CA2057049A1 - Dominant negative members of the steroid/thyroid superfamily of receptors - Google Patents

Abstract

Disclosed are novel trans-repressing analog receptors wherein the ligand-binding domain(s) are modified versus wild type receptor, such novel receptors having repressed trans-activation transcription activation properties. Also disclosed are recombinant methods and means for preparing such receptors and assays using such receptors.

Description

2~5~ `g ~ ~-; PCT/US90/03113 DOMINANT NEGATIVE MEMBERS OF THE STEROID/THYROID
SUPERFAMILY OF RECEPTORS

Related A~lications This is a continuation-in-part application of U S Patent Application Serial No 07/358,696, filed 26 May 1989, which is in turn, a continuation-in-part application of U S Patent Application Serial No 07/289,561 filed 23 December 1988, the entire contents of which are both hereby incorporated by express reference herein Field of the Invention The present invention relates to trans-repressing analog receptors of the steroid/thyroid superfamily In a particular aspect, it relates to the identi~ication and characterization of proteins that ~unction as transcription trans-activation repressors, as well as to their preparation and use, including novel DNA
i~olates encoding same; expression vectors operatively harboring th-~- DNA ~equ-nces; and bo~ts trani~fected with ald v-ctor-In another aspect, the present invention r-lat-s to the use o~ the above-described transcription tran~-activation r-pressors in various assays and cr--ning m-thod~
., .

Backg~ound of the ~y~ LQ~
The characterization and preparation of variou~
hor~ono and hormone-liko receptors, including steroid, , i "

W O 90tl4356 z ~ ~ ; PC~r/US90/03113 . ii q~, thyroid, and retinoid receptors such as those represented by the glucocorticoid, mineralocorticoid, thyroid, estrogen-related and retinoid classes has been subject of considerable research.
It is known, for example, that the glucocorticoid receptor belongs to a large superfamily of ligand-dependent transcription factors that have themselves diverse roles in homeostasis, growth and development. Comparison of complementary DNAs encoding these receptors, as well as mutational analyses of their coding sequences, have identified certain functional domains within the molecule that are thought responsible respectively for DNA binding, hormone binding and nuclear localization. See Evans, et al., Science 240, 889 tl988) for a review of this subject matter.
In the case of the glucocorticoid receptor, the so-called DNA binding domain spans some sixty-six amino acids and is highly conserved among various species. In addition, this domain has been found to be required in order to activate transcription. See Hollenberg, et al., Cell 49, 39 (1987), Miesfeld, et al., Science 236. 423 (1987), Danielsen, et al., Mol.Endo }, 816 ~1987), Kumar, et al., Ç~ 1, 941 (1987), Gronemeyer, EMB0 J. 6, 3985 (1987), and Waterman, et al., Mol.~ndo 2, 14 (1988).
Thi- domaln ha~ b-en found to contain nine invariant ¢yzt-in- re-idues. Although the contribution of each cy~t~lne residue to overall function is unknown, as is the actual ~tructure formed by this domain, it has been propo~od that the~e cysteine residues coordinate two zinc ~0 lon~ to rorm two DNA binding, ~o-called ~inger domains, which re8ult in a ternary structure thought responsible ~or the localization and binding o~ the glucocorticoid receptor to the requisite DNA site. See Xlug, et al., ~ : .
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WO90/14356 ~ PCT/US90/03~13 Tr.Biochem.Sci 12, 464 (1987), Bens, et al., Cell 52, 1 (1988), and Evans, sura.
In a location nearer the carboxyl-terminal end distal from the DNA binding region is the so-called ligand bindi~g domain which has the demonstrated ability to block activity of the receptor in the absence of hormone. Thus, presence of the requisite hormone relieves the inhibition of the receptor to activity.
Deletion of this region has been found to produce a hormone-independent transcription activator. See Godowski, et al., Nature 325, 365 (1987), Hollenberg, et al., supra, Rumar, et al., suDra, Danielsen et al., su~ra, and Adler et al., Cell 52, 685 (1988).
In contrast to these two domains, the seguences lying towards the amino-terminal region from the DNA
binding domain are poorly understood both as to ~tructure, and particularly, function. This region is extremely variable both in size and in composition amongst the various receptors - See Evans, supra - and may contribute to the heterogeneity of receptor function.
See Kumar et al., supra, and Tora et al., 333, 185 (1988).
Despite extensive analysis, some of which has b-en reported ln the ~cientl~ic literature, the region(s) that d-termine(s) trans-activation of transcription inltlatlon r-maln~ poorly characterized. Trans-~ctl~atlon domains can be de~ined a~ polypeptide regions that, when combined with the functional DNA binding domain, increa~e productive tran~cription initiation by RNA polymera~-s. See Sigler, Nature 333, 210 (1988), Br nt t al., S~ , 729 ~1985), Hope et al., ~ell 46, 88S ~1986), Na et al., Cell 48, 847 ~1987), Ma et al., S~L~ ~, 113 ~1987), LQch et al., ÇÇ11 ~, 179 (1988), and Hop- et al., Nature 333, 635 ~1988).

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Previous research of the human glucocorticoid receptor by linker scanning mutagenesis identified two regions outside of the DNA binding region having a role in transcription activation These regions were defined as r1 and r2- Giguere et al , Cell 46, 645 (1986) Further research from these laboratories has also resulted in the report of a co-localization of trans-activation and DNA binding functions See Hollenberg et ~1 , supra, Miesfeld, et al , supra, Danielsen et al , suDra, and Waterman et al , suDra As a result, this research has given rise to an emerging picture of an increasingly modular molecule with discrete domains, each contributing to the identified properties of ligand-binding, DNA-binding and trans-activation of transcription Until recently, the region(s) determining the trans-activation activity were not at all well understood Thus, the picture based upon extant literature lacks an overall appreciation of the dynamic nature of the steroid receptors and how the various domains determine the cascade of events initiated by ligand-binding and consummated by promoter-specific trans-activation Further, although previous research has id-nti~ied runctional "domains", there has been little 2S ~y~t-matic errort to identiry amino acids that contribute to th- pecl~ic a¢tlvitles Or the molecule it~el~ Thus, th- pr-viou- id-ntirication Or steroid receptor trans-activation region~ resulted only rrom a demonstrated loss Or activity via deletion or insertional mutagenesis, but in no ca~- hav- the propertie~ o~ the regions themselve~
been conrirmed in assays that rerlect a dominant gain o~
runction 8ee al80 Ptashne, Nature 335, 683 ~1988) ~hus, Godowski et al , science ~, 812 (1988), report results that ~how that the glucocorticoid receptor . . : .. . - , i - : , . .

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contains at least one "enhancement domain~' other than that overlapping the glucocorticoid response element binding region (i.e., the DNA binding domain) and that the ~econd domain occupies a region near the receptor amino-terminus. Similarly, Webster et al., Cell 54, 199 tl988) report on an inducible transcription activation function of the estrogen and glucocorticoid receptors, and these researchers speculate that the relative positions of the hormone regions (i.e., ligand and DNA-binding domains) are not important for the transcription induction activity of the receptor. Yet, these researchers admit that they have no definition of the exact location and nature of what they call the hormone-inducible activating domain, to say nothing of its characteriz~tion ~nd role in trans-activating potential.
The work by Giguere et al., supra, demonstrated a 1088 of activity in the glucocorticoid receptor based upon an assay measuring transcription activity, as a result of performing random ~ite-mutagenesis at several locations of the molecule. As a follow-up, Hollenberg et al. deleted regions in the molecule, ag~in demonstrating overall 10s5 of transcription activity induced by such removal of ~tr-tche- o~ ~mino ~cld~.
Th- hum~n glucocorticoid receptor (hGR) has -rv-d a~ ~ prototype, model receptor for gene r-gulatlon. As noted above, the DNA-binding and ligand-blnding function~l domains have been defined previously.
Si~llarly, it ha~ be-n tound that thes~ modular domain~
o~ th- hGR r-c-ptor or other r-ceptors may b- moved to oth-r parts o~ the roceptor or attached to heterologous DNA-binding dom~lns and ~tlll maintain functlon.
ln contrast, rolatively little i8 known about nogativo regulation by hGR. This iB 8urpriBing in light W090/143~ ~s '`~ ~ PCT/US90/03113 2C~7~49 of the key role that steroids play in development and negative feedback regulation. Glucocorticoid helps determine neural crest cell fate in the developing sympathoadrenal system, in part by repressing the induction of neural-specific genes ~See Stein et al., E~Y
Bip 127, 316 (1988) and Anderson et al, Cell 47, 1079 (1986)]. Glucocorticoid also modulates the hypothalamic-pituitary-adrenal axis by inhibiting second messenger-induced peptide hormone induction. Recently, Akerb~om et al. (Science 241, 350 (1988)) showed that the hGR
negatively regulates the cyclic AMP-inducible alpha glycoprotein hormone promoter in a steroid and DNA-binding dependent manner. Wild-type expression is exhibited by a promoter of just 168 base pairs (termed alphal68). Basal expression in placental cells is mediated by factors bound to a 36 base pair palindromic cyclic AMP response element (CRE) cooperating with proteins binding to a 25 base pair tissue-specific element (TSE). Expression may be further enhanced through the CRE by the elevation of intracellular cyclic AMP levels. The hGR represses both the basal and cyclic AMP enhanced transcription in a glucocorticoid-dependent ~ashion. ~he transact~ng elements to which the hGR binds have been de~ined and are related to the consensus GRE
2S ~-quence ~or activation. 8imil~r research is reported by Saka~, ot al., Genes and Develo~ment ~, 1144 (1988).

g~y_sl~the Invention The present invention ia the result o~ a thorough analy~i8 Or the structural requirements o~
hormone receptors for repression. ~his analy~is has r-v-aled an absolute requirement ~or the DNA binding do~ain and a role rOr the carboxyl terminus for ropression. Although the DNA-binding domain alone is not ,, ' '. ,~

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sufficient for maximal repression, the addition of polypeptides to the carboxyl terminus or other modifications as described herein at the carboxyl terminus leads to the creation of novel fusion proteins having dominant negative repressor activity.
In accordance with the present invention, we have identified, isolated and characterized the domains of intracellular hormone or hormone-like receptors that can be modified so as to repress trans-activation transcription activity. This information has enabled the further characterization of various receptors of the steroid/thyroid superfamily, both in terms of physical attributes and biological function and effect of various domains, particularly that domain capable of being lS modi~ied to provide repres~ed transcription activity.
The foregoing has in turn enabled the production of novel analog receptors having repressed transcription activation properties.
It has been determined, based upon the information provided herein, that receptors of the steroid/thyroid superfamily contain domains that function in overall trans-activation transcription activity, even though tho three receptor domains, i.e., the DNA-binding, th- llgand-binding, ~nd tho trans-activation tran~cription domains, aro po6itioned independently of on- anoth-r and aro outonomous in runctiOn. In accordance with the present invention, it has been di-covered that the carboxyl terminu~ o~ a given receptor i- that domain re~ponsiblo for modulating the tran~-~ctivation transcription activity of ~aid receptor. Itha~ ~urther been found that the DNA-binding doma~n is a nocessary component in any receptor hereo~ having r-pross-d trans-activation transcription activity.

