CA2004339A1 - Novel receptors: their identification, characterization, preparation and use - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

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

Description

NOVEL RECEPTORS: THEIR IDENTIFICATION.
CHARACTERIZATION. PREPARATION AND USE
Related A~lications Reference i8 made to Patent Cooperation Treaty International Application Publication No.
WO 88/03168 and European Patent Application Publication No. 0 325 849. These published applications refer in various respects to hormone receptors and compositions thereof, and to methods for their preparation, and use, particularly in novel assay systems. The entire disclosures of both of these published applications are hereby incorporated herein by reference.

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200~339 . ~ .

Field of the Invention The present invention relates generally to the identification and characterization of certain polypeptide sequences that function as transcription trans-activation domains, and to their preparation and use, particularly in the preparation of novel intra-cellular hormone or hormone-like receptors, for example, steroid receptor polypeptides, thyroid polypeptides and retinoid polypeptides including those of the human species, where advan~age is provided in terms of trans-activation transcription initiation activity by augmenting the effect of said domains.
More particularly, the present ~nvention is directed to such novel receptor polypeptides, wherein the transcription trans-activation domains have been augmented in effect so as to produce novel entities that exhibit increased transcriptlon initiation activity surprisingly super~or to the parent molecule.
Novel aspects relating to the preparation of such transcription trans-activation molecules, including novel DNA isolates encoding same and the transcription trans-activation domains, expression vectors operatively harboring these DNA sequences and hosts transfected with said vectors are included within the scope of this inventlon.
Most particularly, the present lnvention concerns the use of the novel transcription trans-activation hormone or hormone-like receptors of the present invention in assays for screening various putative materials that may have operative binding affinity for the novel hormone or hormone-like receptors hereof. In a preferred em~odiment, this aspect of the invention provides an assay for screening such putative materials, for example, steroid agonists and antagonists in an enhanced, so-called trans-activation system.

