JPH04506073A - Dominant negative member of the steroid/thyroid superfamily of receptors - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] A member of the steroid thyroid superfamily of receptors related to i1 This application is based on U.S. Patent Application No. 07/35G, 696, filed S. 26, 1989. No. 077, filed December 23, 1988. No. 289.561, the entire contents of both applications are incorporated herein by reference. It is used as a reference.

l bean yu vertical The present invention provides trans-repressive analogs of the steroid/thyroid superfamily. Concerning log receptors. In certain aspects, the invention provides transcriptional transactivating lipids. Identification and characterization of proteins that function as lesser receptors; Preparation and use of said proteins, including isolates of novel DNA that feeds said D an expression vector operably containing an NA sequence; and transfected with the vector. related to the host being treated.

In other aspects, the invention is useful for various assays and screening methods. The present invention relates to the use of the transcriptional transactivating repressor as described above.

1 bean! ! l Glucocorticoids, mineral fluticoids, thyroids, estrogen related, and steroids, thyroids and as represented by the retinoid class. various hormones and hormone-related receptors, including retinoid receptors The characterization and preparation of microorganisms have become an important research topic.

For example, glucocorticoid receptors play a variety of roles in homeostasis, growth, and development. It belongs to a large superfamily of ligand-dependent transcription factors that play a major role in is known.

Comparison of complementary DNAs that feed these receptors and their coding Analysis of mutations in the binding sequence revealed that DNA binding, hormone binding, and nuclear localization within the molecule, respectively. A certain functional domain of the rod, which is thought to be responsible for the survival of animals, has been identified. [:vans See the review of this topic in Nagience et al., 240.889 (1988). and.

In the case of glucocorticoid receptors, the so-called DNA-binding domain consists of approximately 66 It spans several amino acids and is highly conserved among various species. moreover, This domain has been found to be essential for activating transcription.

Hollenberg et al., Cel Yu 49. 39 (1987), Miesf eld et al., 5science236.423 (1987), Danielsen et al., Mo1. Endo L 816 (1987), Kular et al., Ce1l 51. 941 (1987), Gronemeyer, Hill, 6. 39 85 (198)) and Watervan et al., Mo1. Endo 2.14 See (198g). This domain contains nine invariant cysteine residues. It was found that The contribution of each cysteine residue to the overall function Although the actual structure formed by these domains is not known, these Stine residue coordinates two zinc ions and forms two domains called finger domains. form a DNA bond, thereby allowing the glucocorticoid receptor to The triple structure that is thought to be responsible for positioning and binding to the DNA site of It has been suggested that it may be possible. Klug et al., Tr.Biochem, Sci 12.464 (1987), Ben5 et al., Ce1l 52.1 ( 1988), and Evans, supra.

A so-called ligand-binding domain is located at the carboxyl terminal side of the DNA-binding region. This domain blocks receptor activity in the absence of hormones. demonstrate the ability to

Therefore, the presence of the necessary hormone removes the inhibition on receptor activity.

Deleting this region produces a hormone-independent transcriptional activator. was found. G.

dowski et al., Nature 325.365 <1987), Hol Lenberg et al., Kumar et al., supra, Danielsen et al., supra. See also Adler et al., Ce11 52.685 (1988), supra.

In contrast to these two domains, the amino-terminal one from the DNA-binding domain The structure and especially the function of the sequence is poorly understood. This area contains various levels. Scepters are highly variable both in size and composition (Ey ans et al.) and contribute to the heterogeneity of receptor function. Said Ku See Mar et al., and Tora et al., supra, 333.185 (1988).

Despite several, further analyzes reported in the scientific literature, transcription initiation The region that determines transactivation of the protein remains largely uncharacterized. Tran When bound to a functional DNA-binding domain, the activation domain activates the RNA polypeptide. Defined as a region of a polypeptide that increases productive transcription initiation by merase can be done. 51g1er, 'Nature 333.210 (1988), B rent et al., Ce11 43. 729 (1985), Hope et al., 9th stop ,.

885, (1986), Ma et al., Ce1l 48. 847 <1987), Ma et al., II, 1L3 (1987), Lech et al., Ce11 52. 17 9 (1988), and Hope et al., Nature 333.635 (19 See 88).

Previous studies of human glucocorticoid receptors by linker-scanning mutations In this study, two regions other than the DNA-binding region that play a role in transcriptional activation were identified. It was. This region was defined as τ and τ2. Giguere et al., Ce1l See J6.645 (1986). Further research from these laboratories will lead to further research. It was reported that the transactivation function and the DNA binding function are located together. Before Hollenberg et al., Miesfeld et al., Danielsen et al. See, et al., and Waterman et al., supra. As a result, from this study, each It contributes to the confirmed properties of ligand binding, DNA binding and transactivation of transcription. Increasingly, the molecular molecules of different domains that give rise to It rose. Until recently, the regions determining transactivation activity were completely (understood) I wasn't there. Therefore, based on the extant literature (the picture appears to be general recognition of functional properties, and initiation upon ligand binding and promoter specificity. How do the different domains carry out the cascade of events completed by differential transactivation? In addition, previous research has not revealed how the functionality is determined. Despite the identification of ``domains'', there are no domains that contribute to the specific activity of the molecule itself. There has been no systematic effort to identify amino acids. Therefore, steroid receptors Previous identification of the transactivation region of the Only as a result of the loss of sex, the nature of the domain itself reflects a dominant increase in function. In no case was this evidenced in the assay. Ptas hne, Nature 335. 683 <1988).

Therefore, Godowski et al., 5science 24 units, 812 (198g) In this case, the glucocorticoid receptor has at least one "promoting domain". This domain contains the glucocorticoid response element binding region (i.e. that is, the second domain does not overlap with the receptor (DNA-binding domain). It has been reported that the region near the amino terminus of the protein is pre-stale. Similarly, W Ebster et al., Ce1l 54. 199 (L988) contains estrogen and the inducible transcriptional activation function of glucocorticoid receptors have been reported. Therefore, they contain hormonal regions (i.e., ligand and DNA binding domains). We speculate that the relative position of is not important for the transcription-inducing activity of the receptor.

Still, they do not know the exact location and location of the so-called hormone's inducible activation domain. lack of definition of transactivation properties and characterization and transactivation capacity. He admits that there is no defined role.

In the study by Giguere et al., random site mutagenesis was performed at several locations in the molecule. As a result, glucocorticoid receptors based on assays that measure transcriptional activity A loss of receptor activity was demonstrated. Subsequently, Hollenberg et al. region in the child and again induced by removal of such amino acids. showed a loss of global transcriptional activity.

The human glucofluticoid receptor (hGR) is a prototypical model model for gene regulation. It has been used as a scepter. DNA binding and ligand binding, as described above. Functional domains have been previously defined. Similarly, hGR receptor or other Such modular domains of receptors can be moved to other positions of the receptor. or can be bound to a heterologous DNA-binding domain and functions can be retained.

In contrast, negative regulation by hGR is largely unknown. This means that How steroids play an important role in development and negative feedback regulation That being said, this is surprising. Glucocorticoids are partially neurospecifically inherited By inhibiting the induction of neural crest cells in the developing sympathoadrenal system, [5tein et al., Dev Bi.

127, 316 <1988) and Anderson et al., Ce1l 47. I watched 1079 (1986). Glucocorticoids are also a second drug. By inhibiting Senger-induced peptide hormone induction, hypothalamus-pituitary - Regulates the adrenal system. Recently, Akerblom et al. 88)) shows that hGR regulates the cycle in a steroid- and DNA binding-dependent manner. RickAMP - Negatively regulates the inducible alpha-glycoprotein hormone promoter It was shown that Wild-type expression is expressed at exactly 168 base pairs (referred to as α168). ) indicated by the blow motor. Basic expression in placental cells is 25 base pairs A 36-base protein that binds to the tissue-specific element (TsE) of coupled to a paired palindromic cyclic AMP response element (CRE) mediated by factors. Expression is further influenced by intracellular cyclic AMP levels. Elevation can be facilitated through CRE. hGR is glucofluticoid dependent suppresses both basic and cyclic AMP-promoted transcription in a natural manner. Ru. The trans-acting element to which hGR binds has been identified, and this Related to the consensus GRE sequence for activation. A similar study was conducted by 5akai To, Genes and Devel 肚■i, 1144 (19g+1) Therefore, it has been reported.

λmameyu! 1 The present invention provides results from analysis of the structure required of hormone receptors for inhibition. It is a fruit. This analysis consists of 1. Absolutely necessary and inhibitory for the DNA binding domain. revealed the role of the carboxyl terminus in regulating DNA-binding domain alone , addition of the polypeptide to the carboxyl terminus, although not sufficient for maximal inhibition, or by other modifications to the carboxyl terminus as described herein. A novel fusion protein with dominant negative inhibitory activity is produced.