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-:: .. , , '- . , -.-' :, .-WO ~/143~ ,.~ `}. PCT/US90/03113 ZC~7~49 -8-This invention provides for novel hormone or hormone-like analog receptors wherein the trans-activation transcription activity is repressed. Such novel analog receptors contain a DNA-binding domain, optionally an N-terminal domain and a C-terminal domain that has been altered so as to provide repressed trans-activation transcription activity compared with parental or wild-type receptor. The novel analog receptors hereof may be hybrid receptors wherein the DNA-binding domain, N-terminal domain and C-terminal domain are provided from different sources. For example, the C-terminal domain of the glucocorticoid receptor can be replaced herein by a portion of the C-terminus of the v-erbA protein.
Alternatively, the C-terminal domain of the glucocorticoid receptor can be replaced with at least a portion of a polypeptide such as ~-galactosidase.
The present invention is further directed to the preparation of such novel analog receptors hereof via recombinant DNA technology in all relevant aspects, including a DNA molecule that i8 a recombinant DNA
molecule or a cDNA molecule consisting of a sequence encoding said analog receptor or a C-terminal ~odified domain thereof, reguisite expression vectors operatively harborlng such DNA comprising exprs~sion control elements op-rative in the reco~binant host~ selected rOr the xpr---lon, and rocombinant host cells transfected with uch operative xpres~ion vectora.
~ he present invention is also directed to the U8- Or th- novel analog receptors described herein for id-ntirytng the r ~ponse element and/or ~unction o~ an "orphanN r-ceptor, i.e., a receptor for which the a~oclated response element and/or function is not known.
The present invention is further directed to the u~o Or the novel analog receptors described herein in .. . :~ . . .. .
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_g_ an improved assay system for the determination of a specific wild type receptor, wherein more than one response element present in the assay system is capable o~ interacting with the wild type receptor.
An additional utility for such repressed analog receptors hereof lies in the area of cancer therapy.
Certain cells require augmented levels of hormone in order to become tumorigenic. One example is the elevated estrogen requirement observed in mammary tumors. Indeed, estrogen antagonists, such as tamoxifin, are used in therapy so as tc decrease the amount of estrogen available so that the transformation of normal mammary cells into tumorous cells is inhibited. Alternatively, the analog receptors of the present invention can be used rOr such purpose.

Brief Descri~tion of the Fiaures Figure l provides the amino acid sequence of the v-erbA protein.
Figure 2 is a dose response curve for hGR-mediated negative regulation.
Figure 3 illustrates the repression by carboxyl terminal Su~ion proteins.
Figure 4 illu~trates several expression and report-r con~truct-.
Flgur- S ~hows the structure and activity o~
rat TR~/v-orbA chimeric protein~.
Figure 6 shows a comparison o~ T3 induction o~
CAT activity by thyroid receptor, v-erbA, and hybrid Tr/v-erbA receptors.
Figur- 7 shows the competition o~ TR~ or v-erbA
with RA induced transcriptional activation by RAR~.
Figure 8 summarizes the structure and activity prop-rties o~ several RAR hybrids and mutants.

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Figure g shows the competition of RA induction by RAR~-v erbA fusion protein Figure lO shows the competition of RA induced tran~activation of endogenous RARs in F9 tetracarcinoma ~tem cells Figure ll summarizes the structure and activity of several GR hybrids and mutants ~etailed Description of the Invention In accordance with the present invention, there is provided a trans-repressing analog receptor of the steroid/thyroid superfamily of receptors, said analog comprising ~l) a first amino acid sequence which is a DNA-binding domain, through which 6aid analog is capable o~ binding to a hormone response element of a wild type receptor, and (2) a second amino acid sequence which is positioned at the carboxy-terminal end of the DNA-binding domain, wherein said second sequence is selected from (a) a polypeptide which has at least about 90 % as many amino acids as the ligand binding domain of the carboxy-terminal portion of ~aid wild type receptor; wherein ~aid ~5 polyp-ptid- has le88 than about 60 % amino acid identity relativ- to the carboxy-terminal domaln of said wild type receptor over either (i) the entire length of said polypeptide, if shorter than the carboxy-t-rminal domain o~ said wild typ- r-c-ptor, or ~ii) any of said polypeptide segments having the same length as the carboxy-terminal domain of said wild type receptor or -. ~ . . : . - . . :
, ~ ., . . ,, - . . . -W O 90/14356 ~7049 PC~r/US9OJ03113 (b) at least the 84 carboxy-terminal amino acids of the carboxy-terminal portion of the v-erbA protein as defined by amino acid numbers 313-398 (see Figure 1; Note that the erb-A amino acid numbering will vary by about 255 amino acids depending on whether the gag se~uence (the amino-terminal 255 amino acids) is included for purposes of numbering the amino acids of the erb-A protein) In accordance with another embodiment of the present invention, there is (are) provided expression vector(s) operatively harboring DNA molecule(s) which is (are) recombinant DNA molecule(s) or cDNA molecule(s) encoding the above-described receptor analogs;
recombinant host cell~ transfected with such expression vector~ and cell cultures comprising such cells in an extrinsic support medium In accordance with yet another embodiment of the present invention, there i~ provided a non-human transgenic mammal, having disease ~ymptoms due to the inabillty to respond normally to a steroid or thyroid hormon-, ~aid mammal having at lea~t a ~ubaet of it~
cell~ capable o~ ~xpre~sing an analog receptor ~or one o~
aid hormon~ aid analog r-ceptor having tran~-2~ r pr~ on activity gr-ater than that o~ it~ ;
corr--ponding wild typ- r-¢-ptor and tran~-activation ~ctivlty 1--- than that o~ its corresponding wild type r c-ptor In accordanc~ with ~till another embodim-nt of the pr --nt lnv-ntion, th-re i~ provided a m-thod ~or conv-rting a wild type hormon- r-ceptor into a tran~-r-pr-~-ing an~log r-c-ptor, ~aid method compri~ing replacing the ligand bindlng domain Or ~aid Wild type rec-ptor with at lea~t th- 84 carboxy terminal , :
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W O 90/14356 . P~r/US90/03113 2(~57~)49 amino acids of the verbA protein as defined by amino acid numbers 313-398 (see Figure 1) This latter embodiment of the present invention can alternatively be accomplished by replacing the ligand binding domain of said wild type receptor with a polypeptide which has at least 90 % as many amino acids as at least the ligand binding domain of the carboxy-terminus of said wild type receptor; and wherein said polypeptide has less than about 60 % amino acid identity relative to the carboxy-terminus of said wild type receptor over either , (i) the entire length of said polypeptide, if shorter than the carboxy-terminus of said wild type receptor, or (ii) any segment of said polypeptide having the same length as the carboxy-terminus of said wild type receptor In accordance with a further embodiment of the present invention, there is provided a method for blocking the transcriptional activation by a wild type receptor of a hormone response element in a cell by contacting the cell with an effective amount of an analog receptor a5 described hereinabove In accordancQ with a ~till further embodiment Or the pre~ent invention, there is provided a method for blocklng th- tran-criptional activation by a wild type r-c-ptor Or a hormono response element present in a cell, said method comprising ~ ubstantially deleting the ligand binding domain Or said wild type receptor, (b) operatively linking the modified receptor Or ~tep ~a) to ~t lea~t the 84 carboxy torminal amino acids Or the vç~kA protein as - derined by amino acid numbers 313-398 (~ee , . - . . .: .
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Figure l) to produce a fusion protein, and thereafter (c) contacting said cell with an effective amount of said fusion protein This latter embodiment of the present invention c~n alternatively be a~complished by (a') substantially deleting the ligand binding domain of said wild type receptor;
(b') operatively linking the modified receptor of step (a') to a polypeptide which has at least about 90% as many amino acids as the ligand binding domain of said wild type receptor, wherein said polypeptide has less than about 60% amino acid identity relative to the ligand binding domain of said wild type receptor over either (i) the entire length of said polypeptide, i~ shorter tha~ the ligand binding domain of said wild -type receptor, or -(ii) any segment o~ said polypeptide having the ~ame length as the ligand binding domain oS said wild type r-c-ptor, to produc- a Su-ion prot-in, and therea~t-r ~c) oontacting ~aid cell with an r~-ctive amount o~ ~aid Su~ion protein In accordanc- with another embodiment Or the pre--nt invention, th-ro i~ provided a method Sor id-ntiSying th- re~pon~- el-mont and/or Sunction oS a r-c-ptor Sor which th- a~sociated r--pon~e lement and/or Sun¢tion i~ not known, said m-thod ¢omprising comparing the refiponse oS a te~t sy~tem having known r-~pon~iven-s~ to wild-type receptor to the .~ .