Z00~339 Bac~ground of the Invention The patent applications cited su~ra disclose, inter alla.l the characterization and preparation of S various hormone and hormone-like receptors, including steroid, thyroid and re~inoid receptors such as those represented by the gl~cocorticoid, mineralcorticoid, thyroid, estrogen related and retinoid classes, and specifically, the glucocorticoid, estrogen, aldosterone and retinoic acid receptors themselves. These specific receptors have been the subject of considerable research and form the particular bases for the inventions disclosed and claimed in these patent applications.
Similarly, the extant, parallel scientific literature has focused on the specific receptors listed above from among the classes of receptors that exist.
It is known, for example, that the glucocort~coid receptor belongs to a large super-family 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 blnd$ng, hormone binding and nuclear localization. See Evans, et al., Sclence 240, 889 (1988) for a review of this subject matter. In the case of the glucocorticold receptor, the so-called DNA binding domain spans some sixty-six amino acids and is highly conserved among various species and 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 ~ 423 tl987), Danielsen, et al., Hol-Endo 1, 816 (1987), Kumar, et al., 5Ç11 51, 941 (1987), Gronemeyer, EMB0 J. 6, 3985 (1987), and Waterman, et al., Mol.Endo 2, 14 (1988). This region has been found to contain nine invariant cysteines residues and although ~0~339 the contribution of each cysteine residue to overall function is unknown, as is the actual structure formed by this domain, it has been proposed that these cysteine residues coordinate two zinc ions to form two DNA
binding, so-called finger domains which result in a ternary structure thought responsible for its localization and binding to the requisite DNA site. See Rlug, et al., Tr.Biochem.Sci 12, 464 (1987), Bens, et al., Cell 52, 1 tlg88), and Evans, su~ra.
In a location nearer the carboxy-terminal end distal from the DNA binding region is the so-called ligand binding domain which has the demonstrated ability to block activity of the receptor in the absence of hormone. Thus, presence of the requisite bormone 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 ~, 365 (1987), Hollenberg, et al., ~YE~ Kumar, et al., supra, Danielsen ç~
~YPE~, and Adler Ç~ 31., ÇÇl~ ~, 68S (1988).
In contrast to these two domains, the sequences lying towards the amino-terminal region fro~ the DNA
binding domain is poorly understood both as to structure, and particularly, function. This region is extremely variable both in size and in composition amongst the various receptors - See Evans, ~Y~E~ - and may contribute to the heterogeneity of receptor functlon. See Xumar çt al., ~upra, and Tora ç~ ., 333, 185 (1988).
Despite extensive analysis, some of which having been reported in the scientific literature, the region (6) that determines trans-activation of transcription initiation remains poorly characterized.
Trans-activation domains can be defined as polypeptide regions that, when combined with the DNA binding functional domain, lncrease productive transcription initiation by RNA polymerases. See Sigler, Nature 333, 210 (1988), Brent ~_31-, Ç~ll 43, 729 (19~5), Hope et al., Cell 46, 88s (1986), Ma et al., Cell 48, 847 ~1987), Ma et al., Cell 51, 113 (1987), Lech et al., Cell 52, 179 (1988), and Hope ~ ature 333, 635 (1988).
Previous research of t~e 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 r~ and 72. Giguere et al., Cell 46, 645 (1986).
Further research from ~hese laboratories has also resulted in the report of a co-localization of trans-activation and DNA binding functions. See Hollenberg et al., supra, Miesfeld, et al., ~YE~ Danielsen et al., ~Yp~, and Waterman et al., supra. As a composite, this research has given rise merely 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. However, until now, the region(s) determining the trans-activation activity, was not at all well understood. Thus, the picture based upon existing research lacks an 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 hasidentified functional ndomainsn, there has been little systematic effort to identify amino acids that contribute to the specific activities of the molecule itself. ~hus, the previous identification of steroid receptor trans-activation reqions resulted only from a demonstrated loss of activity via deletion or insertional mutagenesis, but in no case have the properties of the regions themselves been confirmed in assays that reflect a dominant gain of function.
Thus, Godowski et al., Science 241, 812 (1988), report results that show that the glucocorticoid receptor ;200~339 contains at least one ~enhancement domain" other than that overlapping the qlucocorticoid response element binding region and that ~he second domain occupies a region near the receptor amino-terminus. Similarly, Webster et al., Cell 54, 199 (1988) 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 characterization and role in trans-activating potential.
As a startinq point for the present invention, Giguere ç~ ., supra, demonstrated 105s of activity in the glucocorticoid receptor based upon an assay measuring transcription activity, when random site-directed mutagenesis was performed at several locations of the molecule. As a followup, Hollenberg Ç_~1- deleted regions w$thin the molecule, again demonstrating overall loss of transcription activity induced by such removal of stretches of amino acids.
It is an object of the present invention to identify and characterize the do~ain~s) responsible for trans-activation transcription activity, and the characterization of such domain~s) in respect of amino acid composition and seguence, to explore the functional interaction of the domain~s), if any, with both the DNA-bindinq and ligand-binding domains of a given receptor, and finally, to exploit such knowledge via the manipulation of such identified and characterized trans-activation transcription domain(s) so as to increase the overall transcription activity of the given receptor so manipulated.

The present invention thus provides novel hormone or hormone-like receptors that have been modified by advantage of ~nowledge of the identity and characterization of the trans-activation transcription activity domain(s), by modifications thereof so as to produce novel, heterologous receptors that have increased activity compared with the parent molecule. It is an object of the present invention to provide novel, heterologous, optionally hybrid receptors having increased trans-activation transcription activity and otherwise having DNA-bindinq and ligand-bindinq domains that may be borrowed from various different receptors.
It is a further object of the present invention to provide novel assays whereby putative receptor agonists and antagonists can be screened and evaluated for potential commercial exploitation. See also Ptasbne, Nature ~, 683 (1988).

Summary of the Invention The present invention is predicated upon the identification, isolation and characterization of the trans-activation transcription domains of intracellular hormone or hormone-like receptor polypeptides that has in turn enabled the discriminate characterizatio~ of the receptor itself, both in terms of physical attributes and the biological function and effect of their various domains. This information has in turn enabled the production of harnessed, recombinant systems useful for preparing the novel receptors hereof having augmented transcription activation properties.

