EP0325849A2
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EP
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Retinoic acid receptor composition and method for identifying ligands
Assignee: Salk Institute for Biological Studies
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Filing: 1988-12-02
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Granted: —
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Expires: 2008-12-02
Abstract
Abstract A novel retinoic acid receptor is disclosed. The novel receptor is encoded for by cDNA carried on plasmid phRAR1, which has been deposited with the American Type Culture Collection for patent purposes. Chimeric receptor proteins are also disclosed. The chimera are constructed by exchanging functional domains between the glucocorticoid, the mineralocorticoid, the estrogen-related, the thyroid and the retinoic acid receptors. In addition, a novel method for identifying functional ligands for receptor proteins is disclosed. The method, which takes advantage of the modular structure of the hormone receptors and the idea that the functional domains may be interchangeable, replaces the DNA-binding domain of a putative novel receptor with the DNA-binding domain of a known receptor such as the glucocorticoid receptor. The resulting chimeric construction, when expressed in cells, produces a hybrid receptor whose activation of a ligand-( e.g. , glucocorticoid) inducible promoter is dependent on the presence of the new ligand. The novel method is illustrated in part by showing that the ligand for the new receptor protein is the retinoid, retinoic acid.
Inventors
- 👤 Ronald Mark Evans
- 👤 Vincent Giguere
- 👤 Estelita Sebastian Ong
- 👤 Prudimar Serrano Segui
- 👤 Kazuhiko Umesono
- 👤 Catherine Caroline Thompson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 10:16
CPC Classifications
C; C12; C12N; C12N15/00; C12Q; C12Q1/00; C12Q1/68; C12Q1/6897; C07; C07K; C07K14/00; C07K14/435; C07K14/705; C07K14/70567; C12P; C12P21/00; C12P21/02; C07K2319/00; C07K2319/61; C07K2319/70; C07K2319/71; C07K2319/715; C07K2319/80; C07K2319/81
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
53 words
A substantially pure double-stranded DNA wherein the sense strand comprises a segment having a sequence of triplets coding for the primary sequence of a protein which has ligand-binding and transcription- activating properties characteristic of retinoic acid receptor protein, said double-stranded DNA segment being capable of being expressed into said retinoic acid receptor protein.
Claim 2
Dependent Claim
53 words
A substantially pure double-stranded DNA wherein the sense strand comprises a segment having a sequence of triplets coding for the primary sequence of a protein which has ligand-binding and transcription- activating properties characteristic of retinoic acid receptor protein, said double-stranded DNA segment being capable of being expressed into said retinoic acid receptor protein.
Claim 3
Dependent Claim
42 words
A substantially pure DNA according to Claim 1 wherein the sequence of triplets in said segment codes for the primary sequence of a protein having substantial amino acid homology with the sequence of amino acids show in Figures 1B-1, 1B-2 and 1B-3.
Claim 4
Dependent Claim
20 words
A substantially pure DNA sequence having substantial sequence homology with the DNA sequence shown in Figures 1B-1, 1B-2 and 1B-3.
Claim 5
Dependent Claim
20 words
A substantially pure DNA sequence having substantial sequence homology with the DNA sequence shown in Figures 1B-1, 1B-2 and 1B-3.
Claim 6
Dependent Claim
38 words
A substantially pure DNA according to Claim 1 wherein the sequence of triplets in the segment codes for the primary sequence of a protein which has ligand- binding and transcription-activating properties characteristic of human retinoic acid receptor protein.
Claim 7
Dependent Claim
5 words
The plasmid phRAR1 (ATCC #40392).
Claim 8
Dependent Claim
5 words
The plasmid phRAR1 (ATCC #40392).
Claim 9
Dependent Claim
17 words
A substantially pure DNA sequence having substantial sequence homology with the DNA of plasmid phRAR1 (ATCC #40392).
Claim 10
Dependent Claim
17 words
A substantially pure DNA sequence having substantial sequence homology with the DNA of plasmid phRAR1 (ATCC #40392).
Claim 11
Dependent Claim
62 words
A substantially pure DNA segment which has (a) one triplet corresponding to a translation stop codon, which is in frame with a triplet corresponding to a translation start codon and downstream of said start codon triplet, and (b) as longest open reading frame, a substantially pure segment with a sequence of triplets coding for the primary sequence of retinoic acid receptor protein.
Claim 12
Dependent Claim
62 words
A substantially pure DNA segment which has (a) one triplet corresponding to a translation stop codon, which is in frame with a triplet corresponding to a translation start codon and downstream of said start codon triplet, and (b) as longest open reading frame, a substantially pure segment with a sequence of triplets coding for the primary sequence of retinoic acid receptor protein.
Claim 13
Dependent Claim
9 words
A substantially pure DNA encoding human retinoic acid receptor.
Claim 14
Dependent Claim
9 words
A substantially pure DNA encoding human retinoic acid receptor.