In accordance with the present invention, we have demonstrated that transactivating transcriptional activity can be modified to suppress transactivating transcriptional activity. identify and isolate domains of intracellular hormones or hormone-like receptors that , and characterized. With this information, steroid/thyroid superfluous Further characterization of the various receptors of the family has become possible. This characteristic Injuries are characterized by physical properties, biological functions, and effects in various domains, especially those that are inhibited. It refers to all domains that can be modified to provide transcriptional activity. this child In turn, novel analogue receptors with suppressed transcriptional activation properties were developed. production of tars became possible.

Based on the information provided herein, steroid/thyroid super The family of receptors generally has domains that function in transactivating transcriptional activity. These three domains, namely the DNA-binding domain, the ligand The transactivation transcription domain and transactivation transcription domain are each located separately. However, it was found that the function was autonomous. According to the present invention, the obtained receipt The carboxyl terminus of the receptor regulates the transactivating transcriptional activity of this receptor. It was discovered that this domain plays a role in Furthermore, D N A bonded The main is required for any receptor with repressed transactivating transcriptional activity. It was found that this is an important element.

The present invention provides a novel hormone or protein that suppresses transactivation transcriptional activity. Providing Lumon-like analog receptors ◎Such new analog receptors contains the DNA-binding domain, optionally the N-terminal domain, and the parental receptor. – or exhibit suppressed transactivating transcriptional activity compared to the wild-type receptor. It contains a C-terminal domain modified to provide This new analog reception Targets have different origins in their DNA-binding domain, N-terminal domain, and C-terminal domain. It may be a hybrid ndresbuter provided by a source. For example, glucocol The C-terminal domain of the ticoid receptor is here defined as the c-terminal domain of the v-erbA protein. Part of the C-terminus may be substituted. Alternatively, the C-terminus of glucocorticoid receptor The end domain is replaced with at least a portion of a polypeptide such as β-galactosidase can be done.

The present invention further provides for the development of such novel methods by recombinant DNA technology in all relevant aspects. We are aiming to prepare a unique analog receptor. For this, this analog receptor - or a recombinant DNA consisting of a modified C-terminal domain of this receptor. A DNA molecule, be it a molecule or a cDNA molecule, a recombinant host selected for expression. An essential element that operably maintains DNA with operable expression control elements in the main body. expression vector, and transfected with such an operable expression vector. included recombinant host cells.

The present invention further utilizes the novel analog receptors described herein. to identify response elements and/or functions of “orphan” receptors. We aim to This orphan receptor has associated response elements and and/or receptors whose functions are unknown.

The present invention further provides an improved 7-nose assay for determining specific wild-type receptors. The use of the novel analog receptors described herein in want to be. The presence of one or more response elements in this assay system type receptors.

Another use of such inhibited analog receptors is in the area of cancer therapy. exist. In certain types of cells, hormone levels must be increased to become neoplastic. There is a need. One example is increased estrogen demand, as seen in breast cancer. . In fact, estrogen antagonists such as spider cow sifuin can help normal breast cells Treatments that reduce the amount of estrogen so that its transformation into cancer cells is inhibited used for. Alternatively, the analog receptors of the present invention may be used for such purposes. can be used.

[i-koi! ! Hoshiseiri FIG. 1 is the amino acid sequence of the v-erbA protein.

Figure 2 shows the dose response curve of hGR-mediated negative regulation.

Figure 3 shows inhibition by carboxyl-terminal fusion proteins.

Figure 4 shows several expression and reporter constructs.

Figure 5 shows the structure and activity of rat TRα/v-erbA chimeric protein. vinegar.

Figure 6 shows thyroid receptors, v-erbAs and hybrid Tr/v - Shows a comparison of T3-induced CAT activity by erbA receptor.

Figure 7 shows the relationship between TRα or V-erbA and RA-induced transcriptional activity by RARa. Indicates conflict.

Figure 8 is a summary of the structures and activities of several RAR hybrids and mutants. Ru.

FIG. 9 shows competition for RARa-verbA fusion protein protein A induction.

Figure 10. 1 in F9 tetracarcinoma stem cells. Figure 3 shows competition for RA-induced transactivation of RAR.

Figure 11 is a summary of the structures and activities of several GR hybrids and mutants. Ru.

1 Saraku”” IL Tigo In the present invention, transcription of steroid/thyroid superfamily receptors is demonstrated. A gas-inhibiting analogue receptor is provided, which analogue includes: (1) A first amino acid sequence that is a DNA binding domain, the analog comprising: A sequence capable of binding to the hormone response element of the wild-type receptor through this sequence. column: and (2) a second amino acid located at the carboxyl terminus of the DNA-binding domain. a amino acid sequence, the second sequence comprising: (a) a carboxylic acid sequence of the wild-type receptor; has at least about 90% of the number of amino acids in the ligand-binding domain of the terminal portion of the of the wild-type receptor, the polypeptide comprising: (i) a copy of the wild-type receptor; the full length of the polypeptide, if shorter than the loboxyl-terminal domain; or ( i) having the same length as the carboxyl terminal domain of the wild type receptor; any of the polypeptide segments; any of the polypeptide segments; have less than about 60% amino acid identity with respect to the syl-terminal domain: yes Yes, (b) A member of the V-erbA protein defined by amino acid numbers 313-398. At least 84 carboxyl-terminal amino acids of the carboxyl-terminal portion (see Figure 1). Reference: The numbering of erb-A amino acids is also based on the erb-A sequence (the amino terminal 255 amino acids). amino acids) are included in the amino/acid numbering of the erb-A protein. (note that the sequence changes by approximately 255 amino acids); , is included.

In another embodiment of the invention, a recombinant DNA encoding a receptor analog as described above Expression that maintains a DNA molecule, which is an A molecule or a cDNA molecule, in an operable manner Vector: a recombinant host cell transfected with such an expression vector; and cell cultures containing such cells in an external maintenance medium are provided.

In another embodiment of the invention, steroid or thyroid hormone Non-human transgenic animals with disease symptoms that cannot respond normally to A mammal is provided.

The mammal has a small number of cells capable of expressing one analog receptor for the hormone. both have one subset, and the analog receptors have a subset of the corresponding wild-type receptors. has greater transrepression activity than the corresponding wild-type receptor - has less transactivation activity than -.

In another embodiment of the invention, the wild-type hormone receptor is trans-repressed. A method is provided for converting to an analog receptor. The method includes The ligand-binding domain of the type receptor is expressed by amino acid numbers 313-398 (Figure 1 at least 8 at the carboxyl terminus of the verbA protein defined in A step of substituting four amino acids is included.

Alternatively, the latter embodiment of the invention provides for the ligand-binding domain of the wild-type receptor to be in the carboxyl-terminal ligand-binding domain of the wild-type receptor. It can be completed by substitution with a polypeptide having at least 90% amino acids. here and the polypeptide has the following ( Less than about 60% amino acid identity across either i) or (ii) (1) If it is shorter than the wild-type receptor, the entire length of the polypeptide is or (ii) has the same length as the carboxyl terminus of the wild-type receptor. Any segment of the polypeptide.

Furthermore, embodiments of the invention provide an effective amount of an analog receptor as described above. By contacting cells with A method of blocking transcriptional activation of a response element is provided.

Furthermore, embodiments of the present invention provide that the field of hormone response elements present in cells is Methods of blocking transcriptional activation by native receptors are provided. to the method includes the following steps (a), (b) and (C): (a) wild type receptor (b) amino acid number 3; 13-398 (see Figure 1) of the verbA protein defined by The modified receptor of step (a) also contains 84 carboxyl-terminal amino acids. to produce a fusion protein; and thereafter (c ) contacting the cell with an effective amount of the fusion protein.

Alternatively, the latter embodiments of the invention include (ao), (bo) and (C) below. can be completed by the steps of: (ao) the ligand-binding domain of the wild-type receptor; a step of substantially deleting the bone; (bo) a small number of amino acids in the ligand-binding domain of the wild-type receptor ( The modified receptor of step (a') is added to the polypeptide having about 90% of both operably linking the protein to produce a fusion protein, wherein the The polypeptide is (i) if it is shorter than the ligand-binding domain of the wild-type receptor, the polypeptide or (ii) the ligand-binding domain of the wild-type receptor. wild-type receptor across any segment of the polypeptide that has the same length. have less than about 60% amino acid identity to the ligand binding domain of the then (c) contacting the cell with an effective amount of the fusion protein; process.

In other embodiments of the invention, for unknown related response elements and/or functions, Provided are methods for identifying response elements and/or functions of receptors that Ru. The method involves determining the response of a test system with known responsiveness to wild-type receptors. and the response of the test system when treated with trans-inhibitory analog receptors. It includes a step of comparing. Here, the trans repression-analog receptor is ( 1) The first amino acid sequence that is the DNA binding domain, through which so that the analog can bind to the hormone response element of the wild-type receptor. and (2) a second column located at the carboxyl terminus of the DNA binding domain. Contains an amino acid sequence; Here, the second arrangement is the following (a) or (b) selected from: (a) the carboxyl-terminal ligand-binding domain of the wild-type receptor; A polypeptide having at least about 90% of the number of main amino acids, The polypeptide has the following structure relative to the carboxyl-terminal domain of the wild-type receptor: have less than about 60% amino acid identity across either: the full length of the polypeptide if shorter than the carboxyl-terminal domain of the receptor; Alternatively, having the same length as the carboxyl-terminal domain of the wild-type receptor, Any segment of the polypeptide; (b) Amino acid numbers 313-398 (Fig. 1 of the carboxyl-terminal portion of the v-erbA protein (defined as collagen). At least 84 carboxyl-terminal amino acids.