It has been determined, based upon the information provided herein, that receptors contain trans-activation transcription domains that are position independent and autonomous in function. ThUs, the present invention provides for novel hormone or hormone-like receptors wherein the trans-activation transcription domains are augmented in their ability to activate transcription. Such novel receptors of this invention contain trans-activation transcription domains additional to the parent molecule, positioned in a manner to provide further increase in transcription activity. These novel receptors may be hybrids wherein the DNA-binding and the ligand-binding domains are provided from receptors of the same or different class and/or species.
The present invention is also directed to the use of such novel receptors for in vitro bio-assays for determininq the functionality of a putative receptor or a putative hormone or hormone-like material. Bio-assays may take the form, for example, of challenging a novel receptor hereof with one or more of a battery of test materials that have putative hormone or hormone-like activity and that can potentially modulate the bio-function of said receptor and monitoring the effect of said material on said receptor an in vitro setting.
The present invention is further directed to the preparation of such novel recep~ors hereof via recombinant DNA technology in all relevant aspects, including a DNA molecule that is a recombinant DNA
molecule or a CDNA molecule consisting of a sequence encodinq said receptor or a trans-activation transcription domain thereof, and to requisite expression vectors operatively harboring such DNA comprising expression control elements operative in the recombinant ~0 host selected for the expression, and to recombinant host cells transfected with such operative expression vectors.
The present invention is further directed to a method for inducing the expression of DNA encoding a reporter molecule or other desired heterologous polypeptide comprising inducing transcription of the DNA
encoding said polypeptide by a complex formed by a novel receptor hereof and a corresponding ligand capable of ~00~33~

binding to said receptor, in an in vitro settinq wherein said receptor and said DNA encoding said polypeptide are produced via recombinant expression in a transfected cell host system.
The present invent$on thus embraces a hormone or hormone-like receptor as a polypeptide having increased trans-activation transcription activity of a promoter with which it is associated, by virtue of its intrinsic ability to bind to a DNA sequence response element of said promoter or by its ability to associate with other polypeptide(s) that bind to said DNA seauence response element, and having trans-activation transcription activity greater than that of its corresponding parent receptor.
The present invention is directed to recombinant DNA technology in all aspects relating to the use of the characterization of the trans-activation transcription domain of a hormone or hormone-like receptor for DNA isolates production, including cross-hybridizable DNA isolates, devising expression vectors therefor, transfected hosts producing therewith and methods comprising a method of use utilizing ~uch information to devise cells or cell lines harboring genetic information sufficient for such cells or cell lines to produce such receptor5 such that they can be used as such or in expression systems or in assays for determining the act~vity of corresponding putative ligands.

Detailed Description of the Invention ~ _f Descri~tion of the Drawinas Figure 1 depicts the identity, and transcription activity, of various human glucocorticoid receptors (hGR) entities hereof that have been modified from the parent molecule in the trans-activation transcription domains (tau1). Wild-type hGR (wt) and taul mutants are schematically represented. Functional regions are hatched (tauO stippled (DNA-binding domain), or indicated by "DEX" (hormone binding domain). Numbers above each receptor define amino acid positions. Heavy vertical bars identify boundaries of an inserted fraqment. Relative luciferase (reporter molecule) activity was measured by MTV-LUC using 10 7 M
dexamethasone (corresponding steroid), except receptors indicated by "C" after the activity which are constitutively active.
Figure 2 sets forth the luciferase activity of various tauz receptors hereof. The wild-type hGR is represented at the top. The tau2 region extends from amino acids 526 to 556 and is represented by a solid rectangle. Replacements of the taul region are indicated by a solid rectangle (tau2) or by hatched rectangles for the amphipa~hic alpha helix ~naah"). Relative luciferase zctivities were measured by MTV-LUC in the presence of 10'7 dexamethasone and are ~ollowed by "(C) n when hormone-independent. Asterisks indicate site of the amino acid end of truncated molecule.
Figure 3 depicts the construction of hybrid steroidal receptor of glucocor~icoid-thyroid hormone receptors whose trans-activation transcription activity i~ increased by the addition of taul domains. A seqment of the human glucocorticoid receptor cDNA encoding amino acid 77 to 262 (encoding the taul domain) was inserted in the rat alpha thyroid hormone receptor cDNA at a position corresponding to amino acid 21, in one or multiple copies. The parental receptor is rTR alpha with a BamHI
linker inserted at the unigue BstEII site in the amino terminus. Constructs were transfected into CV-l cells with TRE-CAT and CAT activity measured in the absence or presence of T~.
Fiqure 4 sets forth the point mutational analysis of the hGR DNA-binding domain. The amino acid sequence of the hGR DNA-binding domain is given. Each line represents information believed to be encoded by part of a separate exon. The consensus se~uence (con) for the steroid hormone receptor super~family is presented below the hGR sequence, with invariant (bold), conserved(standard type) and non-conserved (dashes) amino acids indicated. Amino acids converted to lysine are topped by circles. Transcription activity of mutants assayed with MTV-CA~ and compared with hGR-SY are indicated as greater than 10% (filled circles), 1% to 10%
(half-filled circles), and less than 1% (open circles).
.