Claim 15
Dependent Claim
19 words
A substantially pure DNA having substantial sequence homology with any of the DNA's claimed in any of Claims 1-8.
Claim 16
Dependent Claim
13 words
A mutant of any of the DNA's claimed in any of Claims 1-8.
Claim 17
Dependent Claim
20 words
A substantially pure mRNA transcribed from any of the substantially pure DNA sequences claimed in any of Claims of 1-8.
Claim 18
Dependent Claim
32 words
A substantially pure protein wherein the amino acid sequence of the protein has at least about 50% amino acid homology with the amino acid sequence shown in Figure 1B-1, 1B-2 and 1B-3.
Claim 19
Dependent Claim
32 words
A substantially pure protein wherein the amino acid sequence of the protein has at least about 50% amino acid homology with the amino acid sequence shown in Figure 1B-1, 1B-2 and 1B-3.
Claim 20
Dependent Claim
19 words
A substantially pure protein encoded by any of the substantially pure DNA sequences claimed in any of Claims 1-8.
Claim 21
Dependent Claim
15 words
Cells transformed by any of the substantially pure DNA's claimed in any of Claims 1-8.
Claim 22
Dependent Claim
153 words
Chimeric receptors having an N-termimus domain, a DNA-binding domain, and a ligand-binding domain wherein the N-terminus domain, the DNA-binding domain and the ligand-binding domain originate from known receptors selected from the generic group of parental receptors consisting of GR, MR, TR, ERR and RR, and wherein the chimera have (1) an N-terminus domain selected from the group of parental receptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃α, hT₃β, hRARα and hRARβ, (2) a DNA-binding domain selected from the group of parental receptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃α, hT₃β, hRARα and hRARβ, and (3) a ligand-binding domain selected from the group of parental receptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃α, hT₃β, hRARα and hRARβ, wherein any one chimeric receptor will have an N-terminus domain, a DNA-binding domain, and a ligand-binding domain that originate from at least two different "parental" sources, thereby never being identical to any wild-type receptor.
Claim 23
Dependent Claim
153 words
Chimeric receptors having an N-termimus domain, a DNA-binding domain, and a ligand-binding domain wherein the N-terminus domain, the DNA-binding domain and the ligand-binding domain originate from known receptors selected from the generic group of parental receptors consisting of GR, MR, TR, ERR and RR, and wherein the chimera have (1) an N-terminus domain selected from the group of parental receptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃α, hT₃β, hRARα and hRARβ, (2) a DNA-binding domain selected from the group of parental receptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃α, hT₃β, hRARα and hRARβ, and (3) a ligand-binding domain selected from the group of parental receptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃α, hT₃β, hRARα and hRARβ, wherein any one chimeric receptor will have an N-terminus domain, a DNA-binding domain, and a ligand-binding domain that originate from at least two different "parental" sources, thereby never being identical to any wild-type receptor.
Claim 24
Dependent Claim
36 words
DNA sequences encoding chimeric receptors selected from the group consisting of GRR, GRG, GGR, RGG, RGR, RRG, TTG, GTT, GTG, GGT, TGG, TGT, TTR, TRT, TRR, RTT, RTR, RRT, GTT, GTG, GGT, TGG, TGT, and TTG.
Claim 25
Dependent Claim
36 words
DNA sequences encoding chimeric receptors selected from the group consisting of GRR, GRG, GGR, RGG, RGR, RRG, TTG, GTT, GTG, GGT, TGG, TGT, TTR, TRT, TRR, RTT, RTR, RRT, GTT, GTG, GGT, TGG, TGT, and TTG.
Claim 26
Dependent Claim
81 words
Chimeric receptor proteins made by expression of a chimeric DNA of the invention or translation of an mRNA transcribed from such a chimeric receptor coding DNA wherein said chimeric receptors 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 ligand-binding activity of the corresponding naturally occurring ligand-binding domain.
Claim 27
Dependent Claim
81 words
Chimeric receptor proteins made by expression of a chimeric DNA of the invention or translation of an mRNA transcribed from such a chimeric receptor coding DNA wherein said chimeric receptors 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 ligand-binding activity of the corresponding naturally occurring ligand-binding domain.
Claim 28
Independent Claim
193 words
A method for identifying functional ligands for receptor proteins, said method comprising: (a) isolating DNA sequences having a putative ligand-binding domain and a putative DNA-binding domain; (b) constructing a chimeric gene by substituting the DNA-binding domain region of the DNA sequence of step (a) with a DNA-binding domain region from a known ligand-responsive receptor protein; (c) transfecting into a suitable receptor-deficient host cell: (1) the chimeric gene from step (b), and (2) a reporter gene functionally linked to an operative hormone responsive element wherein the hormone response element is capable of being activated by the DNA-binding domain region of the receptor protein encoded by the chimeric gene of step (b); (d) challenging the transfected host cell from step (c) with a battery of candidate ligands which can potentially bind with the ligand-binding domain region of the chimeric receptor protein encoded by the chimeric gene of step (b); (e) monitoring induction of the reporter gene by means of changes in the protein levels of the protein coded for by the reporter gene; (f) selecting as a functional ligand(s) that ligand(s) which is capable of inducing production of the protein product of the reporter gene.
