sietse
Akzo Nobel N.V.
Pak Michael
Rothwell Figg Ernst & Manbeck P.C.
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