2. General Methods and Definitions Amino acid identification uses the single- and three-letter alphabets of amino acids, i.e.:
Asp D Aspartic acid Ile I Isoleucine 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 X Lysine Ala A Alanine Arg R Arginine Cys C Cysteine Trp W Tryptophan Val V Valine Gln Q Glutamine Met M ~ethionine Asn N Asparagine Steroid receptors hereof are prepared 1) havinq methionine as the first amino acid (present by virtue of the ATG start signal codon insertion in front of the structural gene) or 2) where the methionine is intra- or extracellularly cleaved, having its ordinarily first amino acid, or 3) toqether with either its signal polypeptide or conjugated protein other than its conventional signal polypeptide, the signal polypeptide or a conjugate beinq specifically cleavable in an intra-or extracellular environment. In all events, the thus :

X~0433g produced receptor, in its various forms, is recovered and purified to a level suitable for intended use. See Supra.
The "hormone or hormone-like receptors" of this invention include the receptors specifically disclosed, for all species that cross-hybridization exists, most notably other mammalian receptors, as well as related (e.g., gene family) receptors of the same or cross-hybridizable species that are enabled by virtue of DNA
lo isolation and characterization and use via cross-hybridization techniques from said specific receptors or from identification via immuno cross-reactivity to antibodies raised to determinants in the usual manner known per se. It also includes functional equivalents of all of the above, including interspecies or intraspecies receptors wherein DNA-binding and/or ligand-binding domains are swapped with one another, or otherwise differing in one or more amino acids from the corre~ponding parent, or in glycosylation and~or phosphorylation patterns, or in bounded conformational structure.
"Expression vector" includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operatively lin~ed to other seguences capable of effectinq their expression. It is implied, although not always explicitly stated, that these expression vectors may be replicable ~n the host organisms either as episomes or as an integral part of the chromosomal DNA. nOperative, n or grammatical equivalents, means that the respective DNA sequences are operational, that is, work for their intended purposes.
In sum, "expression vector" is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified DNA sequence disposed therein is included in this term as it is applied to the specified sequence. In general, expression vectors of utility in recombinant DNA techniques are often in the 200~339 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, "plasmid" and "vector" are used interchangeably as the plasmid is the most 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.
Apart from the novelty of the present invention involving the manipulation by means of repositioning or augmentation of the trans-activation transcription domains of a parent steroid receptor, it will be understood that the novel steroid receptors of the present invention may otherwise permissively differ from the parent steroid receptor in respect of a difference in one or more amino acids from the parent entity, insofar as such differences do not lead to a destruction in kind of the basic steroid receptor activity or bio-~o functlonality.
Recombinant host cells" refers to cells whichhave been transfected with vectors constructed using recombinant DNA technigues.
"Extrinsic support medium" includes those known or devised media that can support the cells in a growth phase or maintain them in a viable state such that they can perform their reco~binantly harnessed functlon. See, for example, ATCC Media Handbook, Ed. Cote t al., American Type Culture Collection, Rockville, MD (1984).
A growth supporting medium for mammalian cells, for example, preferably contains a serum supplement such as fetal calf serum or other supplementing component commonly used to facilitate cell growth and division such as hydrolysates of animal meat or milk, tissue or organ extracts, macerated clots or their extracts, and so forth. Other suitable medium components include, for example, transferrin, insulin and various metals.