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response of said test system when treated with trans-repressing analog receptor; wherein said trans-repressing analog receptor comprises (1) a first amino acid sequSence which is S a DNA-binding domain, through which said analog is capable of binding to a hormone response element of said wild type receptor, and (2) a second amino acid sequSence which is positioned at the carboxy-terminal end of the DNA-binding domain, wherein said sequSence is selected from (a) a polypeptide which has at least about 90% as many amino acids as the ligand binding domain of the carboxy-lS terminal portion of said wild type receptor; wherein said polypeptide has less than about 60% amino acid identity relative to the carboxy-terminal domain of said wild type receptor over either the entire length of Said polypeptide if shorter than the carboxy-terminal domain o~ said wild typ- rec-ptor, or any egment o~ said polypeptide h~ving tbo ~ame length as the carboxy-terminal domain o~ said wild type receptor; or (b) at least the 84 carboxy-t-rminal amino tscids o~ the carboxy-termin~l portion Or the v-erbA protein as derin-d by amino ~scid numbers 313-398 (~ee Figure l) For ex~ple, th- ~peciric respon~e element~s) with which the orph~n receptor interacts can be d-t-r~sined by 8creening ~ v~riety Or test systems, each WO90/14356 z~7~49 PCT/US90/03113 $L ~ 6 having a single known response element The response element of the specific test system which is activated by the orphan receptor, but which is repressed by an analog receptor derived from the orphan receptor according to the pre~ent invention, is the response element for the orphan receptor Similarly, the function of an orphan receptor can be determined by comparing the response of a test system when contacted with the orphan receptor, relative to the response of the same test system when contacted with a trans-repressing analog of the orphan receptor according to the present invention Differences in the response in the two side-by-side comparisons provide an indication of the functional role of the orphan receptor lS In accordance with yet another embodiment of the present invention, an improvement is provided for use in assay systems responsive to the presence of a specific wild type receptor, wherein more than one response element i8 capable of interacting with said wild type r-ceptor, the improvement comprising inactivating, with re~pQct to said assay, response element(s) which also respond to wild type roceptor~s) other than said specific r-ceptor wherein said response elements are inactivated by adding to ~aid a~say ~y~tem an eSfective amount of a ~S tran--r-pr--~ing analog roceptor for oach of said other r-c-ptor~s), wh-r-ln oach Or said trans-repressing analog r-c-ptor~ comprise~
(1) a ~irst amino acid seguence which i8 a DNA-binding domain, through which ~aid analog is capabl- oS binding to a hormone re~pon-e element of a receptor other than said ~pecific wild type receptor, and - , ~ ,,, :.
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W090/14356 ~ PCT/US90/03113 2~-5~ -9 ~

(2) a seco~d amino acid sequence which is positioned at the carboxy-terminal end of the DN~-binding domain, wherein said sequence is Relected from (a) a polypeptide which has at least about 90% as many amino acids as the ligand binding domain of the carboxy-terminal portion of said receptor other than said specific wild type receptor;
wherein said polypeptide has less than about 60% amino acid identity relative to the carboxy-terminal domain of said receptor other than said ~pecific wild type receptor over either the entire length of said polypeptide if shorter than the carboxy-terminal domain of said receptor other than said specific wild type receptor, or any ~egment of said polypeptide having the ~ame length as t~e carboxy-t-rminal domain of ~id rec-ptor oth-r than sald spec~ric wild typ- r-c-ptort or ~b) at l-a~t the 84 carboxy-t-rminal amino acid- o~ tho carboxy-terminal portlon of th- v-erb~ protein a~ defined by am~no ~cld nu~ber- 313-398 (~ee Figure 1) A~ ~ploy-d her-in, th- t-rm "dominant n-g~tiv-n, wh-n u--d ln refer-nc- to the analog r-ceptorJ
l o~ th- pr-~-nt inv-ntion, r-f-r~ to species which have a ; n-g~tiv- eff-ct on the transcriptional activation ~otivity of th- ~sociat~d re~pon~- element, even ~n the pr---nce of wild-type r-ceptor and it- associated ligand ': ` . ", :, ' .

W O 90/14356 2~7049 PC~r/US90/03113 ~r ~h-',~,~

- Amino acid abbreviations employed in the present disclosure use the following standard single- and three-letter designations, i e Asp D Aspartic acid Ile I Isoleucine S Thr T Threonine Leu L Leucine Ser S Serine Tyr Y Tyrosine Glu E Glutamic acid Phe F Phenylalanine Pro P Proline His H Histidine Gly G Glycine Lys K Lysine Ala A Alanine Arg R Arginine Cys C Cysteine Trp W Tryptophan Val V Valine Gln Q Glutamine Met M Methionine Asn N Asparagine Receptors employed in the practice of the present invention can be prepared by recombinant techniques, by synthetic chemistry, or the like The thus produced receptor, in its various forms, is recovered and purified to a level suitable for its intended use The existence of a superfamily of ligand-inducible tran~-acting ~actors i~ now recognized, including tho~e ~or steroid hormones, retinoic acid and vit~in D3, two ~ubtype~ o~orms) o~ thyroid hormone r-o-ptor~ (t-r~-d ~ and ~), and the like Mutational analy-i~ and ~tructural comparison~ or these hormone r-c-ptor~ has enabled tho identification Or domains r-sponslbl- ~or hormono-binding, DNA-binding and trans-activation o~ g-n- oxpre~sion See Sap et al , Nature 324, 635 (1986), Weinberg-r et al , Nature ~, 641 ~1986) and Evan~, ~g1~ng~ ~Q, 889 (1988) ~he receptors o~ the pre~ent invention are tran~-repressing analogs o~ hormone or hormone-like receptorJ w~ich are re~erred to broadly as members o~ the .
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WO90/143~ ~ 3r, .~ ?,, ~ PCT/US90/03113 Z(~7~49 ~

steroid/thyroid superfamily of receptors, e g , glucocortoid receptor, mineral~corticoid receptor, progesterone receptor, estrogen receptor, estrogen-related receptors, vitamin D3receptor, thyroid hormone receptor, retinoic acid receptor, aldosterone receptor, androgen receptor, and the like Receptors of the present invention include functional equivalents of all of the above, including receptors differing in one or more amino acids from the corresponding parent, or in glycosylation and/or phosphorylation patterns, or in bounded conformational structure The terminology "functional equivalents thereof" refers to trans-repressing analog receptors which differ from the previously described analog receptor(s) with respect to one or more amino acids, insofar as such differences do not lead to a destruction in kind of the basic repressed receptor activity or biofunctionality It will be understood, therefore, that receptors that are known in the art, whether wild-type, hybrids, or functional equivalents as set forth herein, are ~uitable as starting materials for the practice of the present invention As employed herein, the term "expression v-ctor" lncludes vectors which Are capable of expressing DNA -qu-nce- contained th-r-in, where such sequences are op-r~t$~-1y linked to other s-guences capable of tt-cting their expression It is implied, although not alway~ xplicitly stated, that these expression vectors ~ay b- r-pllcabl- in host organi~ms either as episom-s or ~- ~n int-gral part o~ th- chromosomal DNA As mployed her-in, the t-rm "operative," or grammatical equivalents, m-an~ that the respectiv- DNA ~eguences are operational, that ls, work tor their intended purposes In sum, "expression vector~ is given a functional definition, and .. .. . .. .,.. . ~-,:. - .. .. . . : - ~ , . . . ~ -., .. . . . . ,. . -, ............. . ,: . .: ~ . -~ , : : ,, . . . :., ~ .: ... .. : -, - . .- .- -WO90/14356 2C~ 3 Sgo/03113 .:,,i ~. '.. ` .

any DNA sequence which is capable of effecting expression o~ a specified DNA sequence disposed therein is included in this term as it is applied to the specified~sequence.
ln general, expression vectors of utility in recombinant DNA techni~ues are often in the form of "plasmids~ which refer to circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. In the present specification, the terms "plasmid" and ~'vector" are used interchangeably as the plasmid can be a commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
As employed herein, the term "recombinant host cells" refers to cells which have been transfected with vectors constructed using recombinant DNA techniques.
As employed herein, the term "extrinsic support medium" includes those known or devised media that can support cells in a growth phase or maintain them in a viable state such that they can perform their recombinantly harnessed function. See, for example, ATCC
Media Handbook, Ed. Cot- et al., American Type Culture Coll-ction, Rockville, MD (1984). A growth supporting m-dium ~or mammalian c-118, rOr xample~ prefQrably 2S oontains ~ ~rum upplement such a~ fetal cal~ serum or other supplementing component commonly used to facilitate c-ll growth and division such as hydrolysates of animal m-at or m~lk, tis~u- or organ extract~, macerated clots or their extract~, and 80 ~orth. Other suitable medium compon-nts include, ~or example, tran~ferrin, in~ulin and various metals.
The vectors and methods disclosed herein are suitable for use in host cells over a wide range o~
prokaryotic and eukaryotic organisms.

