The Salk Institute

Other

Stanley M Hollenberg

Ronald Mark Evans

Estelita S Ong

Vincent Giguere

Cary Alan Weinberger

Catherine C Thompson

Jeffrey Arriza

Anthony E Oro

Richard Alan Heyman

Roland M Evans

Klaus Damm

In accordance with the present invention, there are provided expression systems for the production of function glucocorticoid receptor proteins, methods for the recombinant production of glucocorticoid receptor proteins as well as sequences encoding novel members of the steroid/thyroid hormone superfamily of receptors (e.g., glucocorticoid receptor). Invention expression systems comprise host cells containing DNA encoding functional glucocorticoid receptor proteins, wherein the DNA is operably linked to control sequences compatible with host cells, thereby enabling the expression of functional receptor proteins. The invention method is carried out by culturing such recombinant host cells, and recovering the functional glucocorticoid receptor proteins produced thereby. In accordance with the present invention, there are also provided substantially pure DNA's comprised of sequences which encode members of the steroid/thyroid hormone receptor superfamily having the hormone-binding and/or transcription-activating characteristics of a glucocorticoid receptor. The invention also provides various plasmids containing receptor sequences which exemplify the DNA's of the invention. The invention further provides receptor proteins, including modified functional forms thereof, expressed from the DNA's (or mRNA's) of the invention.

Hormone receptor compositions and methods

The present invention provides substantially pure DNA's comprised of sequences which encode proteins having the hormone-binding and/or transcription-activating characteristics of a glucocorticoid receptor, a mineralocorticoid receptor, or a thyroid hormone receptor; various plasmids containing receptor sequences which exemplify these DNA's; receptor proteins, including modified functional forms thereof, expressed from these DNA's (or mRNA's); and a bioassay for determining the functionality of a receptor protein. Use of this bioassay has led to the discovery that a necessary and sufficient condition for activation of transcription of a gene (G), whose transcription is activated by hormones complexed with receptors, is the presence of the hormone and its receptor cell (C) where (G) is located. As a result, two new methods for producing desired proteins in genetically engineered cells were discovered.

Dominant negative chimeras of the steroid/thyroid superfamily of

The present invention provides a recombinant expression system for production of functional thyroid hormone receptor protein(s). The invention also provides a method to produce thyroid hormone receptor protein(s) by culturing the cells of the invention recombinant expression system. Also provided are thyroid hormone receptor protein(s) produced by the invention method. In addition, the present invention provides recombinant DNAs comprised of sequences which encode proteins having the hormone-binding and/or transcription-activating characteristics of a thyroid hormone receptor. The invention also provides various plasmids containing receptor sequences which exemplify the DNAs of the invention. The invention further provides complementary mRNAs, cells transformed with invention DNAs, and nucleic acid probes derived from invention DNAs.

DNA encoding thyroid hormone receptor compositions and methods

Disclosed is an analysis of domains of receptors of the steroid/thyroid hormone superfamily, and particularly of the glucocorticoid receptor, to identify requirements for the trans-acting transcriptional repression activities of the receptors. Based on the analysis, certain novel receptor analogs are provided, as are various novel DNAs, expression vectors, cells and transgenic animals as well as novel methods of using trans-acting transcription-repressing analogs of the receptors in various applications. These applications include gene therapy, screening of cells in culture or transgenic animals for compositions effective to treat various diseases due to inability to properly respond to hormones of the steroid or thyroid hormone group, and screening of cells in culture for ligands that are capable, upon binding to one of the receptors, to activate its trans-acting transcription activating and trans-acting transcription-repressing activities.

Receptor transcription-repression activity compositions and methods

The present invention provides methods for the controlled production of recombinant proteins in cells. Cells employed in the invention method contain a gene encoding the desired recombinant protein, with transcription of the gene maintained under the control of a transcriptional control element which is activated by a ligand/receptor complex. The ligand/receptor complex is formed when a ligand (which is a hormone or/and analog thereof) is complexed with a receptor (which is a hormone receptor or functional analog thereof which has the transcription activating properties of the receptor). Receptor is produced by the expression of non-endogenous DNA which is also present in the cells used for production of recombinant protein.

Controlled expression of recombinant proteins

The present invention provides substantially pure DNA's comprised of sequences which encode proteins having the hormone-binding and/or transcription-activating characteristics of a glucocorticoid receptor, a mineralocorticoid receptor, or a thyroid hormone receptor. The invention also provides various plasmids containing receptor sequences which exemplify the DNA's of the invention. The invention further provides receptor proteins, including modified functional forms thereof, expressed from the DNA's (or mRNA's) of the invention. In addition to the novel receptor DNA, RNA and protein compositions, the present invention involves a bioassay for determining the functionality of a receptor protein. By using our bioassay system we have discovered that a necessary and sufficient condition for activation of transcription of a gene (G), whose transcription is activated by hormones complexed with receptors, is the presence of the hormone and its receptor in the cell (C) where (G) is located. As a result of that discovery we have also invented new methods for producing desired proteins in genetically engineered cells. Two of these methods are methods of the present invention. The first is a method for inducing transcription of a gene whose transcription is activated by hormones complexed with the receptors. The second is a method for engineering a cell and increasing and controlling production of a protein encoded by a gene whose transcription is activated by hormones complexed with receptor proteins.