, ;~1339 The vectors and methods disclosed herein are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms.
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 for carrying out basic techniques encompassed by the present invention. See, for example, Maniatis, et al, Molecular ~lonina: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1982 and the various references cited therein, and in particular, Colowick et al., Methods in Enzymoloqv Vol 152, Academic Press, Inc.
(1987). All of the herein cited publications are by this reference hereby expressly incorporated herein.
The foregoing description and following experimental details set forth the methodology employed initially by the present researchers in identifying and characterizing and preparing particular receptors. The art skilled will recognize that by supplying the present information lncluding the location and ma~eup of the trans-activation transcriptional domain of a given receptor and how it can be manipulated to produce the novel receptors hereof, it is not necessary, or perhaps even scientifically advisable, to repeat these details in their endeavors 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 similar 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 and others, and fragments thereof, using means within the skill of the art having benefit of the present disclosure. All of such means are 20(~4339 ~5 lncluded within the enablement and scope of the present invention.

3. ,Detailed Description of Particularlv Preferred Embodiments The present invention was premised upon use of the glucocorticoid receptor as a model herein for the preparation of novel modified entities thereof including hybrids of the glucocorticoid receptor with other receptors such as the thyroid receptor, particularly in the swapping of DNA-binding and ligand-binding domains to make up such hybrids. In each case the essence of this invention, namely, the repositioning and/or augmentation of the trans-activation transcription domain(6) so as to create novel receptors whose trans-activation transcriptional activity i8 increased over the parent, is illustrated herein using the glucocorticoid receptor as the parent or hybrids thereof upon which comparisons were made for the novel trans-activation transcription domain modified versions.
It will be unde~stood therefore that for receptors that are known in the art, whether wild-type, hybrids, or functional equivalents a8 set forth herein, they are suitable as starting materials for the trans-activation transcriptional domain(6) modifying aspects of the present invention.

4. Examples The following examples detail materials and methods employed in the experimental procedures that follow:

Point Mutaqenesis and ~ranscriptional Activation To define systematically the role of the DNA
binding domain in the function of the receptor, research Z00~339 employed extensive site-directed mutagenesis of this conserved region. By testing the role of individual amino acids this method has the potential to determine whether mutants which affect trans-activation are independent from those which influence DNA binding.
The sequence of the hGR DNA binding region is given in Figure 4, followed by the consensus sequence for the steroid hormone receptor superfamily. Among members of the superfamily, this domain contains 20 invariant and lo 12 conserved residues. All invariant amino acids, as well as eight conserved and eight non-conserved residues, were changed to glycine. The ability to stimulate transcription from the GR-responsive MTV promoter and to complex specifically with a glucocorticoid response element (GRE) DNA fragmen~ in vitro was measured.
The steroid-dependent enhancement of transcription from MTV-CA~ for each mutant is given in Table I and is indicated above each altered amino acid in Figure 4. A11 activities are relative to the wild type receptor hGR-SB, which yields an average induction of approximately 1000 fold. Thus, even 1% residual activity is significant. The deletion mutants listed in Table I, delta420-451 and delta450-487, re~ove the first and second halves of the DNA binding domain, respectively, with the break point corresponding to an exon-exon boundary within the human mineralocorticoid and chicken progesterone receptor cDNAs. Both have no ~easurable activity indicating that neither n finger" alone is sufficient for activation.
Of the glycine point mutants, all of those which alter one of the nine invariant cysteine residues destroy activity, consistent with the idea that they are critical for function. An additional, non-conserved cysteine at position 431 is not necessary for function.
Surprisingly, only five of the remaining 24 glycine mutants are completely inactive. The five non-functional allelee are all invariant residues, suggesting that they 200433~
-are involved in general aspects of DNA binding, not in determining sequence specificity. However, not all invariant residues are critical for function. For example, when aspartic acid 426 is converted to glycine there is almost no loss in activity. Seven out of eight mutants with conserved amino acids changed to glycine are functional, with half of this group retaining greater than 40~ activity. Similarly, mutation of non-conserved amino acids within the DNA binding domain in no instance produces a receptor with less than 10% activity and generally causes little reduction in function. Thus, despite some significant exceptions, a good correlation exists between the conservation of an amino acid and the extent of functional loss when converted to glycine, with all invariant cysteines playing a critical role for f~nction.
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Table 1. Comparison of Transcriptional Activity and DNA
Binding f~r hGR Glycine Mutants Relative CATaRelative DNA Bindingb hGR Mutant Activity in vitro hGR-SB 100 100 A 420-451 <1 ~450-48~<1 G421 <1 <1 G423 <1 <1 G424 <1 G438 <1 <1 G441 <1 <1 G442 <1 60 G444 <1 G445 <1 <1 G447 <1 2 G453 go 60 G457 <1 2 G463 <1 G473 <1 <1 G476 <1 ~1 G477 ~1 1 G481 <1 3 a Activity was measured with MTV-CAT in CV-l cells.
b The DNA binding assay is described elsewhere (Hollenberg et al., 1987). The value given was derived from total immunoprecipitated counts but reflects specific binding as determined by gel electrophoresis.

