US4981784A
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Retinoic acid receptor method
Assignee: Salk Institute for Biological Studies
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Filing: 1988-11-30
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Granted: 1991-01-01
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Expires: 2008-11-30
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 M. Evans
- 👤 Estelita Ong
- 👤 Prudimar S. Segui
- 👤 Catherine C. Thompson
- 👤 Kazuhiko Umesono
- 👤 Vincent Giguere
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 04:36
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: 13
Claim 1
Independent Claim
177 words
A method for identifying functional ligands for receptor proteins, said method comprising: (A) isolating DNA sequences having a ligand-binding domain and a DNA-binding domain; (b) constructing a chimeric gene by substituting operative portions of the DNA-binding domain region of the DNA sequence of step (a) with operative portions of a DNA-binding domain region from a known ligand-responsive receptor protein; (c) introducing 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 response 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 at least one compound to be evaluated for ligand bidning activity with the chimeric receptor protein encoded by the chimeric gene of step (b); (e) monitoring induction of the reporter gene; (f) identifying as a functional ligand(s) that ligand(s) which is capable of inducing production of the protein product of the reporter gene.
Claim 2
Independent Claim
177 words
A method for identifying functional ligands for receptor proteins, said method comprising: (A) isolating DNA sequences having a ligand-binding domain and a DNA-binding domain; (b) constructing a chimeric gene by substituting operative portions of the DNA-binding domain region of the DNA sequence of step (a) with operative portions of a DNA-binding domain region from a known ligand-responsive receptor protein; (c) introducing 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 response 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 at least one compound to be evaluated for ligand bidning activity with the chimeric receptor protein encoded by the chimeric gene of step (b); (e) monitoring induction of the reporter gene; (f) identifying as a functional ligand(s) that ligand(s) which is capable of inducing production of the protein product of the reporter gene.
Claim 3
Dependent Claim
47 words
A method according to claim 1 (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 4
Dependent Claim
15 words
A method according to claim 1 (c) wherein the host cell is a COS cell.
Claim 5
Dependent Claim
29 words
A method according to claim 1 (c) (2) wherein the reporter gene is selected from the group consisting of a chloramphenicol acetyltransferase (CAT) gene and a firefly luciferase gene.
Claim 6
Dependent Claim
53 words
A method according to claim 1 (c) (2) wherein the hormone response element is selected from the group consisting of wild-type, recombinantly produced 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 D 3 response element.
Claim 7
Dependent Claim
37 words
A method according to claim 5 wherein the glucocorticoid response element is encompassed within the mammary tumor virus long terminal repeat sequence (MTV LTR), and the thyroid response element is encompassed within the growth hormone promoter sequence.
Claim 8
Independent Claim
144 words
A method for identifying functional ligands for receptor proteins in a cell wherein said cell contains, (a) an expressible chimeric DNA sequence (c) comprised of operative portions of a DNA-binding domain of a first receptor sequence linked to 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 said chimeric DNA sequence is expressed and wherein the DNA-binding domain of the chimeric receptor protein thus produced 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 compound to be evaluated for ligand binding activity wherein said compound to be evaluated is not known to be a functional ligand for the chimeric protein encoded by said chimeric DNA sequence (c).
Claim 9
Independent Claim
144 words
A method for identifying functional ligands for receptor proteins in a cell wherein said cell contains, (a) an expressible chimeric DNA sequence (c) comprised of operative portions of a DNA-binding domain of a first receptor sequence linked to 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 said chimeric DNA sequence is expressed and wherein the DNA-binding domain of the chimeric receptor protein thus produced 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 compound to be evaluated for ligand binding activity wherein said compound to be evaluated is not known to be a functional ligand for the chimeric protein encoded by said chimeric DNA sequence (c).
Claim 10
Dependent Claim
12 words
A method of claim 7 wherein said cell is a COS cell.
Claim 11
Dependent Claim
26 words
A method according to claim 7 wherein the reporter gene is selected fromthe group consisting of a chloramphenicol acetyltransferase (CAT) gene and a firefly luciferase gene.
Claim 12
Dependent Claim
51 words
A method according to claim 7 wherein the hormone response element is selected from the group consisting of wild-type, recombinantly produced 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 D 3 response element.
Claim 13
Dependent Claim
37 words
A method according to claim 10 wherein the glucocorticoid response element is encompassed within the mammary tumor virus long terminal repeat sequence (MTV LTR), and the thyroid response element is encompassed within the growth hormone promoter sequence.
References Cited (Backward Citations)
No citation records indexed.
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