For example, the specific response elements that an orphan receptor interacts with are Screening various test systems each with one known response element It can be determined by activated by orphan receptors, but the book Characteristics inhibited by analog receptors derived from orphan receptors of the invention Different test response elements are orphan receptor response elements. be.

Similarly, the function of an orphan receptor is determined by contacting the orphan receptor. The response of the test system when Determined by comparing the response of the same test system when exposed to the log. It can be done. When comparing these two side by side, the difference in response is due to the orphan receptor. provide an indication of the functional role of the

In other embodiments of the invention, assays responsive to the presence of specific wild-type receptors Improvements are provided for use in the system.

wherein more than one response element is capable of interacting with the wild-type receptor; The improvement includes using a wild-type receptor in addition to the specific receptor for this assay. including the step of inactivating a response element that is also responsive to -. Here, the corresponding The response element provides the assay system with an effective amount of target for each receptor. It is inactivated by adding a lance-inhibiting analog receptor. here Each trans-inhibitory analog receptor includes (1) and (2) below. Having: (1) A first amino acid sequence that is a DNA binding domain, which sequence, the analogue can be used to bind receptors other than the specific wild-type receptor. a sequence capable of binding to a hormone response element; and (2) A second amino acid sequence located at the carboxyl terminus of the DNA-binding domain. A sequence selected from the following (a) or (b).

(a) Ligation of the carboxyl-terminal portion of a receptor other than the specific wild-type receptor; having at least about 90% of the number of amino acids in the Gund-binding domain; a polypeptide that binds to a receptor other than the specific, wild-type receptor; less than about 60% of the following for the carboxyl-terminal domain of having amino acid identity: a variant of the receptor other than the specific wild-type receptor; the full length of the polypeptide if it is shorter than the loboxyl-terminal domain; or Same length as the carboxyl-terminal domain of a receptor other than the specific wild-type receptor Any segment of the polypeptide having: (b) v-erbA defined by amino acid numbers 313-398 (see Figure 1) At least 84 carboxyl-terminal abutments of the carboxyl-terminal portion of the protein Mino acids.

As used herein, as used in reference to analog receptors of the invention The term “dominant negative” refers to the presence of the wild-type receptor and its associated ligand. Even if there is a type that has a negative effect on the transcriptional activation activity of the related g: kettle element. It is about.

Amino acid abbreviations used in this disclosure are standard one-letter and three-letter designations. is used. In other words, input sp D a person lgu 1000 degrees Thr T%v7>Lau L Roy Logon HlsHl:/enter) 7Gly G Gu ° Soji 7 LYS X: Son λ1a containing alanine containing rqR full ↑° giant Mat M 7-te;rsn Enter sn N Asharagun (shichi lower b) Receptors used in the practice of this invention can be prepared by recombinant technology, synthetic chemistry, etc. can be manufactured. In this way, receptors produced in various types can be recovered and purified to a level suitable for its intended use.

The existence of a superfamily of ligand-inducible trans-acting factors is now emerging. It is recognized. This includes steroid hormone receptors, retinoic acid and and vitamin D3 receptor, two subtypes (isomers) of thyroid hormones. These include receptors (called α and β).

Through mutational analysis and structural comparison of these hormone receptors, hormone binding is possible. identification of domains responsible for DNA binding and transactivation of gene expression. I became Noh. Sap et al., Nature 324. 635 (1986), We Inberger et al., Nature 324. 641 (1986> and Ev See ans, 5science 240.889 (198g).

The receptors of the invention are transrepressors of hormones and hormone-like receptors. is an analogue of the steroid/thyroid superfamily of receptors. members, e.g. glucocorticoid receptors, mineralocorticoid receptors ter, progesterone receptor, estrogen receptor, estrogen-related receptor, vitamin D3 receptor, thyroid hormone receptor, retinotopic receptor Ino acid receptor, aldosterone receptor, androgen receptor, etc. Show widely. The receptors of the invention may have one or more receptors for the corresponding parent type. Differences in the above amino acids, or differences in the mode of glycosylation and/or phosphorylation, or functions with the above, including receptors with limited conformational differences. Includes equivalent items. The term "their functional equivalents" means one or differs from previously described analogue receptors with respect to more amino acids , a trans-inhibitory analog receptor. The difference here is the basic suppression does not lead to destruction of receptor activity or biological functionality.

Thus, receptors known in the art may be wild-type, hybrid, or Any functional equivalent as described herein may be considered as the starting material of the present invention. It's suitable.

As used herein, the term "expression vector" refers to a vector that expresses a DNA sequence contained in the vector. Includes vectors that can be used. Here, such a sequence shows an effect on its expression. operably linked to other sequences to obtain. It's not always obvious However, these expression vectors can be used in the host organism as ebisomes or by staining. It can be replicated as part of the body's DNA. As used herein, the term "operational" ', or its grammatical equivalent, means that each DNA sequence is in a working state. that is, that they work for their intended purpose. in short , an “expression vector” has a functional definition and the characteristics placed therein. Any DNA sequence that can affect the expression of a given DNA sequence is included in this term; Applies to a specific array. Expression vectors commonly used in recombinant DNA technology is in the form of a "plasmid" which, in its vector form, Refers to a circular double-stranded DNA loop that is not attached to a chromosome. In this specification, The terms "plasmid" and "vector" mean that plasmid is generally a form of vector. can be used interchangeably. However, the present invention has no equivalent functionality. expression vectors, and then other expression vectors known in the art. It is intended to include the form as well.

As used herein, the term "recombinant host cell" refers to a cell that has been constructed using recombinant DNA technology. A cell that has been transfected with a vector.

As used herein, the term "external maintenance medium" includes any known or devised medium. It is possible to maintain cells in the proliferative phase or perform functions that are useful for recombination. It also includes a medium that can be maintained in a viable state. For example, ATCCMedi a Randbook, Ed, Cote et al., American Type Cu 1ture Co11ection, R.

See ckville, MD (1984). For example, growth fibers for mammalian cells. The maintenance medium preferably contains a serum supplement, such as fetal bovine serum, or animal meat. or milk hydrolysates, tissue or organ extracts, dissociated blood sacs or Supplemental formulations commonly used to promote cell proliferation and differentiation, such as extracts, etc. minutes are included. For example, suitable media components for ponds include transferrin, insulin, Includes phosphorus and various metals.

The vectors and methods described herein are applicable to a wide range of prokaryotes and eukaryotes. Suitable for use in biological host cells.

In addition to the foregoing discussion and various citations to existing article techniques, the present invention incorporates Standards of molecular biology, including defined definitions and methods and means of performing basic techniques. Quasi-textbooks are cited. For example, Maniatis et al. 2 units 1 unit orator Manual, Col1d Spring) Iarbo r Laboratory, New York, 1982 and here. Various references have been made to Colowick et al., Methods in E. nz molo Vol 152. Academic Press, Inc. (1987). All publications mentioned herein are particularly It is incorporated in the detailed description.

Non-human transgenic organisms contemplated by the present invention include biological documents (e.g. , mouse, rat), pig, sheep, lower eukaryotes (e.g. Drosophila melanogaster) , Xenopus), etc.

Transcriptional activation by steroid receptors and thyroid hormone receptors is , was found to be dependent on the presence and binding of each ligand. but , deletion analysis shows that glucocorticoids and estrogens lacking the hormone binding domain receptors for progesterone and progesterone recognize specific response elements. , was disclosed to be able to function as an essential active factor. Therefore, the ligand that Either itself or its binding domain must be directly involved in DNA recognition. There isn't.

The v-erbA protein appears to contain an apparently complete NA-binding domain. It was brought out. However, the carboxyl-terminal domain (the thyroid hormone of its origin) As a result of amino acid changes and deletions in the thyroid hormone receptor lacks the ability to bind to Analog receptors for mutant thyroid hormones -, these mutations are associated with high expression of ``C/l/'', resulting in thyroid Change the hormone receptor to v-erbA, a hormone-independent transcription factor. proposed to replace it.

Using an electrophoretic retardation experiment (get retardation assay), Both V-erbA and thyroid hormone receptors share cognate response elements. It was confirmed that it recognized and bonded to the target. As a result of this binding, the ligand Reporter of thyroid hormone responsiveness by unbound receptor The gene was suppressed. By adding thyroid hormone, the level of A 10-100-fold transcriptional enhancement was obtained. Surprisingly, v-erbA It does not function as an activator to the extent that Functions as a repressor. Expressed with thyroid hormone receptor , and in addition, in the presence of thyroid hormone, v-erbA suppression is dominant and block hormone-stimulated regulation, thereby causing V-erbA to It has been shown that it can function as a potent receptor antagonist.