Claim 29
Independent Claim
193 words
A method for identifying functional ligands for receptor proteins, said method comprising: (a) isolating DNA sequences having a putative ligand-binding domain and a putative DNA-binding domain; (b) constructing a chimeric gene by substituting the DNA-binding domain region of the DNA sequence of step (a) with a DNA-binding domain region from a known ligand-responsive receptor protein; (c) transfecting into a suitable receptor-deficient host cell: (1) the chimeric gene from step (b), and (2) a reporter gene functionally linked to an operative hormone responsive element wherein the hormone response element is capable of being activated by the DNA-binding domain region of the receptor protein encoded by the chimeric gene of step (b); (d) challenging the transfected host cell from step (c) with a battery of candidate ligands which can potentially bind with the ligand-binding domain region of the chimeric receptor protein encoded by the chimeric gene of step (b); (e) monitoring induction of the reporter gene by means of changes in the protein levels of the protein coded for by the reporter gene; (f) selecting as a functional ligand(s) that ligand(s) which is capable of inducing production of the protein product of the reporter gene.
Claim 30
Dependent Claim
49 words
A method according to Claim 18 (b) wherein the known ligand-responsive receptor protein is selected from the group consisting of glucocorticoid receptor, mineralocorticoid receptor, human thyroid receptors alpha and beta and rat thyroid receptor alpha , estrogen-related receptors hERR1 and hERR2, and retinoic acid receptors alpha and beta .
Claim 31
Dependent Claim
15 words
A method according to Claim 18 (c) wherein the host cell is a COS cell.
Claim 32
Dependent Claim
31 words
A method according to Claim 18 (c) (2) wherein the reporter gene is selected from the group consisting of a chloramphenicol acetyltransferase ( CAT ) gene and a firefly luciferase gene.
Claim 33
Dependent Claim
51 words
A method according to Claim 18 (c) (2) wherein the hormone response element is selected from the group consisting of wild-type, engineered or synthetic (1) glucocorticoid response element, (2) thyroid response element, (3) mineralocorticoid response element, (4) estrogen-related response element, (5) retinoic acid response element, and (6) vitamin D₃ response element.
Claim 34
Dependent Claim
37 words
A method according to Claim 22 wherein the glucocorticoid response element is part of the mammary tumor virus long terminal repeat sequence (MTV LTR), and the thyroid response element is part of the growth hormone promoter sequence.
Claim 35
Dependent Claim
38 words
A method according to Claim 18 wherein the ligand(s) identified in step (f) are corroborated by evaluating the binding properties of the expressed product of the DNA sequence of step (a) and the ligand(s) identified in step (f).
Claim 36
Independent Claim
131 words
A method for identifying functional ligands for receptor proteins in a cell wherein said cell contains, (a) a chimeric DNA sequence (C) comprised of (A) operative portions of a DNA-binding domain of a first receptor sequence linked to (B) operative portions of a ligand-binding domain of a second receptor sequence, and (b) a reporter nucleic acid 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 functionally linked to the reporter sequence, said method comprising challenging the cell with at least one candidate ligand and monitoring induction of the reporter nucleic acid sequence by means of changes in the amount of expression product of the reporter sequence.
Claim 37
Independent Claim
131 words
A method for identifying functional ligands for receptor proteins in a cell wherein said cell contains, (a) a chimeric DNA sequence (C) comprised of (A) operative portions of a DNA-binding domain of a first receptor sequence linked to (B) operative portions of a ligand-binding domain of a second receptor sequence, and (b) a reporter nucleic acid 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 functionally linked to the reporter sequence, said method comprising challenging the cell with at least one candidate ligand and monitoring induction of the reporter nucleic acid sequence by means of changes in the amount of expression product of the reporter sequence.
Claim 38
Dependent Claim
12 words
A method of Claim 25 wherein said cell is a COS cell.
Claim 39
Dependent Claim
29 words
A method according to Claim 25 wherein the reporter gene is selected from the group consisting of a chloramphenicol acetyltransferase ( CAT ) gene and a firefly luciferase gene.
Claim 40
Dependent Claim
49 words
A method according to Claim 25 wherein the hormone response element is selected from the group consisting of wild-type, engineered, or synthetic (1) glucocorticoid response element, (2) thyroid response element, (3) mineralocorticoid response element, (4) estrogen-related response element, (5) retinoic acid response element, and (6) vitamin D₃ response element.
Claim 41
Dependent Claim
37 words
A method according to Claim 28 wherein the glucocorticoid response element is part of the mammary tumor virus long terminal repeat sequence (MTV LTR), and the thyroid response element is part of the growth hormone promoter sequence.
References Cited (Backward Citations)
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