In vitro DNA Binding of Point Mutants The CAT activity measured in the transcription assay is the sum of multiple individual functions including nuclear localization, DNA binding, dimerization and perhaps the allosteric events and protein-protein interactions that ultimately result in activation. If more than one essential function is encoded by the DNA
binding domain, some of the non-functional point mutants may still retain their ability to bind DNA but fail to activate. To explore this possibility, each mutant protein was produced by transfection of the corresponding expression vector into COS-l cells and assayed for its ability, in crude extracts, to form a specific DNA-protein complex with radiolabeled DNA. The activity ofeach mutant in this immunoprecipitation DNA binding assay is given $n Table I; the value shown represents specific binding of the GRE fragment. In general, there is good correspondence between the ability to activate transcription ~nd to bind DNA in vitro. Mutants for which DNA binding activity is significantly lower than transcriptional activity, such as G446 G455, G467 and G486, may be unstable in vitro. The transcriptional activation measured for these mutants is a result of specific interaction with D~A in vivo, since deletion of GREs from the MTV promoter abolishe~ their activity.
Only a single mutant, G442, wh$ch converts t~e lysine directly following the first putative finger to glycine, has lost the capacity to eff~ciently stimulate transcription while maintaining affinity for DNA in vitro. The specificity of binding to GREs relative to non-specific seguences is only minimally affected, indicating that its dramatic }oss in activity is not due to an inability to bind promoter sequences in vivo. This is an intriguing mutant because it suggests that DNA
binding is not sufficient for activation. Perhaps G442 prevents a secondary event, ~uch as an allosteric change, 20~4339 following the prima~y protein-DNA interaction. Despite this exception, these results demonstrate the cysteine-rich 66 amino acid region to embody the DNA binding domain. Indeed, although DNA binding i6 necessary for trans-activation this function must be located in other regions of the receptor.
The G442 species has particular significance in terms of utility because it is the single species prepared that fails to result in activity but ~till shows lo evidence of substantial DNA binding. It is contemplated, for exa~ple, that an assay can be devised exploiting the property of both G442 and the I550* species. The IS50*
species lacks completely a ligand-binding domain, and as such, is not responsive to the presence or absence of hormone. The hormone to a specific ligand-binding site relieves the inhibition of the molecule to act as a trans-activation transcription factor. Lacking this domain mean~ that the I5S0* ~pecies will alway~ produce activity in an assay with or wlthout presence of steroid.
On the other hand, the G442 species in ~he same assay will always be inactive with or without steroid. By devising an appropriate assay where both receptors G442 and I550* are present in the system, initially there will be 100% activity based upon the contribution of the I550*
species alone. As the appropriate steroid is added to the system, the activity observed will fall increasingly toward zero wit~ time. Administration of a putative antagonist along with the appropriate steroid, if active as such, would restore the activity as the antagonist would interfere with the action of the steroid thus reducing the overall activity and a rebound in activity would be seen.