Therefore, the present invention provides a method for binding promoters or externally operable promoters. Hormones or hormones that have the ability to suppress the transactivating transcriptional activity of including receptors that are similar to Such repression can be achieved, for example, by Due to the inherent ability of analog receptors to competitively bind to response elements or this or a similar DNA response element. The ability of analog receptors to competitively position other polypeptides to bind to Ru. Therefore, there is an overall increase compared to suppression of the corresponding parental or wild-type receptor. The added transactivation transcription is suppressed.

(Margin below) K Watago The following experiments include the identification, characterization, and investigation of specific novel analog receptors. The method used for manufacturing is specified.

The organization and location of the trans-acting transcriptional repression domain of a given receptor; By manipulating receptors such as By providing information of the invention, including methods by which Greceptor may be produced, There is no need to repeat what is described herein to reproduce this work; Those skilled in the art will appreciate that it is probably scientifically expedient not to repeat the recognizes. Instead, they choose alternative, reliable and well-known methods It is possible. For example, they can be used in similar or pond-appropriate expression vectors or Certain novel receptors described herein can be co-coated for use in cell and culture systems. Basic NA sequences can be synthesized that code. That is, according to the disclosure herein, , in addition to providing the content actually disclosed, may benefit from the disclosure herein. The specific receptors disclosed, as well as other receptors, can be determined using the means available in the art. This allows for the production of proteins, as well as fragments thereof. All of these methods are available in books. within the scope and possibilities of the invention.

X Example 1:) Transfection 1 J EG-3 human placental cell transfectin was described by Dalegeane et al. Mo1. Ce11. Biol, troll, 7. pp, 3994-400 2 (19J17) by the calcium phosphate precipitation method. Dalbe Modified Eagle's medium (DMEM), 1 O% adjusted bovine serum (CB) S), and JE G-3mf & maintained at 0.4% glucose were transduced. 5% activated charcoal-treated blood/l1lCBS + glucose (Akerblom et al., 5science 241.1) l), 3 50-353 (1988)). Typically 2 μg of reporter The plasmid and 4 μg of receptor plasmid were transferred to transfer silane efficiency. Rous Virus Virus (RSV)-β-galactocida as an internal control. construct (Hollenberg et al., Ce1l 49.pp, 39-46 (19g?)) was used together with 2μg. After calcium phosphate treatment, Dexa Metacin and aldosterone (to-7M) were added.

For β-galact7dase fusion experiments, RSV-luciferase was used as an internal control. It was used as a tool.

Transfection of CV-1 cells was also performed by calcium phosphate precipitation method. Ru. CV- cells were maintained in DMEM supplemented with 5% calf serum and 30% to A total of 20 μg DNA is transfected at 50% ninfluency.

5 μg expression plasmid and 2.5 μg report per 10 cm dish. plasmid DNA, and internal control for transfection efficiency. 12.5 μg of RSV-β-ga is co-transfected. 10” M3. 5. 10 with or without 3' triiodothyronine (T3) % of resin-treated fetal calf serum [5 Huels et al. Vol, 105. pp, 80-85 (1979), transfection Proliferate the cells. After 40 hours of addition of T3, cells were harvested and then , β-galactosidase and CAT assay were performed using the method described in Example 2. It will be done.

Example 2: Revota Anosei Chloramphenicol acetyltransferase (CAT) assay was performed at 3 p.m. Holle nberg et al., Ce11. Vol, 49゜pp, 39-46 (198? ). Thin layer chromatography (TLC) plate and Econo fl containing 5% dimethyl sulfoxide (DMSO). Count in uor.

β-galactosidase (β-gal) a.7sei was described by Herbomel et al., C. e11. Vol, 29. Those described in pp. 653-662 (1984) be done in accordance with the law.

Example 1 hGR-mediated negative regulation alpha 168 promoter (chorionic gonadotropin repressed by GR hG for the promoter of the alpha subunit gene encoding To explain the R-mediated repression, hGR expression proteins in human placental J EG-3 cells are shown. Regarding the negative regulation of the alpha168-CAT reporter plasmid by lasmids. A dose-response study will be conducted. By the calcium phosphate precipitation method, various amounts of h GR expression plasmid (controlled by R3V promoter) and alpha 168-CAT (reporter) expression plasmid is co-transfected . Co-transfection under conditions that increase the receptor-to-promoter ratio. Run the option. Next, the presence of the steroid hormone dexamethacin or In the absence, the resulting transient CAT activity is measured.

Through this study, we demonstrated that by adjusting the amount of RSV control plasmid, The total amount of R5V promoter DNA transfected is kept constant. In other words , controls the possibility that transcription factors are removed by RSV DNA. child The CAT activity of the reporter construct is measured according to the method described above.

The alpha168-CAT reporter plasmid was described by Delegeane et al., supra. Constructed using the method described in .

The hGR expression plasmid controlled by the RSV promoter is Hollenbe rg et al., Ce11. Vol, 49. pp. 39-46 (1987) Constructed according to the method described.

The control plasmid RSV contains rat thyroid hormone receptor. The region to be coded is included in the antisense direction. This is based on Thompson et al. , 5science, Vol, 237. pp.

1610-1614 <1987).

Figure 2 shows hGR cDNA in the presence and absence of dexamethacin. The effect of transfection on reporter gene expression is shown. O in the diagram is Represents a medium without addition of dexamethacin, * indicates a medium with addition of dexamethacin (10”M) represents. In the particular experiment shown in Figure 2, 2 μg of promoter plasmid is used. Arrows indicate receptors for promoters used in subsequent experiments. Indicates the ratio of

Figure 2 shows that steroid-dependent suppression increases as the amount of receptor expression plasmid increases. It has been shown to increase. Receptor c When DNA is absent, 1 A maximum inhibition of less than 0% is measured. The ratio of receptor to promoter is 1 From 5 to 5, more receptor plasmids cause further steroid-dependent inhibition. When no control is applied, a plateau of inhibitory activity appears. RSV promoter amount is one This plateau indicates that the active sites of the receptor are probably saturated. . In subsequent experiments, the receptor:promoter ratio was 2: It is l. Steroid-dependent suppression varies between 6-fold and q20-fold; The average is 9 times as represented in Figure 2. This assay uses wild-type hGR repression. Suppressions as low as 10% of control can be reliably measured.

Figure 3 shows wild-type hGR1 partially deleted hGR fusion protein and various hG Comparison of CAT activities of R fusion proteins is shown. hGR fusion protein for those listed above. Assay by method. l582*, wild type hGR (hgrwt), I53:)s and l532*-β-galactosidase fusion, the hormone used was Dexa Metacin, and the hormone used for the fusion protein GGM was aldosterone. It is.

In Figure 3B, a schematic diagram of the production of various hGR-derived proteins is shown. . That is, l582* is a partially deleted hGR with only 582 amino acids. l532* is a partially deleted hGR with only 532 amino acids: l532*-β-galactosidase is derived from the l-532 amino acid of hGR white, and A fusion protein containing 8-1025 amino acids derived from β-galactosidase. There is a 3030 bp BamHI of pBG-1, which is a derivative of psK105. Created by inserting the LacZ fragment into the BamH1 site of 532 so that the reading frame fits. (Ca5adaban et al., Methods in Enz mol.

Vol, too, 293. Academic Press (1983)) : and GGM are fusion proteins that first contain an additional Xho I site. , hGR nucleotide position I 596 (Hollenberg et al. 18, 635 (1985)) and human mineralocorticoid receptor (h MR) nucleotide position 2233 (Arriza et al., 1ence 237 ．． 268 (1987)), and then the appropriate Xhol of hMR. Amino acids 1-489 of hGR and hMR constructed by inserting the fragment into hGR It is a receptor having amino acids 671-984 of .

Figure 3B shows the alpha 168 promoter of the carboxyl-terminal fusion protein. and the mouse mammary tumor virus (MTV) promoter. The activations are compared. Percentage of wild-type activity compared to RSV control plasma Calculated for each experiment assuming Sumid activity as 0% and wild type hGR activity as 100%. and the standard error of the mean is expressed. Control 1 and Control 2 indicates transfection using RSV control plasmid. Conte Roll 1 and the next four constructs used dexamethacin as the steroid and Control 2 and GGM used aldosterone as the steroid. l53 Number 21 of the β-galactosidase constructs is from Ca5adaban et al. Kamikou 1 (company) 1. vol, 100. pp, 298-308 (1983 ) indicates the amino acid number of β-galactosidase numbered, and the number of GGM The numbers indicate the hMR amino acid numbers assigned by Arriza et al., supra (1987). vinegar. “*” indicates activity lower than 10% of wild-type inhibitory activity, and “**” indicates wild-type activity. It exhibits an activity lower than 1% of the sexualizing activity.