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In addition to the above discussion and the various references to existing literature teachings, reference is made to standard textbooks of molecular biology that contain definitions and methods and means ~or carry$ng out basic technigues encompassed by the present invention. See, for example, Maniatis, et al, Molecular Clonina: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982 and the various references cited therein, and in particular, Colowick et al., Methods in Enzymoloav Vol 152, Academic Press, Inc.
(1987). All of the herein cited publications are by this reference hereby expressly incorporated herein.
Non-human transgenic organisms contemplated by the present invention include rodents (e.g., mice, rats), lS pigs, sheep, lower eukaryotes (e.g., Drosophila, Xenopus), and the like.
Transcriptional activation by the steroid receptors and the thyroid hormone receptors has been found to be dependent on the presence and binding of the respective ligand. However, deletion analysis has disclosed that glucocorticoid, estrogen, and proqesterone receptors lacking the hormone binding domain still r-cognize the ~peciSic response elements and may function ; a- con~titutiv- activator~. Thu~, neither the ligand it~ nor lt- binding domain need to participate dlrectly in DNA recognition.
Th- v-erbA protein has been found to contain an apparently intact DNA-binding domain; however, as a r--ult o~ a~ino-acid changes and a deletion in the carboxy-terminal domain ~re~ative to it~ progenitor, the thyroid hormone receptor), it lac~s the ability to bind thyroid hormone. ~y analogy to mutated steroid hormone r-ceptors, it has been proposed that these mutations, in coniunction w1th th- high l-v-1 Or xpr s-ion, conv-rt i I

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W O 90/14356 ~S70 - PC~r/US90/03113 the thyroid hormone receptor into v-erbA, which is a hormone independent transcription factor.
Utilizing a gel retardation assay, it has now been demonstrated that both v-erbA and the thyroid S hormone receptor recognize and bind to a cognate response element in the absence of ligand. The consequence of this binding is the suppression of a thyroid hormone responsive reporter gene by the non-liganded receptor.
Addition of thyroid hormone results in a 10-100 fold stimulation of transcription from the repressed level.
Surprisingly, v-erbA does not function as an activator as one might expect, but rather as a constitutive repressor of T~ responsive genes. When co-expressed with the thyroid hormone receptor, and in the further presence of thyroid hormone, the v-erbA repression is dominant, blocking hormone stimulated modulation, thereby demonstrating that v-erbA can function as a potent receptor antagonist.
The present invention thus embraces hormone or hormone-like receptor analogs having the ability to repress trans-activation transcription activity of a promoter with which it is associated, or an extrinsic, operativs promoter. Such repression is due, for example, to th- intrin~ic ability o~ the analog receptor to 2~ oomp-titiv-ly bind to a DNA re~ponso element of said pro~ot-r or by th- ability o~ th- analog receptor to co~p-titiv-ly displace other polypeptide~s) that bind to aid DNA response element, or a proximate DNA response lem-nt, thus creating an overall repression of trans-~0 otlYation tran~cription activity compared with that o~lt~ corresponding par-nt or wild-type receptor.

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WO90/14356 -. ~ PCT/USgO/03113 2(:~704~.. `. . ~

EXAMPLES
The following experimental details set forth the methodology employed in identifying, characterizing and preparing particular novel analog receptors. The art ~illed will recognize that by supplying the present inSormation including the location and ma~eup of trans-activation transcription repression domain(s) of a given receptor and how such receptor can be manipulated to produce the novel analog receptors hereof having repressed trans-activation transcription activity, it is not necessary, or perhaps scientifically advisable, to repeat the details described herein to reproduce this work. Instead, they may choose to employ alternative, reliable and known methods. For example, they may synthesize the underlying DNA sequences encoding a particular novel receptor hereof for deployment within ~imilar or other suitable, operative expression vectors and culture systems~ Thus, in addition to supplying details actually employed, the present disclosure serves to enable reproduction of the specific receptors disclosed, as well as others, and fragments thereof, using means wit~in the skill o~ t~e art having benefit of the pre~ent disclosure. All oS ~uch means are included within the nabl-ment and ~cope of the present invention.
Exampl- l: Tran~-ct~on~
Tran~Section~ in JEG-3 human placental cells ar- performed via the calcium phosphate precipitation m-thod described by Delegeane et al, Mol. Cell. Biol., Vol. 7, pp. 3994-4002 ~1987). JEG-3 cells, maintained in Dulb-cco'- modiSied E~gle's medium (DMEM), lO percent de~ined c~l~ bovine serum (CBS), and 0.4 percent glucose are split 24 hours prior to transfection into 5 percent CBS charcoal-stripped serum plus glucose (A~erblom et .. . ... ... ... . . . ..

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al., Science 241, pp. 350-3~3 (1988)). Typically, 2 ~g of reporter plasmid and 4 ~g of receptor plasmid were used along with 2 ~g of a Rous sarcoma virus (RSV)-B-galactosidase construct (Hollenberg et al., Cell 49, pp.
39-46 (1987)) as an internal control for transfection ef~iciency. Dexamethasone and aldosterone (10~ M) were added after calcium phosphate treatment. For the B-galactosidase fusion experiments, the internal control was RSV-luciferase.
Transfections in CV-l cells are also performed via the calcium phosphate precipitation method. CV-l cells are maintained in DMEM supplemented with 5% calf bovine serum and transfected at 30%-50% confluency with a total of 20 ~g DNA. 5 ~g expression plasmid and 2.5 ~g reporter plasmid DNA, together with 2.5 ~g RSV-~-gal as an internal control for transfection efficiency, are cotransfected per 10 cm dish. Transfected cells are grown in 10% resin-treated fetal calf serum [Samuels et al., J. Endocrinoloov, Vol. 105, pp. 80-85 (1979)~ in the presence or absence of 107M 3,5,3' triiodothyronine ($3).
Cells are harvested 40 hours after the addition of T3; ~-galactosida~e and CAT-assays are then performed as de~crib-d in Exa~pl- 2.

Exampl- 2: R-porter A~ay~
chloramphQnicol acetyltransferase ~C~T) assays ar- performed as de~cribed by Hollenberg, et al, ÇçLl, Vol. 49, pp. 39-46 ~1987), but with 25 ~q of total cell xtr~ct protein for 3 hour~ or le~s. Thin-layer chro~atography ~TLC) plates are cut and counted in Econofluor containing 5% dimethyl sulfoxide ~DMS0).
~-Galacto~idase ~-gal) assays are performed as de~cr~bed by Herbomel, et al. Cell, Vol. 29, pp. 653-662 ~1984).

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WO 90/14356 PCI'/US90!03113 2~7~ 24-Example 3 hGR Mediated Negative Regulation To demonstrate hGR-mediated repression of the alphal68 promoter (a pro~oter for the alpha-subunit gene encoding chorionic gonadotropin; which is repressed by GR), a dose response study is conducted of negative regulation of the alphal68-CAT reporter plasmid by the hGR expression plasmid in human placental JEG-3 cells Varying amounts of the hGR expression plasmid (driven by the RSV promoter) and alphal68-CAT (reporter) expression plasmid are cotransfected by the calcium phosphate precipitation method Cotransfections are carried out so as to provide increasing receptor to promoter ratios The resultant transient CAT activity in the presence or absence oS the steroid hormone dexamethasone is then measured Throughout the study, the total amount of transfected RSV promoter DNA is kept constant by substituting an RSV control plasmid, thus controlling for possible titration of transcription factors by RSV DNA
The CAT activity of the reporter construction is measured as described above The alphal68-CA~ reporter plasmid i~
con~tructed a~ de~cribed by Delegeane et al , supra The hGR xpr--~ion plasmid, driven by the RSV
2S promot-r, i- con~tructed ~8 described by Hollenberg et ~1, C-ll, Vol ~9, pp 39-46 ~1987) Control plasmid RSV contains the rat thyroid hormone receptor coding region in the antisen~e dlr-ction, and i~ described by Thompson et al, in Sci-nce, Vol 237, pp 1610-1614 ~1987) Figur- 2 shows the e~Sect oS the transfection o~ hG~ cD~A on reporter gene expression in the presence and absenc~ o~ dexamethasone open circles in the Figure indic~te ~edia w~thout added dexamethasone, while solid : , ;, , -: -. . . . ' -. :. . - -. . . ,, , - , . - .................... : . . . ., , - -' ' '.''' ' ' ` .; ',' ,' ' "'.,,''.., .' . ' ' ' .' ,' "

W O 90~14356 PC~r/US90/03113 2(~,~, ,, ~

circles indicate media with added dexamethasone (107M) In the particular experiment presented in Figure 2, 2 ~g of promoter plasmid is used The arrow indicates the r~tio of receptor to promoter used in subsequent experiments Figure 2 shows that increasing amounts of the receptor expression plasmid yield a correspondingly higher steroid-dependent repression In the absence of receptor cDNA, less than 10 percent of maximal repression is measured Beginning at a receptor to promoter ratio o~ 1 and continuing to a ratio of 5, a plateau of repression activity emerges where more receptor plasmid yields no additional steroid-dependent repression since the amount o~ RSV promoter is held constant, this plateau indic~tes probable ~aturation of the site of receptor action For subsequent experiments, a receptor to promoter ratio of 2 1 is used The steroid-dependent repression varies between 6 and 20 fold with an average o~ 9 fold as typified in Figure 2 This assay can reliably measure as low as 10 percent of wild type hGR
repression.
Figure 3 shows the relative CAT activity Or wild-typ- hGR, trunc~t-d hGR'- and several hGR ~usion prot-in- hGR ~u-ion prot-in- ar- assayed as described 2S ~bo~o For I5~2*, wlld-typ- hGR ~hgrwt), I532* and the IS32~-~-gal~ctosid~ usion, the hormone used is dexamethason-~ ~or the ~usion protein GGM, the hormone u--d i~ aldo~teron-In F~gure 3B, the gen-ral make-up o~ th-~ariou- hGR-d-riv-d protein- i~ illustrated Thus, I582*
i~ ~ truncated hGR h~ving only 582 amino acids; I532* is ~ truncat-d hGR h~ving only 532 amino acids; I532*-~-gal~cto-ida~- is a fusion protein comprising amino acids I-S32 ~ro~ hGR, plu- amino acid~ 8-1025 ~rom ~-.

WO90/143~ ~, PCT/US90/03113 ZC~49-; ~

galactosidase, made by inserting the 3030 bp BamHI LacZ
fragment of p8G-l, a derivative of pSKlos, in frame into the BamHI site of I532 (Casadaban et al., Methods in ~P~ymLoloav Vol 100, 293, Academic Press (1983)); and GGM
is a fusion protein constructed by first introducing an additional XhoI site into both the hGR at nucleotide position I596 (Hollenberg et al., Nature 318, 635 (1985)) and the human mineralocorticoid receptor (hMR) at nucleotide position 2233 (Arriza et al., Science 237, 268 (1987)) and then inserting the appropriate XhoI fragment of the hMR into the hGR. This gave a receptor with amino acids 1-489 of hGR and 671-984 of hMR.
Figure 3B compares the repression activity of the carboxy terminal fusion proteins on the alphal68 promoter to activation on the mouse mammary tumor virus (M~V) promoter. The percentage of wild type activity is calculated by assigning RSV control plasmid as zero activity and the wild type hGR as 100 percent in each experiment, plus or minus the standard error of the mean.
Control 1 and control 2 refer to transfection with RSV
control plasmid. Control 1 plus the next 4 constructions used dexa~ethasone as the stero~d, while control 2 and GGM u~ed aldo~t-rone as the steroid. The numbers on the I532~-~-galactosida-- construct re~er to ~-galactosidase a~lno acld numb-ring ~rom Ca~ad~ban et al., Meth.
Enzymol., Vol. 100, pp. 298-308 ~1983), and the numbers on GCN r-fer to bNR amino acid numbering from Arriza et al., ~u~ra (1987). An n~n indicates that activity is ~ than 10 perc-nt wlld typ- repre~sion activity, "~"
indicat-~ tb~t activity iJ les~ tban 1 percent wild type activatlon activity.
Novel sequence specific repressor~ can be cr-ated by attacbing heterologous protein ~equences to tbe carboxyl terminal side of th- bGR DNA binding domain.