GAL4/hGR Chimeras If DNA binding and ~ functions are truly separable, the possibility exists to replace the hGR DNA
binding domain with a non-receptor DNA binding domain to produce a hybrid activator protein with a new promoter specificity. To test this possibility, the hGR cysteine-rich region was substituted with the $irst 74 amino acids of yeast GAL4. These GAL4 amino acids are sufficient for sequence-specific DNA recognition, but have no transcriptional activation capability. The ability to trans-activate lies outside the DNA binding domain and is encoded in two separate regions of the protein.
Therefore, in order to produce a functional transcription factor fusions between the GAL4 DNA binding domain and hGR must contain trans-activation functions contributed by the hGR.
To assay function of hGR-GAL4 hybrids, a GAL4-responsive promoter, deltaMTV-GAL-CAT, was constructed from MIV-CAT. Thi~ fusion gene wa~ rendered GR-non-responsive and GAL4-inducible by replacement of GRE
sequences with a synthetic GAL4 binding ~ite. When measuring the activation of hGR and GAL4 on th~s promoter, hGR cannot produce measurable stimulation from this promoter, whereas GAL4 can increase CAT expression about 20-fold relative to a control expression vector.
This is consistent with previous reports that GAL4 can function in higher eukaryotic cells. The stimulation by GAL4 i8 clearly mediated through the GAL4 synthetic binding site: deletion of this 6ite renders the promoter non-responsive to GAL4.
Fusions between the GAL4 DNA binding domain and potential hGR activation domains were examined for their ability to ~timulate the GALA-responsive reporter. To demonstrate that the hGR DNA binding domain is not required for the activity of this hybrid, the GAL4 DNA
binding domain wa6 substituted for the hGR DNA binding domain to produce the hybrid GgalG. (~he hGR, including amino terminus (G-), DNA binding domain (G), and carboxy terminus (G), is referred to as G-G-G, respectively.
Replacement of the hGR DNA binding domain with that of GAL4 generates G-gal-G.) The resultant hormone-dependent hybrid clearly can function in the absence of the hGR DNA
binding domain and actually acts as a more potent transcription factor than GAL4 in this assay system, giving a 500 fold increase in CAT activity with addition of hormone. Unexpectedly, GgalG can also stimulate delta MTV-CAT without a GAL4 binding site, but only about 5% of that measured on delta MIV-GAL-CAT. The plasmid delta MTV-AT may contain a cryptic GAL4 recognition site that is revealed only with the stronger GgalG activator and not the weaker GAL4. Indeed, activation is dependent on the GAL4 DNA binding domain; GgalG is functional on deltaMTV-CAT whereas GGG is not.
The trans-activation capability of GgalG must be determined by the amino- or carboxy-terminal regions of hGR, since the GAL4 DNA binding domain alone i8 inactive. Accordingly, each of the~e hGR regions was individ~ally tested for its ability to complement the GAL4 DNA binding function. Both hybrids are functional with Ggal(delta) displaying constitutive activity while (delta)galG is fully hormone dependent. Therefore, autonomous trans-activation functions are embodied in both the N-terminal and C-terminal segments of the hGR, although sub~ect to different constraints.