Disclosed are novel hormone and hormone-like receptors Wherein the trans-activation transcription domain(s) is modified in terms of position and/or copy number or otherwise versus parent receptor, such novel receptors having increased trans-activation transcription activation properties surprisingly superior to the parent receptor. Also disclosed are the recombinant methods and means for preparing such receptors and assays based upon the use of such receptors for screening and identifying putative materials that can affect such receptors and/or for the expression via induced transcription of a reporter or other desired, preferably heterologous gene or DNA product.

Receptors: their identification, characterization, preparation and use

Bioassays are disclosed which are useful for determining whether a compound is a hormone receptor agonist (i.e., is capable of promoting the transcription-activation activities of such receptors) or a hormone receptor antagonist (i.e., is capable of blocking the transcription-activation activities of such receptors). The invention bioassay is conducted by culturing test cells in the presence of at least one compound whose ability to function as a ligand for said receptor protein (or functional engineered or modified forms thereof) is sought to be determined. Alternatively, test cells are cultured in medium containing increasing concentrations of at least one compound whose ability to inhibit the transcription activation activity of hormone receptor agonists is sought to be determined, and a fixed concentration of at least one agonist for the receptor protein. Test cells employed in the practice of the present invention contain non-endogenous DNA which expresses hormone receptor (or functional modified forms thereof) and a DNA sequence encoding a hormone response element operatively linked to a reporter gene. The cultured cells are monitored for evidence of transcription of the reporter gene as a function of the concentration of test compound in the culture medium. The variation in transcription levels of the reporter gene as a function of concentration of test compound indicates the ability of test compound to promote or inhibit activation of transcription.

Bioassay for identifying ligands for steroid hormone receptors

The present invention relates to bioassays for evaluating compounds as potential antagonists for members of the steroid hormone superfamily of receptor protein(s). The invention bioassay comprises culturing test cells expressing steroid receptor protein in the presence of increasing concentrations of at least one compound whose ability to inhibit the action of agonists on members of the steroid hormone superfamily of receptors is sought to be determined, and a fixed concentration of at least one agonist for said steroid hormone receptor protein(s). Test cells employed in the practice of the present invention contain DNA which expresses a member of the steroid hormone superfamily of receptor protein(s) in a form having improved trans-activation transcription activity, relative to wild-type receptor, and a DNA sequence comprising a response element for said hormone receptor protein operatively linked to a reporter gene. Improved trans-activation transcription activity is provided by receptor having a plurality of at least one parental receptor trans-activation domain selected from .tau..sub.1 or .tau..sub.2, located outside the DNA-binding and ligand-binding domains of the receptor protein. The invention assay is completed by monitoring the level of expression of reporter gene product as a function of the concentration of potential antagonist compound in the culture medium, thereby indicating the ability of potential antagonist compound(s) to inhibit activation of members of the steroid hormone superfamily of receptors.

Disclosed are novel hormone and hormone-like receptors wherein the trans-activation transcription domain(s) is modified in terms of position and/or copy number or otherwise versus parent receptor, such novel receptors having increased trans-activation transcription activation properties surprisingly superior to the parent receptor. Also disclosed are the recombinant methods and means for preparing such receptors and assays based upon the use of such receptors for screening and identifying putative materials that can affect such receptors and/or for the expression via induced transcription of a reporter or other desired, preferably heterologous gene or DNA product.

The present invention discloses two hormone receptor-related bioassays. The first bioassay is useful for determining whether a protein suspected of being a hormone receptor has transcription-activating properties of a hormone receptor. The second bioassay is useful for evaluating whether compounds are functional ligands for receptor proteins. According to the first bioassay, cells that contain non-endogenous DNA which expresses a protein suspected of being a hormone receptor and which contain a DNA sequence encoding an operative hormone responsive promoter/enhancer element linked to an operative reporter gene, are cultured, the culturing being conducted in a culture medium containing a known hormone, or an analog thereof. The cultured cells are then monitored for induction of the product of the reporter gene as an indication of functional transcription-activating binding between the hormone or hormone analog and the protein suspected of being a hormone receptor. According to the second bioassay, cells that contain non-endogenous DNA which expresses hormone receptor or a functional engineered or modified form thereof, and which also contain a DNA sequence encoding an operative hormone responsive promoter/enhancer element linked to an operative reporter gene, are cultured, the culturing being conducted in culture medium containing at least one compound whose ability to functionally bind the receptor protein is sought to be determined. The cultured cells are then monitored for induction of the product of the report gene as an indicator of functional binding between the compound and the receptor.

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Seattle, WA, United States of America

Stanley M Hollenberg