Linking a heterologous protein sequence to the carboxyl terminal side of the DNA-binding domain of hGR can be used to generate novel sequence-specific repressors. In some cases, E. coli β-galactosidase (β-ga I) is inserted into the hGR DNA so that the reading frame matches. can be fused to the carboxyl-terminal side of the A-binding domain and assayed for regulatory properties. . This hybrid protein was directed against the mouse mammary tumor virus (MTV) promoter. The protein is a constitutive protein with properties not different from those of the type-partially deleted receptor. Acts as an activator. For the alpha168 promoter, the fusion The protein is a constitutive repressor whose activity is similar to the partially deleted receptor, l This is a significant increase compared to 532*. In other words, it creates a functional transcriptional repressor. To achieve this, a heterologous E. coli protein sequence was attached to the DNA-binding domain of hGR. It is sufficient to add

The results of this study provide various means to identify positive and negative regulatory effects of hGR. I will provide a. First, an amino-terminal chain containing a strong activator sequence, τ1, Main is not necessary for trance suppression. In fact, by deleting τ1, It was found that a stronger plenosaur was produced. From this fact, the repressor Some positive activity remains in the amino-terminal region of hGR even when it functions as a It is thought that

By making certain modifications to hGR, it is possible to retain its normal binding function. This results in receptors that lack virtually all positive activation capacity, despite the fact that . This fact indicates that the process of activation may be mechanistically different from the process of inhibition. suggests. This observation further suggests that the function of the DNA-binding domain is dependent solely on the appropriate regulatory arrangements. Show more than just arranging columns. Moreover, this result suggests that the binding of DNA Another subsequent event that is apparently not important for negative regulation is activation. indicates that it is necessary.

Another difference between activation and repression is that the β-galactosidase molecule only in repression It functionally replaces the carboxyl terminus of hGR. carboxyl terminal Deletion of , results in a mutant receptor with minimal inhibitory activity.

Example 4: Thyroid receptor-mediated negative regulation in the absence of binding ligands , thyroid receptors (TRs) can bind to innate response elements. In this respect, it differs from the glucocorticoid receptor (GR). Considering these differences Then, if a modification similar to the modification studied for GR is made to TR, a similar problem can be obtained. It will be of interest to determine whether they provide inhibitory derivatives of ences.

Vectors used in these studies are prepared as follows: The expression vector RS-rTRa was obtained from Thoa+pson et al., Proc. he Natl, Acad, of Sci, Vol, 86. pp, 3494-3498 (1989).

The expression plasmid RS-v-erbA contains the cloned gag-v-erbA gene. Genes [Vennstriim et al., J. Virol, Vol.

36, pp. 575-585 (1980)]. and then modify the 5' and 3° ends appropriately to create vectors KpnI and and BamH1 site.

Hind I I I adapter sequences flanked by Response element [TRE,;TCAGGTCATGACCTGA;Glass et al., Ce1l Vol. 54. Coat Pp, 313-323 (198g) Synthetic oligonucleotides containing pBL-CAT2 [Luckow et al., Nuc , Ac1ds Res, 15.5490 (L987)] only Hfnd Insert into the III cloning site. Has single or multiple copies of TRE The plasmid containing the plasmid is identified by restriction enzyme rubbing and sequence analysis. rTR α gene [Thompson et al., 5science, Vol. 237. p. p, 161G-1614 (19g?)] and v-erbA gene [Dams et al., EMBOJ, Vat, 6.375 (1987) and I/ennst. r5++ et al., J. Virol, 36.575 (1980)] A hybrid gene is constructed using the restriction enzyme cleavage site. rTRa A schematic of the protein sequence composition of and v-erbA is listed in Figure 3A. these The cDNA encoding the protein is cloned into an R3V expression vector.

[DNAJ and rTs/Ta] in the figure are the DNA binding domains, respectively. In and thyroid hormone binding domain.

The 12 amino acids at the 7-minoterminus of chicken c-erbA/TRα) PI3 gmg-v-0rbA hybrid Synthesize proteins. In addition, v-erbA is chicken c-erbA/ What makes TRa unique is the two amino acids in the DNA-binding domain and the hormone-binding The nine amino acids in the domain are different, and the hormone-binding domain is different. The difference is that nine amino acids have been removed. The la shown in Figure 3A Comparison of TRα and ′ revealed an additional 17 amino acid differences. ing. These amino acid differences are species-specific and between chicken and rat. A comparison is made with the amino acid sequence of TRα. Represented above the construct in Figure 3A The numbers shown indicate the amino acid positions.

The PstI fragment of the ligand-binding domain of rTRa was removed to create TR(Δ154/ 317). The carboxyl terminal of rTRa is v-e r bA-ne Ps of the o construct [Sap et al., Nature 324.635 (1986) Substitute with tI-XbaI fragment to create TR(Δ154/317)erbA . The Pst11 fragment of either rTRa or v-erbA was inserted into each plate. Plasmid TR(317)erb was reinserted into the unique Pst1 site of Smid. Create A% TR (154erbA) and TR (154/3t7).

All modifications are subcloned fragments derived from the ligand binding domain of rTRa. was carried out to replace the Xba fragment of RS-rTRa with the corresponding chimeric fragment. Generate the hybrid expression construct.

CO8 cells were cultured in DMEM containing 5% T3-free bovine serum and DEAE dextrose. JGiguere et al., Ce1l 46. 645 (1986)] o After 36 hours, the cells were collected and the buffer was added to 20 m M Hepes (pH 7,8), 0.4M KCI, 2mM dithiothreitate Kumar et al., Ce11. The extract was prepared as described in Vol. 55, 145 (198B). Prepare.

DNA binding reactions and gel electrophoresis were performed substantially as described by Glass et al., Ce11. Va t, 54.313 (1988). 6-10 μg of total titanium Dilute the sample solution containing protein so that the final KCI concentration is 80 mM, 2 μg polydeoxycytidylic acid (POIY [ACL]) for 20 minutes at room temperature. 1 batch of ink. At this time, a palindrome of 25 to 50 fmol A 32P-labeled oligonucleotide encoding a TRE was added. reaction mixture Incubate the ink at 22°C for 30 minutes, add 50mM Hepes (pH 7, 8) The sample was placed on a 5% polyacrylamide gel containing . labeled oligonucleotides Competitor DNA was added before adding DNA.

rTRa was partially digested with PvuII to retain the open reading frame and BamH I linker (12mer) was added to construct the DNA binding mutant 195TR. Ta. The position of the linker was confirmed by restriction enzyme mapping and sequence analysis.

TR(154)erbA was partially digested with PvuII to preserve the open reading frame. and added a Bam Hr linker (12mer) to create a DNA-binding mutant. , i95 (154) erbA was constructed. Restriction enzyme 7 noping and sequence analysis I checked the position of the linker.

First, the tothyroid hormone α receptor (rTRa) [:Thomps on et al., 5science 237.1610 (1987)] and v-ert ) Evaluate the transcriptional activity of both A oncogene products. The method is to We measured their ability to modulate the expression of a lumon-responsive reporter gene (see Figure 4A). This is done by determining the The constructs shown in this figure include previously identified Thyroid hormone response element [GIass et al., Nature.

738 (1987) and Glass et al., Ce1l 54.313 (198 8)] is an oligonucleotide corresponding to thymidine kinase-chloramphenicol. acetyltransferase (tk-CAT) fusion gene [Luck ow et al., Nuc, Ac'ds, Li, 5490 (1987)] (see FIG. 4B). The reporter genes used are shown in Figure 4B, Oligonucleotides encoding each T3 response element (THE) Inserted into the HindIII (H) site upstream of the tk promoter-CAT construct. is included.

Encodes rTRa or V-erbA, which is under transcriptional regulation by RSV LTR. The expression plasmid and one reporter plasmid were Significantly lower Cvl cells were co-transfected.

Reporter constructs tk-CAT, tk-TRE, -CAT or tk-TR E-ClO2-CAT and each expression plasmid (R9-rTRa or R 3-v-erbA) and internal reference plasmid R3V-βGAL were V-1M cells are co-transfected. In control experiments, rTRa A construct with the coding sequence in the reverse direction (RS-3'-5') is used.

tk-TRE, -CAT or molded vector, tk-CATl Transfection showed high basal BellCAT activity, and this activity was found in the silo. It increases slightly with the addition of idohormones. In contrast, TR expression vector -, RSV-rTRa, and tk-TREo-CA A significant effect on T expression is produced. If thyroid hormone is not present It has been observed that basal CAT activity decreases by 80% (referred to as “low basal level activity”). be noticed. This phenomenon indicates that TR expression induces a ligand-independent inhibitory effect on transcription. It suggests that something is going to happen. Thyroid hormone, triiodothyronine [T3] , when added to a final concentration of l100n, tk-TRE, -CAT becomes 2 0 times accelerated. This is 3 to 3% of the basal level activity obtained in the absence of TR expression. This corresponds to a five-fold promotion.