W090/143~ ~ ~ ~5~,~49 PCT/US90/03113 In one case, E coli B-galactosidase (B-gal) can be fused in frame to the carboxyl terminal side of the h~R DNA-binding domain and assayed for regulatory properties On the mouse mammary tumor virus (MTV) promoter, this hybrid functions as a constitutive activator with properties unchanged from that of the parental truncated receptor On the alphal68 promoter, the fusion protein is a constitutive repressor whose activity is dramatically increased when compared to the truncated receptor, I532*
Thus, the addition of a heterologous E coli protein sequence to the DNA binding domain of the hGR is sufficient for generation of a functional transcriptional repressor The results of this study provide several means to distinguish positive and negative regulatory effects o~ the hGR First, the amino terminal domain that contains a potent activator sequence, t ~ is not necessary for trans-repression Indeed, it has been found that deletion of ~ engenders a more potent repressor This argues that even when functioning as a repressor, the amino terminal region of the hGR retains some residual positivo activity The fact that certain modifications of the hGR
produce a receptor that retains normal repressor function but h~ lo-t virtually all positive activation capability d-mon-trat-- that th- proc-~s o~ activation can be m-ohani~t~cally distlnguish-d from that of repression $hi~ observation ~urther indicates that the function of th- DNA-binding domain i~ mor- than simply to locate an ~ppropriate regulatory s-quQnce Moreov-r, the re~ult al80 implies that activation requir-s an additional ev-nt ubseguent to DNA-binding that is apparently not critical ~or n-gative control ... . . . . . ..... . . . . . .

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W O 90/14356 ~ ~ PC~r/US90/03113 . ~

Yet another distinction between activation and repression is that a ~-galactosidase moiety functionally replaces t~e hGR carboxyl terminus only in repression Removal of the carboxyl terminus results in a receptor variant with minimal repression activity Example 4 Thyroid Receptor Mediated Negative Regulation Thyroid receptor (TR) differs from the glucocorticoid receptor (GR) in that the TR is able to bind to its cognate response element even in the absence of its associated ligand In view of these differences, it is of interest to determine if modifications to the TR
analogous to the modifications investigated for the GR
will provide similar trans-repressing derivatives The vectors employed for these studies are prepared a8 follows Expression vector RS-rTR~ is constructed as described by Thompson et al, Proc of the Natl Acad of Sci , Vol 86, pp 3494-3498 (1989) The expression plasmid RS-v-erbA is generated ''' by excising the coding sequence of the cloned gag-v-erbA
gene lVennstrom et al , J Virol Vol 36, pp 575-585 ~1980)~, ~ollowed by appropriate modi~ic~tion of the 5' and 3~ nds and insertlon between the Xpnl and BamHl 2S ~it-- o~ th- voctor pRS-hG ~ [Giguere et al , Nature ,330, 624 ~1987)~ Synth-tic oligonucleotides encod~ng a pallndromlc r~spons- elemQnt [TREp; TCAGGTCATGACCTGA; see Clas~ t al , Cell Vol 54, pp 313-323 (1988)] ~lanked by ~indII~ ad~ptor s-guences are inserted ~nto the unique HlndIII cloning ite in pBL-CAT2 [Luckow et al , ~yQ, i91~a_E~ , 5490 ~1987)~ Plasmids containing one or multipl- copios o~ the ~RE are identi~ied by restriction nzyme mapping and sequQnce analysis Hybrid genes are con~tr,ucted using restrlction sites common to both r,TR~

.. , .. - - .

WO90/143~ z~7~49 PCT/US90/03113 ~ ~, ;; 3~
[Thompson et al , Scie~ce, Vol 237, pp 1610-1614 (1987)~ a~d v-erbA genes [Damm et al , EMB0 J Vol 6, 375 ~1987) and Vennstrom et al , J Virol 36, 575 (1980) A schematic organization of the rTR~ and v-erbA
protein seguences is given in Figure 3A cDNAs encoding thes- proteins are cloned into an RSV expression vector The "DNA" and "TJT4"designations in the Figure refer to the DNA- and thyroid hormone binding domain, respectively The 12 amino-terminal amino acids of chicken c-erbA/TR~ are replaced by part of the retroviral gag-gene, resulting in the synthesis of a P759~o-v-er~
hybrid protein In addition, v-erbA differs from chicken c-erbA/TR~ in 2 amino acids in the DNA binding domain and 9 amino acids plus a 9 amino acid deletion in the hormone binding domain The comparison to rat TR~ shown in Figure 3A reveal~ an additional 17 amino acid differences that are species specific and also found in comparison between chicken and rat TR~ deduced amino acid seguences The numbers on top of the constructs shown in Figure 3A
indicate amino acid positions TR(~154/317) is created by deleting the Pstl 2ragment in the ligand binding domain of rTR~
Replac-ment oS th- rlR~ carboxy-terminus by a Pstl-Xbal ~ragmont Srom a ~--rbA-neo construct ~Sap et al , Nature 324, 63S ~1986)~ g-nerate~ TR~154/317)erbA The pl~-~id- TR~317)-rbA, TR~154erbA and TR~154~317) are g-n-r~t-d by r-in--rting the Pstl fragment~ from either rTR~ or v-~rbA into the unique Pstl ~ite of either plasmid All modificatlon~ are perSormed on a subcloned fragm-nt Srom th- ligand binding domain Or rTR~ and hybrid xpr--~ion con~truct~ ar- g~n-ratsd by r~placing th~ Xba fragm~nt oS RS-rTR~ with ths corresponding chim ric fragm~nts . .. .- , ~ ........................................ : . . .
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WO90/14356 $'~?~ PCT/US90/03113 2C-57~)49 Cos cells are grown in DMEM with 5% T3 free bovine serum and transfected using DEAE dextran [Giguere et al , Cell 46, 645 (1986) ~ . After 36 hours, cells are harvested and extracts prepared as described by Kumar et S al , in Cell, Vol 55, 145 (1988), except that the buffer was 20 mM Hepes (pH 7 8), 0 4 M KCl, 2 mM dithiothreitol (DTT) and 20~ glycerol DNA binding reactions and gel electrophoresis are performed essentially as described by Glass, et al , in Cell, Vol 54, 313 (1988). Aliquots containing 6-10 ~g of total protein are diluted so that the final concentration of RCl is 80 mM, then incubated with 2 ~g of polydeoxycytidylic acid (poly[dC]) for 20 min at room te~perature At this time, 25-50 fmoles of a ~2P-labeled oligonucleotide encoding the palindromic TRE
was ~dded The reaction mixture was incubated at 22 C
~or 30 min and then loaded on a 5% polyacrylamide gel containing 50 ~M Hepes pH 7 8 Competitor DNAs were added prior to the addition of the labeled oligonucleotide ~he DNA binding mutant i95~R was constructed by partial digestion of r~R~ with PvuII and adding a BamHI
linker (12mer) to restore the open reading frame The position o~ the linker was veri~ied by restriction enzyme mapping ~nd equence an~lysis Th~ DNA binding mutant i95(154)erbA was con~truct-d by partial dig-stion of TR(154)erbA with PvuII and adding a BamHI linker (12mer) to restore the open re~ding ~rame The position o~ the linker w~s v-ri~ied by restrlction enzyme mapping and sequence ~naly~i-rirst, tbe transcriptional activity o~ both the rat thyroid hormone ~ receptor (rTR~) [~hompson, et al , Science ~1~, 1610 (1987)] and the v-erbA oncogene product i- asses~ed by determining their ability to regulate WO90/14356 2~57~49 PCT/US9o/03113 ~ ? ~ l t '~

expression of thyroid hormone responsive reporter genes (see Fig 4A) The constructs shown in the Figure contain oligonucleotides corresponding to previously identified thyroid hormone response elements ~Glass et al , Nature 329, 738 ~1987) and Glass et al , S~ll 54, 313( 1988)~ lin~ed to a thymidine kinase-chloramphenicol acetyltransferase (tk-CAT) fusion gene [Luckow et al , Nuc Acids Res 15, 5490 (1987)~ (see Fig 4~) The reporter gene constructs used, and shown in Figure 4B, contain oligonucleotides encoding the respective T3 response elements (TRE) inserted into the HindIII site (H) upstream of the tk promoter-CAT construct Expression plasmids encoding rTR~ or v-erbA under the transcriptional control of the RSV long terminal repeat are cotransfected with one of the reporter plasmids into CVl cells, which lack significant levels of endogenous TR
CV-l cells are co-transfected with the reporter construct tk-CAT, tk-TREp-CAT or tk-TRE-C~ CAT; the respective expression plasmid (RS-rTR~ or RS-v-erbA) and the internal reference plasmid RSV-~GA~ In the control experiments, a construct carrying the rTR~ coding ~eguences in reverse orientation (RS-3'-5') is used Tr~ns~ection oS tk-TREp-CAT or the parental 2S v-otor tk-CAT r--ult~ in a high b~al level of CAT
actlvlty that i~ only marginally stimul~ted by th-addltlon o~ thyrold hormone In contrast, cotransfection wlth the TR expression vector RSV-rTR~ result~ in marked ~ cts on tk-TREp-CAT expres~ion In the ab~ence o~
thyrold hor~one, ther- 1~ ob~erved an 80% decr~a~- ln ba-al CA~ actlvity (re~erred to a~ "low basal l-v-l activityn), indicating that TR expres~ion provoke~ a llgand-lndependent inhibitory e~ect on transcription Addltion or a thyroid hormone, triiodothyronine ~T3~, to a . . .