The regulatory function of rTRα is not limited to THE. For example, rat growth hormone genetics Offspring-derived TREs can also sustain hormone-independent and hormone-dependent transcriptional responses . This functional assay demonstrates the putative transcription of the v-erbA oncogene product. It is possible to determine activity directly. v-erbA interacts with thyroid hormone Although it has lost its binding ability, it still has a complete NAmAm domain, so v-e It can be expected that rbA functions as a constitutively active TR. However, v-erb Co-transfection of A with either reporter plasmid results in transcription is not promoted, but rather an effect similar to negative regulation of rTRα in the absence of the hormone. shows. That is, in cells expressing V-erbA, CAT activity is at a high basal level. It decreased by 80% from 100% to 100% and cannot be recovered by the addition of T3.

Various mutations in v-erbA result in altered properties of this protein. prototypical, compared to TR) and activation and repression. To elucidate the regulatory process in more detail, we constructed a chimera of the v-erbA oncogene. Receptor [Damm et al., EMBOJ, Vol. 6. pp, 375-3 82 (1987) and Vennstri et al., J. Virol, Vo. l, 36. pp. 575-585 (1980)] and rat T Ra [Construct Thompson et al. (198?), supra. Rat TRα/ A schematic representation of the structure of the v-e r b A chimeric protein is displayed in Figure 5A. . The numbers depicted above each construct refer to the amino acid positions. The black bar is Deletion of 9 amino acids of v-erbA resulting in complete carboxyl terminus A 401 amino acid fusion compared to a 410 amino acid protein with ends. Protein is obtained. Figure 5B shows tk-TR in the indicated expression vectors. Positive and negative regulation of EoCAT on CAT activity is shown. this stick Graph shows average values from 2-6 independent transfection experiments. bar graph The stippled bar on the left is the value without hormone, and the diagonal bar is the value when T3 of loonM is present. shows.

For amino acids 154 to 410, v-erbA has rTRα and 26 amino acids. No acid appears, and 9 amino acids near the carboxyl terminus are deleted. They differ in some respects. By replacing this region of rTRα with v-erbA, This results in a hybrid TR(154)erbA (Figure 5A). Its properties are similar v- substantially identical to erbA (Fig. 5B). That is, the DNA-binding domain Mutations in the and flanking amino acids are not important for the v-erbA phenotype.

Internal region of rTRα ligand-binding domain (amino acid portion from positions 154 to 316) ) replacement of the T3-responsive hybrid (TR(1 54/316) e rbk'). In contrast, the carboxyl group of rTRα The syl-terminal 93 amino acids were replaced with the corresponding sequence of v-erbA (this sequence consists of 9 amino acids). The deletion of one amino acid and the replacement of 11 other amino acids with The hybrid, deTR, has suppressive properties similar to those of viral oncogene products. (317) Obtain erbA.

To investigate the effect of v-erbA on endogenous thyroid receptor function, Co-transfection studies in Cv1 cells are performed. to CV-1 cells , reporter gene tk-TRE, -CAT (0.5μg), lμg rTR a Whole Ra vector and internal control plasmid R3V-β-GAL were simultaneously transfect. Additionally, a 10-fold excess of the expression plasmid shown in Figure 6 jl (10 μg) was co-transfected with rTRα. Displayed as (-) In this experiment, 10 μg of Funtrol plasmid R5-3″-51 was used.

The average values of 2 to 6 independent transfection experiments are represented in the bar graph in Figure 6. ing. All experiments are performed in the presence of 1100n of T3.

As shown in Figure 6A, a 10-fold molar excess of v-erbA stimulates rTRα. The thyroid hormone-dependent transcriptional induction of the reporter gene is reduced by 90%.

Hybrid constructs TR(154)erbA and TR(317)erbA also v - exhibits erbA-like activity and is consequently induced by T3 and rTRα Substantially completely suppresses promotion of the reporter gene. However, this activity is completely The reason for this depends on the presence of a DNA-binding domain, which is the presence of a DNA-binding domain variant195 (154) This is because erbA shows no competition.

Similarly, placing TRE within the MTV promoter inhibits T3 induction from v-erb A and chimeric constructs.

These results demonstrate that there is an inverse relationship between TR activity and the concentration of competitor products. It is expected that there will be. To investigate this conjecture, we need TR(154)erbA and r TRa was co-transfected with varying molar ratios of total Ra vector and hormone The responsiveness of the engine was evaluated. In other words, the reporter gene tk-TRE is added to CVI cells. , -CAT (0.5 μg), 1 μg of expression vector RS-rTRα, and At the same time, increasing the hormone non-binding competitor TR(154) erbk Transfect. In all transfections, Funtrolp Add lasmid R5-3'-5' to keep RSV promoter amount constant do. Average values of three independent transfection experiments are shown. looo Cells were grown in the presence of nM T3.

As expected, as TR(154)erbA increases, rTRα activity increases. decreases (see Figure 6B). In this assay, a small amount of TR(154)erbA But it is effective. A 3:1 plasmid ratio completely eliminates the hormone-induced response. to disturb.

The DNA binding ability of hormone receptors is ligand dependent [Evans, Beni et al. Despite the fact that it is generally assumed that First, the results disclosed herein and previous observations [Lavin et al., J.B. iol, Chew, 263.9418 (1988) contradicts this conjecture. . Down-regulation of transcription by responsive promoters is probably due to hormonal In the absence of , the receptor recognizes the original response element and interacts with it. It is the result of the ability to combine. Two lines of evidence support this presumption. One piece of evidence The complete NA-binding domain was not present in gel electrophoresis retardation experiments and observed transcriptional effects. It is necessary for both. The second piece of evidence is when the response element is not present. , no sufficient repression is observed, but tandem linking of THE results in positive and negative transcription. It is about being able to act. glucocorticoid receptors and estrogens positive synergism on receptors and their respective response elements [5chi le et al., 5science 242. 1418 (1988) and 5trih le et al., EMBOJ, L 3389 (19+38)] has already been observed. However, there is no clear precedent for the negative synergy observed in the present invention.

Example 5: Retinoic acid receptor-mediated negative regulation RARα and TRa and V- To investigate positive and/or negative interactions with erbA, TRa or v -Cv-1 cells with RARα and reporter plasmid in the presence of erbA transfect at the same time (Figure 7). In the figure, TRa or v-erbA competes with RA-induced transcriptional activation by RAR (!). RARα expression vector (1,0 μg) was added to TRa (5, Ottg> or v-erbA (5° reporter plasmid ΔMTV-TREp-CAT (1,0 μg) with Qμg).

Plasmids were added to 5 x 105 CV-1 cells (5% offspring) per 10.0 cm dish. Calcium phosphate precipitated (maintained in DME medium supplemented with bovine calf serum) introduced by law.

Previously, Damm et al., Nature, Vol. 339.593 (1989), and U+mesonO et al. NatureSVol 335.262 (1988 ), the expression plasmids RARaTRα and v-erbA is under the control of the R3V promoter. Cells were eluted from activated carbon resin for 36 hours. Dulbecco's modified Eagle's medium containing 10% calf serum [5aIIIuel S et al., Endocrinology, Vol. 105.80 (1979)], Cultures were grown in the presence or absence of hormones as indicated. Next, Gor man et al.Mo1. Ce11. Biol, Vol. 2, 1044 (1982). Prepared for CAT activity by three cycles of freeze-thaw as described above. Ta. Aliquots of cell extracts were labeled for β-gal activity prior to performing the CAT assay. Standardized. Hormones were added to a final concentration of ionM. These de Data are the average of four separate experiments.

A 5-fold molar excess of TRa reduced RA-dependent induction of CAT activity by 90%. T No inhibition of transcription was seen when both 3 and RA were added simultaneously. subordinate Therefore, in the absence of T3, TRa inhibits RA-induced gene expression by RARα. whereas in the presence of T3, activation by <TRa was observed. It will be done. Similarly, TR inhibition of RAR activation was reported by Graupner et al., Natu re, 1101340.653 (1989) and 13rent et al., The New Biologist, Vol. 1, 329 (1989) It has been observed closely. When v-erbA was co-transfected with RARα , a similar competition of RA-induced gene activation is observed.

To investigate whether a series of R Mutant RA consisting of partial deletion of AR and hybrid RARα-erbA fusion Create Rα.

RAR functions as a ligand-dependent transcription factor and therefore has a ligand-binding domain. Mutants containing alterations or deletions in the DNA-binding domain but with an intact DNA-binding domain are the dominant Can give a negative phenotype. In fact, from the results described in Example 4, the carboxylic acid of TRa The ligand binding domain located at the end of v-erbA is attached to the carboxyl terminus of v-erbA. Upon substitution, the hybrid TRα- An erbA molecule is formed. Therefore, a similar fusion of RARα and v-erbA , the ligand-binding domain of RARα located at the carboxyl terminus was It is constructed by substituting the carboxyl terminus of A (Figure 8). Hybrid RAR The α-erbA fusion is the N-terminus of TR317-erb-A and DNA binding. Remove the domain (see [)aim et al., supra) and replace that part with the corresponding RARα This method was constructed by replacing the N-terminus and DNA-binding domain with sea urchin, RARα-erbA hybrid protein is produced). Partial deletion mutant Now insert the last amino acid of RARα at the indicated position before insertion of the translation stop signal. Leave it there. Partially deleted mutants of RAR185 and 203 contain wild-type receptors. RAR153 was constructed with only one restriction enzyme site present in 153 By creating a unique Xho site and inserting a stop codon at the RARα position. So, I built it. Retinoic acid was added to a final concentration of 1100n.

In transactivation experiments, by the addition of retinoic acid, a positive response was induced by CAT This corresponds to a 25-fold increase in activity. Addition of retinoic acid promotes transactivation. There is no negative response to this. In competition experiments, the positive response is due to retinoic acid-induced trauma. 80% of the activity, whereas a negative response Therefore, there is no conflict.

The RARα-erbA protein itself is active in the presence or absence of hormones. It does not act as an activator of transcription. However, the RARα-erbA fusion If the body is co-transfected with RARα, this will result in an imbalance of RARα. Functions as an agonist. See Figure 9.

CV-1 cells are RAR-expressing Tuctor 1. 0 u g), ΔMTV-TR Ep-CAT reporter plasmid (see Umesono et al., supra) (5,0 μg), gradually increase to 20.0mg with reference plasmid (5°0mg) RAR (!-co-transfected with erbA and carrier plasmid) tion. The R3V-promoter is added by adding a carrier plasmid. for all transfections. The maximum response % is lOO represents the CAT induction observed with the RAR wild-type receptor in the presence of nM RA. ing. This activation corresponds to a 25-fold induction of RA. All data are 4 times is the average of separate experiments.

RARa-erbA can antagonize RA-induced activation of RARβ and RAR7 . Therefore, the carboxyl terminal m of RARa is placed at the carboxyl terminal of V-erbA. Replacement confers a dominant negative phenotype on RARa. In contrast, a series of RAR mutants consisting of loboxyl-terminus deletions are themselves transfectable. When activated, it does not act as an activator of transcription and does not co-act with RARa. does not act as a competitor of RARa when transfected with stomach. These data for RAR partial deletions are from Espeseth et al., Ge nes and Dev, Vol. 3.1647 (1989). There are some discrepancies with the research. They found that the RARα mutant, which is essentially the same as RAR-185, , give rise to a small proportion of stable F9 clones that do not differentiate in response to RA, and therefore This RAR partially deleted mutant may function as a dominant negative RAR. I assumed.

The nature of the RAR (!-erbA fusion) with v-erbA and TRa is It is further investigated by examining its ability to suppress/antagonize endogenous RAR.

F9 cells were established as a cellular model of RA-dependent differentiation. This cell is RA Includes receptors of the α, β, and γ subtypes of the R family. RARa and and RARγ are present in undifferentiated stem cells, whereas RARβ is present in RA induced by treatment. For example, 5trickland and Mahda vi Ce1l, Vol 15, 393 (1978): 5porn et al. Th e Retinojds, Vol. l-2, Academic Press ( Orlando, F.L., 1984); and Hu and Gudas M. o1. Ce11. Biol., Vol. 10.391 (1990).

Injection of either RARa-erbA, TRa or V-erbA into F9 cells. Transfection results in strong inhibition of RA-induced transactivation ( Figure 10). F9 cells (maintained in DMEM supplemented with 10% CBS) were Porter ΔMTV-TREp-Luc (5,0) μg), 5.0 mg of Con Troll plasmid (RSV-CAT), a Ll! R3V-TRa, RSV Calcium release with either -verbA or RSV RARa-erbA. Transfection is carried out by the acid method. 5. 0Mg Reference Plus Mid and 5°0 Mg carrier plasmids. cells for 24 hours, 11 Culture in the presence or absence of 00n RA. The reporter construct is a firefly The gene encoding luciferase [:D+4et et al., Mo1. Ce11. B iol, Vol. 7.725 (1987)], the reporter construct de It is exactly the same as ΔMTV-TREρ-CAT except that it is replaced with CAT.

Cells were cultured for 24 hours after hormone addition, then harvested and Hollenberg Lucif et al., Ce1l, Vol. 55.899 (1988). erase assay was performed. Data are the average of 4 separate measurements.

Implementation fFj16: Glucofluticoid receptor-v e r bA and G R-βGalGal fusion tan The fact that the Baku Ra-erbA fusion protein functions as a dominant negative RAR; The ability of the TRa-erbA hybrid, 7d, to function as an inhibitor of TRa. The foregoing discussion of the power of steroid-erbA hybrid receptors suggests that the steroid-erbA hybrid receptor may provide a general approach to making hormone receptor antagonists. Ru. Therefore, the fusion of GR and erbA (GR-erbA, Figure 11) By replacing the carboxyl terminus of rbA with the ligand-binding domain of GR, It was made.

In the figure, the wild-type GR is shown first and is numbered to indicate the amino acid position. be. The DNA and ligand binding domains are also shown. Hybrid G The R-erbA fusion, like the RAR-erbA fusion protein described above, The variant gives the last amino acid before the carboxyl-terminal nonsense peptide. I can do it. To obtain GR-532βgal, OrO et al., in Ce11. Vol. 55.1109 (1988), E. coli βgal is fused to position 532 of GR to fit the frame. It is β Galactosidase and the glucocorticoids reported by Oro et al. It encodes a protein that exhibits receptor properties.

For transactivation experiments, CV-1 cells were incubated with expression vector (1,0 μg), Porter-1MTV-Luc (5,0Mg), RSV as internal control -CAT (5,0 Mg), and carrier plasmid total maximum 20.0 Transfect with up to Mg. Data are shown as % of best response. Ru. Therefore, the presence of a synthetic glucocorticoid, dexamethasone lXl0-7M, , refers to the luciferase induction observed by GR wild type. This activation is This corresponds to a 3000-fold induction.

For competition experiments, cv-i cells were transfected with GR expression vector 1.　0Mg, MTV Lipo 5.0μg1 of RSV-CAT5. as an internal control. Oμg Crab Competitor 5. 0 Mg, and a total maximum of 2 of the Bovine Yaria plasmids Up to 0.0 Mg was co-transfected. Dexamethacin final concentration txto-7M. This response is a non-specific competitor. In the presence of It corresponds to the percentage when The data in Figure 11 are the average of four separate experiments.

The fusion protein is a glucocorticoid in the presence or absence of gluticoids. Does not activate coid-responsive reporter genes. However, GR-erbA is When co-transfected with R, it functions as an antagonist of GR.

A 5-fold molar excess of GR-erbA inhibited dexamethacin induction of the reporter gene by 8 7% decrease.

For comparison, the properties of a series of GR partially deleted mutants were compared to dominant negative competitors. - and by acting as a transcriptional activator. Partial deletion at carboxyl terminus A mutant receptor that does not activate transcription (GR, 487) or an essential activating receptors (GR515, GR532).

When partially deleted for GR, when co-transfected with wild-type GR, dexamethas have no, or at best, only a slight inhibitory effect on zone-induced transcriptional activity. O has only a suppressive effect The properties brought out by positioning the carboxyl terminus of GR are unique to other polypeptides. To test whether β-Cat could be replaced by It is fused to the carboxyl terminus of GR in frame. This G R-5 32βGalM synthesis protein functions as a negative regulator of GR transcription This was previously shown (see Example 3). However, this β-Gal fusion protein The protein has essentially the same activity as the parent part 1 deletion, and increases GR activation by 60%. only decreases.

Therefore, it acts as a dominant negative receptor for GR partial deletions and βGal fusions. The ability and utility to use The ability to do so is initially reduced by the lower level. In contrast, GR-erbA The only mutant receptor that has no transcriptional activity without ligand, Functions to block xamethacin-induced transcriptional activity. Furthermore, GR-e rbA is highly reactive to intrinsically active, GR receptors such as GR532. Can act as a strong competitor and also act as a dominant negative inhibitor maintains the possibility of

The foregoing description sets forth specific methods that may be used to practice the present invention. Specification Initially used for the identification, isolation, characterization, preparation and use of receptors for The details of the specific method used, as well as the specific properties and sequence thereof, have been disclosed. , those skilled in the art will be able to obtain the same information and further investigate other intracisternal as well as interspecies relationships. We are well aware of how other robust methods can be devised to extend this information to receptors. know. Therefore, no matter how detailed the aforementioned text is, the overall scope is should not be construed as limiting. Rather, the scope of the invention is It should be governed only by legal interpretation of the appended claims.