.. . . .. .. . . - . . ~, .

WO90/14356 ~ PCT/US90/03113 ~ 7()49 `

final concentration of loO nM results in a 20 fold stimulation of tk-TREp-CAT This corresponds to a 3-5 fold stimulation over the basal level of activity obt~ined in the absence of TR expression S The regulatory function of the rTR~ is not restricted to the TREp; the TRE from the rat growth hormone gene is also able to sustain hormone-independent and dependent transcriptional responses This functional assay enables a direct determination of the putative transcriptional activity of the v-erbA oncogene product Since v-erbA has lost its ability to bind thyroid hormone but retains an intact DNA binding domain, it is reasonable to expect that it would function as a constitutively active TR Unexpectedly, cotransfection Or v-erbA with either reporter plasmid does not stimulate the transcription, but rather resembles the negatlve regulatory effects of rTR~ in the absence of hormone Thus, in cells expressing v-erbA, CAT activity is reduced by 80% Srom the high basal level, and can not be relieved by the addition o~ T3 To identiSy the eSfect oS different mutations in v-erbA on the altered properties oS this protein ~r-latlve to its progenitor, TR), ~nd to Surth-r dissect th- proce-~e~ o~ activat$on and ropresaion, chimeric 2S r-c-ptor- Or th- v--rbA oncogene [8e- Damm et al , EMB0 J Vol 6, pp 375-382 (1987) ~nd Vennstrom et ~1 J
Virol Vol 36, pp 575-585 (1980)], and the rat ~R~
[Thompson et al , (1987), supra] are constructed A
ch-matlc repre~entation o~ the structure Or the rat TR~/v-erbA chi~-ric prot-in~ i~ present-d in Figure 5A
Numb-rg on top Or each construct indicate amino acid po~itions Th- black bar indicates the d-letion oS 9 ~mino acids in v--rbA, r-sulting in ~usion proteins o~
401 a~ino acid- co~par-d to the 410 a~ino acid oS the ~: . .` ., . . ,, : ', :. . ' ,` :' `' '~'" ':. ` ' ' ' ' ' ~ ' .' ".., , ' .~' ' ~ ' ': ' .-` . .
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W090/l4356 PCT/US90/03113 2C57~)49 protein with intact carboxy-terminus In Figure 5B, positive and negative regulation of CAT activity from tk-TR~p-CAT is shown, using the indicated expression vector6 The histogram summarizes the average values of 2-6 independent transfection experiments Stippled bars ln the histogram indicate the presence of no hormone;
while strlped bars in the histogram indicate the presence o~ 100 nM T3 From amino acids 154 to 410, v-er~A differs from rTR~ in 26 amino acids and a deletion of 9 amino acids close to the car~oxy-terminus Swapping this region of rTR~ with v-erbA qives rise to the hybrid TR(154)erbA (Fig 5A), whose properties are virtually identical to the v-erbA homologue (Fig 5B) Thus, the mutations in the DNA binding domain and flanking amino acids are not crucial to the v-erbA phenotype Substitution of an internal region of rTR~
ligand binding domain (at amino acids 154-316), creates a T3 responsive hybrid (TR(154/316)erbA) that functions like the natural receptor In contrast, replacement of the carboxy-terminal 93 amino acids of rTR~ with the corresponding seguence of v-erbA, containing the 9-amino-acid doletion and additional 11 amino acid differences, yield~ the bybrid protein TR(317)erbA with suppressor prop-rtie~ identic~l to th- viral oncogene product To xamin- the ~rect of v-erbA on the function ot ndog-nou- thyro~d receptor, cotran~ection studies in CVl c-ll~ are per~ormed The reporter gene tk-TREp-CAT
~0 S ~g) i~ cotrans~ected into CV-l cells with 1 ~g of rTR~ xpr-~ion v-ctor and the internal control plasmid RSV-~-GAL In ~ddition, ~ 10-~old exces~ (10 ~g) o~ the xpr-~-ion pl~Jm~d~ indicated in Figure 6 iB
cotr~n-r-cted with rTR~ In the run designated by (-), 10 ~g o~ th- control plasmid, RS-3'-5', is used The .. . . . .......... . .

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woso/l43s6 PCT/US90~3~13 2C57~)49 ~ ~ ~ 1 s- ` ! t average values of 2-6 independent transfection experiments are shown in the histogram in Figure 6 All experiments are performed in the presence of 100 nM T3 As shown in Figure 6A, a 10-fold molar excess o~ v-erbA reduces by 90% the thyroid hormone dependent induction Or the reporter gene transcription by rTR~
The hybrid constructs TR(154)erbA and TR(317)erbA also exhibit v-erbA-like activities, provoking a virtually complete repression of the T3 and rTR~ induced stimulation of the reporter gene The activity is, however, dependent on the presence of an intact DNA-binding region since the DNA-binding domain mutant i95(154)erbA, fails to compete Similarly, T3 induction is blocked by v-erbA and the chimeric constructs when the TREp is placed in the context o~ the MTV promoter One prediction of these re~ult~ i5 that an inverse relationship exists between the activity of the TR and the concentration of the competitor product To examine this prediction, varying molar ratios of TR(154)erbA and the rTR~ expression vector were cotransfected and hormone responsiveness was asses~ed ~hus, the reporter gene tk-TREp-CAT (0 5 ~g) wa8 cotransfect-d into CV1 cells with 1 ~g of expression v-ctor RS-rTR~ and increasing guantitie~ o~ the non-hormon- binding compotitor TR(154)erbA The amounts of RSV-promot-r w~- h-ld con~tant in ~11 transfection~ by th- addition o~ the control pl~-mid, RS-3'-5' Shown are the av-rage v~luo- o2 three independent transfection xp-rimont~ Cell~ were grown in the presence of 100 nM
T~ -A~ one might expect, increasing quantities o~~R~lS4)-rbA lead to docreasing activity o~ the rTR~ (see Fig. 6B). In this as~ay, ven sm~ll amounts of .. ...

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- . . , .. -WO90/143~ PCT/US90/03113 Z(~57 `. ~ ?
-35~
TR ( lS4 )erbA are potent, with a 3 1 plasmid ratio completely blunting the hormone induced response Although it is generally assumed that the ability o~ a hormone receptor to bind DNA is ligand dependent ~Evans, Science 240, 889 (1988)], the results pre~ented herein, as well as previous observations [Lavin et al , J Biol Chem 263, 9418 (1988)] challenge this convention The downregulation of transcription from the responsive promoter is presumably a consequence of the ability of the receptor to recognize and bond to its cognate response element in the absence of hormone Two pieces of evidence support this proposal First, an intact DNA binding domain is necessary for both the gel retardation experiments and the observed transcriptional lS effects Second, in the absence of the response element no significant repression is observed, whereas a tandem copy of the ~RE potentiates both positive and negative transcriptional effects Although positive synergism has been observed by glucocorticoid and estrogen receptors and their respective response elements tSchule et al , ~QiQnÇ~ , 1418 ~1988) and Strahle et al , EMB0 J 7 3389 ~1988)] the negative synergism observed here i~
without apparent pr-cedent 2~ Exampl- S R-tinoio A¢id Receptor Mediated Negative R-gulation To investigate the possible positive and/or n-gat$v- intoraction o~ RAR~ with TR~ and v-erbA, CV-l c-lls ar- cotran9rect-d with the RAR~ and reportor pla-mid in th- pre~onc- o~ TR~ or v-erbA ~Fig 7) In the Flgure, TR~ or v--rbA compete with the RA induced transcriptional ~ctivation by RAR~ RAR~ expression v-ctor ~1 0 ~g) i~ cotr~n~ected with reporter pl~smid ~NIV-TREp-CAT (1 0 ~g) along with TR~ ~5 0 ~g) or with v-.. . . .. .. . . ..

WO90/143~ PCT/US90/03113 Z~7049 -36-erbA (5 0 ~g) Plasmids are introduced into CV-1 cells (maintained in DME medium supplemented with 5% calf bovine serum), 5 x 1~5 per 10 0 cm dish, by calcium phosphate precipitation The expression plasm~ds, RAR~ TR~ and v-erbA
are under control of the RSV promoter as previously described by Damm et al in Nature, Vol 339, 593 (1989) and ~mesono et al, in Nature, Vol 335, 262 (1988) The cells were cultured for 36 hours in Dulbecco-modified Eagle medium containing 10% resin-charcoal stripped calf bovine serum [Samuels et al, Endocrinology, Vol 105, 80 (1979)~ with or without hormone as indicated and subsequently prepared for CAT activity through three cycles of freeze thawing as described by Gorman et al, Mol Cell Biol , Vol 2, 1044 (1982) An aliquot of the cell extracts was normalized by ~-gal activity prior to carrying out the CAT assay The hormone is added at a final concentration of lOOnM These data are the average of 4 independent experiments A 5 fold molar excess of TR~ reduces the RA
dependent induction in CAT activity by 90~ No inhibition of transcription i8 observed when both T3 and RA are added ~imultaneously Therefore, in the absence Or T3, th- T~ prevent~ th- RA induced activation of gene xpr-~-ion by th- RAR~, wher~as in the presence o~ T3, on- ob~-rv-~ activation, presum~bly through the TR~
Simllar ob-ervations for the TR inhibition of RAR
activation have recently been observed by Graupner et ~l , N~ture, Vol 340, 653 (1989) and 8rent et al , The N-w Biologi-t, Vol 1, 329 (1989) A ~imilar competition of the RA induced gene activation i8 observed when v-erbA
i~ cotransfected along with the RAR~
To investigate whether one could confer a domin~nt n-gativ- e~fect directly onto the RA~, mutant ... _ . .. . . . ~ . ., :
; : . . ... , ,' ' :' ;, :' .