7'7-: A0ya- T-7° Utomi F'phr Tomoe FIG, 2 Kasutei CAT nine works FIG-5B - 12yoLyoL"2"--FIG, 6A FIG. 6 [3 (irereshi) RARcx + TR (I RARcz + v-erbAFIG, 7 EAFoCaz M/ZcyC-e, rbA figRARa(1,0μ9)+ ++10 RARa-erbA (μg)” 1.0 5.0 10.0F9! FFB stutter L force raw shakuju 9 pieces Copy and translation of amendment) Submission (Article 184-8 of the Patent Law) November 26E, 1991 Team T

Claims (1)

[Claims] 1. Transrepression of steroid/thyroid superfamily receptors A analog receptor, the analog is the amino acid of the following (1) and (2). Contains an acid sequence: (1) a first amino acid sequence that is a DNA binding domain; The analog then interacts with the hormone response element of the wild-type receptor through this sequence. and (2) a sequence capable of binding to the carboxyl terminus of the DNA-binding domain; a second amino acid sequence selected from (a) and (b) below; array; (a) Access to the ligand-binding domain of the carboxyl-terminal portion of the wild-type receptor. A polypeptide having at least about 90% of the number of amino acids, (i) shorter than the carboxyl-terminal domain of the wild-type receptor; is the full length of the polypeptide, or (ii) the carboxylic acid of the wild type receptor. any of the polypeptide segments having the same length as the terminal domain. which is less than about 60% with respect to the carboxyl-terminal domain of the wild-type receptor. have no amino acid identity, or (b) v-erbA protein defined by amino acid numbers 313-398 (see Figure 1) At least 84 carboxyl-terminal amino acids in the carboxyl-terminal portion of proteins noic acid; when the wild-type receptor is a glucocorticoid receptor, Second amino acid sequence. 2. The hormone response element is attached to a promoter that is subjected to trans-repression. 2. The analog receptor of claim 1, wherein the analog receptor is operably linked to . 3. Substantial transcriptional activation activity in the presence or absence of ligand The analog receptor according to claim 1, which has no sex. 4. the first amino acid sequence is a DNA binding domain of the wild type receptor; 2. The analog receptor of claim 1, which is. 5. The wild-type receptor may be a retinoic acid receptor or a thyroid hormone receptor. ceptor, vitamin D3 receptor, glucocorticoid receptor, mineral corticoid receptor, estrogen receptor, estrogen related receptor -, aldosterone receptor, androgen receptor, or progesterone 5. The analog receptor according to claim 4, wherein the analog receptor is selected from the receptor receptors. 6. The DNA binding domain is (1) a glucocorticoid receptor; (2) derived from thyroid receptor; or (3) retinoic acid receptor; The analog receptor according to claim 5. 7. the DNA binding domain is a human glucocorticoid receptor; The analog receptor according to claim 6. 8. The DNA binding domain is a silo 7. The analog receptor according to claim 6, which is derived from an id receptor. 9. 6. The DNA binding domain is derived from a retinoic acid receptor. Analog receptors described in . 10. the first amino acid sequence is derived from the wild-type receptor, and the first amino acid sequence is derived from the wild-type receptor; When the analog receptor is present or absent, the The analog receptor according to claim 1, which has no transcriptional activation activity. 11. Claim 1, wherein said carboxyl terminal domain is derived from a polypeptide. The analog receptor described in 0. 12. the polypeptide is at least a portion of a β-galactosidase polypeptide 12. The analog receptor of claim 11, wherein the analog receptor is minutes. 13. The carboxyl-terminal domain is derived from the v-erbA protein. 11. Contains at least a C-terminus that is 84 C-terminal amino acids. analog receptor. 14. 2. A method of preparing the analog receptor of claim 1, comprising: Expressed in a recombinant host transfected with DNA encoding the A method comprising the steps of: 15. 15. The method of claim 14, further comprising circulating the analog receptor. The analog receptors are isolated and purified to reveal the externally derived biological mechanisms of the analog receptors. 1. A method for use in an assay for measuring potency. 16. A recombinant DNA molecule encoding the analog receptor according to claim 1. A DNA molecule is a cDNA molecule. 17. An expression vector operably comprising the DNA according to claim 16. 18. A recombinant host cell transfected with the expression vector according to claim 17. Cell. 19. A cell according to claim 18 and an external maintenance medium ensuring the survival of the cell. A cell culture containing. 20. Diseases that do not respond normally to steroids or thyroid hormones a transgenic mammal, other than a human, having a at least one subset expresses one analog receptor for the hormone and the analog receptor is larger than the corresponding wild-type receptor. It has strong transrepression activity and is smaller than the corresponding wild-type receptor. A mammal with lance activating activity. 21. Converts wild-type hormone receptors to trans-repressive analog receptors A method for converting the ligand-binding domain of the wild-type receptor into an amino acid verbA proteins defined by numbers 313-398 (see Figure 1). a step of substituting both 84 carboxyl-terminal amino acids; A method that encompasses. 22. Converts wild-type hormone receptors to trans-repressive analog receptors A method, The ligand-binding domain of the wild-type receptor is linked to the carboxy has at least 90% of the number of amino acids in the terminal ligand-binding domain A step of substituting with a polypeptide, wherein the polypeptide is located below the carboxyl terminus of the wild type receptor. (i) or (ii) have less than about 60% amino acid identity: ( i) if shorter than the carboxyl terminus of the wild-type receptor, the polypeptide full length; or (ii) having the same length as the carboxyl terminus of the wild-type receptor. any segment of said polypeptide. 23. 1. A method of blocking transcriptional activity of a hormone response element within a cell, the method comprising: , comprising the step of contacting the cell with the analog receptor of claim 1. Method. 24. Transcription of hormone response elements present in cells by wild-type receptors A method of blocking activation comprising: (a) ligand binding of said wild-type receptor; substantially deleting the domain; (b) verbA protein defined by amino acid numbers 313-398 (see Figure 1) At least 84 carboxyl-terminal amino acids of the protein are modified in step (a). operably linking the modified receptor to produce a fusion protein; And after that (c) contacting the cell with an effective amount of the fusion protein; Method. 25. Translated by wild-type receptors, which are hormone response elements present in cells. A method of blocking photoactivation comprising: (a) ligand binding of the wild-type receptor; substantially deleting the binding domain; (b) a minimum number of amino acids in the ligand-binding domain of the wild-type receptor; is capable of activating the modified receptor of step (a). a step of producing a fusion protein by combining Here, the polypeptide is directed against the ligand-binding domain of the wild-type receptor. The de is (i) If it is shorter than the ligand-binding domain of the wild-type receptor, the polypeptide or (ii) the same length as the ligand-binding domain of the wild-type receptor. any segment of the polypeptide having less than about 60% After that step with no amino acid identity, (o) contacting said cell with an effective amount of said tactile protein; A method that encompasses. 26. Receptor responses to unknown associated response elements and/or functions A method for identifying elements and/or functions, the method comprising: When the test system is treated with (I) or (II) below, the wild type receptor comparing the responses of the test system in response to the Including, the method: (I) wild type receptor; or (II) trans-repressive analog receptor; where the trans-repressive analog receptor The scepter contains the following (1) and (2): (1) A first amino acid sequence that is a DNA binding domain, the analog being can bind to the hormone response element of the wild-type receptor through the sequence of ; and (2) a second amino acid located at the carboxyl terminus of the DNA binding domain. An acid sequence selected from (a) or (b) below: (a) Access to the ligand-binding domain of the carboxyl-terminal portion of the wild-type receptor. A polypeptide having at least about 90% of the number of amino acids, wherein the wild type For the carboxyl-terminal domain of the receptor, the polypeptide is ) or (ii) having less than about 60% amino acid identity; (i) if shorter than the carboxyl terminus of the wild-type receptor, the polypeptide or (ii) identical to the carboxyl-terminal domain of the wild-type receptor. any segment of the polypeptide having the same length; or (b) v-erbA protein defined by amino acid numbers 313-398 (see Figure 1) At least 84 carboxyl-terminal amino acids in the carboxyl-terminal portion of proteins noic acid. 27. In an assay system that measures the presence of specific transgenic receptors, here , more than one response element can interact with the wild-type receptor, and the receptor For example, it responds to wild-type receptors other than the specific wild-type receptor. An improvement has been made that includes the step of inactivating the responsive element, in which the responsive element is inactivated. The response element provides the assay system with an effective amount of target for each receptor. is inactivated by adding a lance-inhibiting analogue receptor, where , each trans-repressing analog receptor contains (1) and (2) below. do: (1) A first amino acid sequence that is a DNA binding domain, the analog being Through this sequence, hormone responses of receptors other than the specific wild-type receptor can be stimulated. a sequence capable of binding to an element; and (2) A second amino acid sequence located at the carboxyl terminus of the DNA-binding domain. and a sequence selected from the following (a) or (b); (a) Ligation of the carboxyl-terminal portion of a receptor other than the specific wild-type receptor; has at least about 90% of the number of amino acids of the Gund binding domain a polypeptide, the polypeptide comprising a receptor other than the specific wild-type receptor; Approximately 60% or more of the following is present for the carboxyl-terminal domain of the receptor: (i) have low amino acid identity; (i) said receptor other than said specific wild-type receptor; the full length of the polypeptide if shorter than the carboxyl-terminal domain of the receptor; or, (ii) the carboxyl-terminal domain of said receptor other than said specific prophylactic receptor; any segment of the polypeptide having the same length as the in; or (b) v-erbA protein defined by amino acid numbers 313-398 (see Figure 1) At least 84 carboxyl-terminal amino acids of the carboxyl-terminal portion of the protein. noic acid.