.. : ~ .
. .
- ,:: ., , -- , -WO90/14356 z~7~49 PCT/US90/03113 RAR~s are created consisting of a series of RAR
truncations as well as a hybrid RAR~-erbA fusion Since the RAR functions as a ligand dependent transcription ~actor, A mutant that contains an alteration or deletion in the l~gand binding domain but with an intact DNA
binding domain may confer a dominant negative phenotype In fact, the results set forth in Example 4 above demonstrate that replacing the ligand binding domain, located in the carboxyl terminus of the TR~, with the carboxyl terminus of v-erbA results in a hybrid TR~-erbA
molecule that functions as a dominant negative mutant of the TR~ Therefore, a similar fusion between RAR~ and v-erbA was constructed by replacing the ligand binding domain Or the RAR~, located in the carboxyl terminal, with the carboxyl terminus of v-erbA (Fig 8) The hybrid RAR~-erbA fusion was constructed by removing the N-terminus and DNA binding domain of ~R317-erb-A, see Damm et al, Supra, and replacing it with the corresponding N-terminus and DNA-binding domain the RAR~ (thus creating a RAR~-erbA hybrid protein) Truncation mutants give the la~t amino acid o~ RAR~ before the insertion of a tr~n~l~tion ~top ~ign~l ~t the indic~tod position The RAR 185 ~nd 203 trunc~tion mut~nt~ wero constructed by u~inq unigu- r-~triction ~ites pr-s-nt in th- wild type r-oeptor wh-r-~ RAR lS3 w~s con~tructed by inserting top codon ~t position 153 RAR~ rollow~ng creation of unique Xho site at thi- position Retinoic ~cid w~s ~dded ~t a rin~l concentr~tion Or lOOnM
In th- tr~n-~ctiv~tion exp-riments, ~ po~itiv-r--pon~- corre-pond- to ~ 2S rold incre~-e in CAT
activity upon r-tinoic ~oid ~ddition A n-g~tive r-~pon~- correapond- to no tr~ns~ctiv~tion upon retinoic ~cid ~ddition In th- competition experiment~, ~
po-itiv- r-~pons- corre~ponds to gre~ter th~n ~n 80%

.. . . , ~ . . , ., . ,, . . ,. ....... . , . ,. : .

W O 90/14356 ~ P~r/US90/03113 2~-57049`- . ~

antagonism of the retinoic acid induced transactivation, while a negative response corresponds to no competition upon retinoic acid addition.
The RAR~-erbA protein does not act by itself as a transcriptional activator whether hormone is present or absent. However, when the RAR~-erbA fusion is cotransfected with the RAR~, it functions as a RAR~
antagonist. See Figure 9.
CV-l cells are cotransfected with RAR
expression vector (1.0 ~g), ~MTV-TREp-CAT reporter plasmid (see Umesono et al, SuDra) (5.0 ~g), reference plasmid (5.0 mg), along with increasing amounts of RAR~-erbA and carrier plasmid up to 20.O mg. The addition of RSV-promoter is held constant in all transfections with the lS addition of the carrier plasmid. The % maximal response refers to CAT induction observed with the RAR wild type receptor in the presence of 100 nM RA. This activation corresponds to a RA induction of 25 fold. All data are the average of 4 independent experiments.
RAR~-erbA is also capable of antagonizing the RA induced activation of the RAR~ and RARy. Therefore, replacement of the carboxyl terminus of RAR~ with the carboxyl terminus o~ v-erbA confers a dominant negative phenotype onto the RAR~. In contra~t, RAR mutants con~i-ting oS a -ries oS carboxyl torminal truncations do not act a~ tran-criptional activators when transfected by th-m~elv-s nor do they Sunction aQ RAR~ competitors when transSected in con~unction with the RAR~. These data on th- RAR truncation~ are in part at odds w~th the r-o-nt ob~orvation of E~pes-th et al., Genes and Dev., Vol. 3, 1647 ~1989), who reported that an RAR~ mutant, virtually identical to RAR-185, gave rise to a small percontago oS stable F9 clones that did not differentiate in re~ponse to RA and thorofore, hypothesized that this , . . , : :

. .. . . .
- . . . . . . . .
' ~ ' . ~, ' ' ' :
.. . . . . ..
. .
'' . ' 2(~7~)49 `
-39~ '}~
RAR truncation mutant functioned as a dominant negative RAR.
The properties of the RAR~-erbA fusion along with the v-erbA and TR~, are further investigated by examinlng their ability to repress/antagonize an endogenous RAR(s) F9 cells have been established as a cellular model for RA dependent differentiation They contaln receptors for the ~,~, and y subtypes of the RAR
family; RAR~ and RARy are present in undifferentiated stem cell whereas RAR~ is induced upon RA treatment See, for example, Strickland and Mahdavi, in Cell, Vol 15, 393 (1978); Sporn et al, in The Retinoids, Vol 1-2, Academic Press (Orlando, FL, 1984) and Hu and Gudas in Mol Cell Biol , Vol 10, 391 (1990) Transfection of elther RAR~-erbA, TR~ or v-erbA into F9 cells results in a ~trong inhibition Or the RA induced transactivation (Fig 10) F9 cells (maintained in DMEM supplemented with 10% C~S) are transfected by the calcium phosphate method with the reporter ~MTV-TREp-Luc (5 0) ~g), 5 0 mg of either the control plasmid (RSV-CAT), or RSV-TR~, RSV-verbA or RSV RA~-erbA 5 0 ~g of reference plasmid and 5 0 ~g Or carrier plasmid Cells are cultured for 24 hour~ in the presence or ~bsence of 100 nM RA as indicated The r-porter construct is exactly the same as 2S ~MTV-TREp-CAT exc-pt that the gene ncoding firerly lucl~-ra~ D W-t et al , Mol Cell ~iol , Vol 7, 72S ~1987)~ ha- b-~n substituted for CAT in the reporter pla~mid C~ wer- cultured for 24 hour~ rollowing the addition of th- hormone and ~ubseguently harvested and a--ay-d rOr lucif-ra~- a8 described by Hollenb-rg et al, ln C-ll, Vol SS, 899 ~1988) Data are th- av-rag- of 4 lnd-pendent m-a-urem-nt~

.. . . .

WO90/143~ ~O; PCT/US90/03113 2~-57049 ~40-Example 6 Glucocorticoid Receptor-v erbA and GR-~Gal Fusion Proteins The above observations that the RAR~-erbA
rusion protein ~unctions as a dominant negative RAR, in con~unction with the ability of TR~-erbA hybrid to ~unction as a TR~ inhibitor, cuggests that steroid-erbA
hybrid receptors may provide a general approach for creating specific hormone receptor antagonists Therefore, a fusion between GR and erbA was created (GR-erbA, Fig ll) by substituting the carboxyl terminus of v-erbA for the ligand binding domain of the GR
In the Figure, wild type GR is shown at top with numbers indicating amino acid positions The DNA
and ligand binding domains are also indicated The hybrid GR-erbA fusion is constructed by removing the ligand binding domain of the GR and replacing it with the carboxyl terminus of v-erbA, similar to the RAR-erbA
fusion protein described above ~he three truncation mutants give the last amino acid before the carboxyl terminal non~ense peptides For GR-532~gal, E. colf ~gal was ~used in frame to position 532 of the GR as described by Oro et al in Cell, Vol 55, llO9 ~l988) It encodes a prot-in that xpre~es ~-galactosidase as well as the glucooorticoid r-c-ptor properties reported by Oro et al, 2S Supra, For th¢ tran~activation experiment~, CV-l cells ar- cotran~ect-d with expression vector (l O ~g), the report-r, MTV-Luc ~5 0 ~g), RSV-CAT (5 0 ~g) as th-int-rnal control and carri-r pla~id up to a total o~
20 0 ~g Data i- r-ported a~ % maximal re~pon~e and r-~er~ to lucir-rase lnduction observ-d with the GR wild typ- in th- pr-~enc- o~ the ~ynth-tic glucocorticoid, dexam-tha~on- at l x l0~M Thi~ activation corresponds to an induction o~ 3000 ~old . .: . . .` ~ ' ' ' .. ~'. ., . ' 6 Z~57~4~ PCT~US90~03ll3 For the competition experiments, CV-1 cells are cotransfected with 1 0 ~g of the GR expression vector, 5 o ~g of MIV reporter, 5 0 ~g of competitor along with 5 0 ~g of RSV-CAT as the internal control and carrier plasmid up to a total of 20 0 ~g Dexamethasone was added at a final concentration of 1 x 10~ M The response refers to a % antagonism of luciferase activity with the wild type receptor in the presence of a nonspecific competitor being equal to 0~ All data in Figure 11 are the average of four independent experiments The fusion protein does not activate a glucocorticoid responsive reporter gene when glucocorticoida are present or absent However, when GR-erbA i5 cotransfected with GR, it functions as a GRantagonist; a 5-fold molar excess of GR-erbA reduces by 87% the dexamethasone induction of a reporter gene For comparison, the properties are examined of a series of GR truncation mutants to act as dominant negative competitors as well as transcriptional activators Truncations in the carboxyl terminus result in ~ mut~nt receptor that either does not ~ctivate transcriptlon ~GR487), or result in ~ut~nts that ~re con-titutlv-ly ~ctiv- r-c~ptors ~GR515, GR532) When the 2S Gr truncation- ~re cotran-~-ct-d ~long wlth the wild type Gi- th-y hav- lther no lnhlbitory e~ect or ~t best a ~lig~t ~uppr-s~lve e~ect upon dexamethasone induced tr~n-~ctiv~tion To xamln- whether th- propertie- exert-d by pl~cing the carboxyl t-rmlnu8 o~ GR could be ~ub~tltut-d by anoth-r polypeptide, B-Gal w~s ~u-ed in ir~me to the c~rboxyl t~rminus o~ th- GR at posltlon 532 Thl8 GR-S32pC~l ~u~ion protein has previously been ~hown to iunction ~ ~ neg~tive regul~tor o~ GR tr~nscription ~See .. . , . . ., , . , - . . -, ., . .:

. ~ , . . . , . -: .
!. . .;,;' . ~ . ' : : , ` ' .
~ ~ ... . .. .

~' .

W090/14356 2 ~ ~7 04 g ~ PCT/US90/03113 . ,; ~

Example 3) However, this ~-Gal fusion protein is constitutively active similar to the parental truncation and it reduces the GR activation by only 60% Therefore, the ability and usefulness of the GR truncations and ~-Cal ~uslons to act as dominant negative repressors isdi~inished primarily by their constitutive activity as well as their weak ability to act as GR competitors In contrast, GR-erbA is the only mutant receptor that contains no transcriptional activity in the absence of ligand and functions to block the dexamethasone induced transactivation In addition, GR-erbA is able to act as a very potent competitor against a constitutively active GR receptor, such as GR532, and further supports its potential to function as a dominant negative inhibitor ~he foregoing description details specific methods that can be employed to practice the present inVQntion Having detailed specific methods initially used to identify, isolate, characterize, prepare and use the receptors hereof, and a further disclosure as to specific entities, and ~eguences thereof, the art skilled w~ll well enough know how to devise alternative reliable ~Qthods ~or arriving at the same information and for extending th~ 8 in~ormation to other intraspecies and int-r~p-ci-~ related receptor~ ~hus, however detailed th- ~or-golng may app-ar in text, it should not be oon-tru-d a- llmiting th- ov-rall ~cope hereo~; rather, th- amblt o~ the pre~ent invention i~ to be governed only by the law~ul con~truction of the appended claims .

- . ;:, . . .: . : .: -: ... . .
,.t,., ~r. ,. ' ~ ~

Claims (27)

CLAIMS:

1. A trans-repressing analog receptor of the steroid/thyroid superfamily of receptors, said analog comprising:
(1) a first amino acid sequence which is a DNA-binding domain, through which said analog is capable of binding to a hormone response element of a wild type receptor, and (2) a second amino acid sequence which is positioned at the carboxy-terminal end of the DNA-binding domain, wherein said second sequence is selected from:
(a) a polypeptide which has at least about 90 % as many amino acids as the ligand binding domain of the carboxy-terminal portion of said wild type receptor; wherein said polypeptide has less than about 60 % amino acid identity relative to the carboxy-terminal domain of said wild type receptor over either (i) the entire length of said polypeptide, if shorter than the carboxy-terminal domain of said wild type receptor, or (ii) any segment of said polypeptide having the same length as the carboxy-terminal domain of said wild type receptor, or (b) at least the 84 carboxy-terminal amino acids of the carboxy-terminal portion of the v-erbA protein as defined by amino acid numbers 313-398 (see Figure 1);
provided that when said wild type receptor is a glucocorticoid receptor, said second amino acid sequence.

2. An analog receptor according to claim 1 wherein said hormone response element is operatively associated with a promoter which is subject to trans-repression.

3. An analog receptor according to claim 1 having substantially no transcriptional activation activity in the presence or absence of ligand.

4. An analog receptor according to claim 1 wherein said first amino acid sequence is the DNA-binding domain of said wild type receptor.

5. An analog receptor according to claim 4 wherein said wild type receptor is selected from a retinoic acid receptor, a thyroid hormone receptor, a vitamin D3 receptor, a glucocorticoid receptor, a mineralocorticoid receptor, an estrogen receptor, an estrogen-related receptor, an aldosterone receptor, an androgen receptor, or a progesterone receptor.

6. An analog receptor according to Claim 5 wherein said DNA-binding domain is derived from: 1) a glucocorticoid receptor, 2) a thyroid receptor, or 3) a retinoic acid receptor.

7. An analog receptor according. to Claim 6 wherein said DNA-binding domain is derived from the human glucocorticoid receptor.

8. An analog receptor according to Claim 6 wherein said DNA-binding domain is derived from the thyroid receptor.

9. An analog receptor according to Claim 6 wherein said DNA-binding domain is derived from the retinoic acid receptor.

10. An analog receptor according to Claim 1 wherein said first amino acid sequence is derived from said wild type receptor, and wherein said analog receptor has substantially no transcriptional activation activity in the presence or absence of ligand.

11. An analog receptor according to Claim 10 wherein said carboxy-terminal domain is obtained from a polypeptide.

12. An analog receptor according to Claim 11 wherein said polypeptide is at least a portion of the beta-galactosidase polypeptide.

13. An analog receptor according to Claim 10 wherein said carboxy-terminal domain comprises at least the C-terminal most 84 C-terminal amino acids from the v-erbA protein.

14. A process for preparing an analog receptor according to Claim 1, said process comprising:
expressing, in a recombinant host cell, transfected DNA encoding said receptor.

15. A process according to Claim 14 further comprising:
recovering and purifying said analog receptor to enable its use in assays that measure extrinsically induced biofunctionality of said analog receptor.

16. A DNA molecule that is a recombinant DNA
molecule or a cDNA molecule encoding an analog receptor according to Claim 1.

17. An expression vector operatively harboring DNA according to Claim 16.

18. A recombinant host cell transfected with an expression vector according to Claim 17.

19. A cell culture comprising cells according to Claim 18 and an extrinsic support medium assuring the viability of said cells.

20. A non-human transgenic mammal, having disease symptoms due to an inability to respond normally to a steroid or thyroid hormone, wherein at least a subset of the cells of said mammal are capable of expressing an analog receptor for one of said hormones, and wherein said analog receptor has trans-repression activity greater than that of its corresponding wild type receptor and trans-activation activity less than that of its corresponding wild type receptor.

21. Method for converting a wild type hormone receptor into a trans-repressing analog receptor, said method comprising:
replacing the ligand binding domain of said wild type receptor with at least the 84 carboxy terminal amino acids of the verbA protein as defined by amino acid numbers 313-398 (See Figure 1).

22. Method for converting a wild type hormone receptor into a trans-repressing analog receptor, said method comprising:
replacing the ligand binding domain of said wild type receptor with a polypeptide which has at least 90% as many amino acids as the ligand binding domain of the carboxy-terminus of said wild type receptor; and wherein said polypeptide has less than about 60% amino acid identity relative to the carboxy-terminus or said wild type receptor over either:
(i) the entire length of said polypeptide, if shorter than the carboxy-terminus of said wild type receptor, or (ii) any segment of said polypeptide having the same length as the carboxy-terminus of said wild type receptor.

23. A method for blocking the transcriptional activation of a hormone response element in a cell, said method comprising contacting said cell with an analog receptor according to claim 1.

24. A method for blocking the transcriptional activation by a wild type receptor of a hormone response element present in a cell, said method comprising (a) substantially deleting the ligand binding domain of said wild type receptor, (b) operatively linking the modified receptor of step (a) to at least the 84 carboxy terminal amino acids of the verbA protein as defined by amino acid numbers 313-398 (see figure 1) to produce a fusion protein, and thereafter (c) contacting said cell with an effective amount of said fusion protein.

25. A method for blocking the transcriptional activation by a wild type receptor of a hormone response element present in a cell, said method comprising (a) substantially deleting the ligand binding domain of said wild type receptor, (b) operatively linking the modified receptor of step (a) to a polypeptide which has at least about 90% as many amino acids as the ligand binding domain of said wild type receptor, wherein said polypeptide has less than about 60% amino acid identity relative to the ligand binding domain of said wild type receptor over either (i) the entire length of said polypeptide, it shorter than the ligand binding domain of said wild type receptor, or (ii) any segment of said polypeptide having the same length as the ligand binding domain of said wild type receptor to produce a fusion protein, and thereafter (c) contacting said cell with said fusion protein.

26. A method for identifying the response element and/or function of a receptor for which the associated response element and/or function is not known, said method comprising:
comparing the response of a test system which is responsive to wild type receptor when said test system is treated with:
(I) wild type receptor, or (II) trans-repressing analog receptor, wherein said trans-repressing analog receptor comprises:
(1) a first amino acid sequence which is a DNA-binding domain, through which said analog is capable of binding to a hormone response element of said wild type receptor, and (2) a second amino acid sequence which is positioned at the carboxy-terminal end of the DNA-binding domain, wherein said second sequence is selected from:
(a) a polypeptide which has at least about 90 % as many amino acids as the ligand binding domain of the carboxy-terminal portion of said wild type receptor; wherein said polypeptide has less than about 60 % amino acid identity relative to the carboxy-terminal domain of said wild type receptor over either (i) the entire length of said polypeptide, if shorter than the carboxy-terminal domain of said wild type receptor, or (ii) any segment of said polypeptide having the same length as the carboxy-terminal domain of said wild type receptor; or (b) at least the 84 carboxy-terminal amino acids of the carboxy-terminal portion of the v-erbA protein as defined by amino acid numbers 313-398 (see Figure 1).

27. In an assay system for the determination of the presence of a specific wild type receptor, wherein more than one response element is capable of interacting with said wild type receptor, the improvement comprising inactivating, with respect to said assay, response element(s) which also respond to wild type receptor(s) other than said specific wild type receptor; wherein said response elements are inactivated by adding to said assay system an effective amount of a trans-repressing analog receptor for each of said other receptor(s) wherein each of said trans-repressing analog receptors comprises:
(1) a first amino acid sequence which is a DNA-binding domain, through which said analog is capable or binding to a hormone response element of a receptor other than said specific wild type receptor, and (2) a second amino acid sequence which is positioned at the carboxy-terminal end of the DNA-binding domain, wherein said second sequence is selected from:

(a) a polypeptide which has at least about 90 % as many amino acids as the ligand binding domain of the carboxy-terminal portion of said receptor other than said specific wild type receptor;
wherein said polypeptide has less than about 60 % amino acid identity relative to the carboxy-terminal domain of said receptor other than said specific wild type receptor over either (i) the entire length of said polypeptide if shorter than the carboxy-terminal domain of said receptor other than said specific wild type receptor, or (ii) any segment of said polypeptide having the same length as the carboxy-terminal domain of said receptor other than said specific wild type receptor; or (b) at least the 84 carboxy-terminal amino acids of the carboxy-terminal portion or the v-erbA protein as defined by amino acid numbers 313-398 (see Figure 1).