Rearranged and Partially Duplicated hGR Hutants Fusion with the GAL4 DNA binding domain demonstrated the presence of distinct trans-activation properties in the hGR amino-terminal 420 amino acids and carboxy-terminal 300 amino acids. Mutants of the hGR
with tau1 duplications were assayed on the luciferase derivative of MTV-CAT, MTV-LUC. Activity values determined with the MTV-luciferase fusion gene and MTV-CAT are equivalent for previously described deletion mutants. Figure 1 shows the series of ~ mutant derivatives and their luciferase activity relative to wild type receptor. Absence of ~ reduces activity to 5~, while the tandem duplication mutant GRll acts as a "super" receptor with 310~ activity. Mutants GR12, GR14, GR17 and GR18 reveal that rl can function between the DNA
and hormone binding regions, as well as on the amino terminal side of the DNA binding domain, giving rise in each case to a hormone-dependent activator (Figure 1).
This indicates a remarkable flexibility of receptor structure. The ability of rl to increase activity is independent of both the amino and carboxy termini, as shown in Figure 1 by comparison of GR13 and GR14 activities, and GR15 with I515* and GR16. The r, region may not account for all trans-activation ability in the amino terminus as shown by the-4-fold greater activity of GR18 relative to GR14. Also in support of this proposition, deletion of both r~ and the carboxy terminus in GR15 leaves 1% residual activity, a ten-fold induction, which c~n be abolished by deletion of the majority of the amino terminus (compare GR15 and GR26, Figure 2).
A second region with potential trans-activation character is r2, located at the very amino terminal end of the hormone binding domain (amino acids 526-556). This region has five negatively charged residues in a stretch of 18 amino acids and is implicated in receptor activity by the two-fold difference in activation of truncation mutants I550* and Isls* (Figure 3). To determine whether T2 constitutes an activator domain it was introduced adjacent to, or in place of rl. Figure 3 shows that this region acts to give a 3-fold increase in activity when introduced into the amino terminus independent of ~
(compare GR20 with "wt", GR21 with ~77-262). A second copy gives a further 2-fold increase, so that a pair of r2 regions gives an overall increase in activity of six-fold (GR22). Therefore, like rl, the position Of ~2 in the ~ .~
.

~00~339 receptor is flexible, its activity is cumulative and its function can be constitutive (e.g., GR25).
Constructions similar to the T2 mutants GR21 and GR22 wsre constructed using the synthetic amphipathic helix, "aah," containing 20% acidic residues and demonstrated to possess trans-activation properties in the context of yeast GAL4 (Giniger et al., Nature 330, 670(1987). The size and charge characteristics of the "aah" sequence are similar to the ~2 region, which led us lo to explore its potential activity in the context of the hGR. Indeed, a similar increase in activity of mutants with single or multiple copies of T2 and aah is observed (Figur~ 3; compare GR21 with GR23, GR22 with GR24~, suggesting that these regions may perform equivalent functions. These results support and extend the notion of the modular nature of trans-activation domains.

Trans-activation ( T ) Domains We hAve defined discrete hGR trans-activation regions according to the following two criteria: deletion decreases activity and duplication lncreases it. By these standards two regions of the receptor, of 200 and 30 amino acids, encode trans-activation functions. The localization of these two regions does not exclude a role for additional activator seguences within the hGR.
Examination of the primary sequences of r~ and ~2 fails to reveal any obvious homoloqy with the exception that both regions have acidic character. This property is noteworthy because activation domains in the yeast transcription factors GAL4 and GCN4, although lacking obvious seguence identity, are rich in acidic residues.
This apparent similarity does not demonstrate that any of the identified activator regions in either GAL4, GCN4, or the glucocorticoid receptor are functioning through a common mechanism, slthouqh this seems likely. The potentiAl for a common mechanism is further supported by 200~39 the observation that the synthetic amphipathic ~ helix ("aah") sequence can functionally replace T2 and to some extent ~. The lacX of obvious sequence and size relatedness of r~, and r2 and the yeast activation ~equences leads to the view that trans-activation functions might be embodied by the net context of negatively-charged residues on the surface of the DNA-bound protein.

hGR Modularity The modular nature of the hGR has emerged not only from primary sequence comparisons within the steroid receptor superfamily, but also by the ability to exchange functional domains to create novel chimeric activators.
The DNA-binding domain of the hGR haæ been replaced with that of the human estrogen receptor (Green et al., Nature 325,75 and Chambon, (1~87) and with the unrelated DNA-bindin~ domain from GAL4. Its position is not critical since it can function at the amino terminus. In addition, the DNA-binding domain can be placed amino or carboxy-terminal to both Tl and r2 without compromising function of these domains. Consistent with modular properties, the position of rl and r2 is not critical and their multimerization leads to increased receptor function. Thus, we have been able to generate receptors with activity of up to four times that of the wild-type receptor or with altered DNA binding specificity. Hybrid receptors are still hormonally inducible indicating a non-specific mechanism whereby the hormone binding domain imposes a ligand-dependent effect on the rest of the molecule. Experimental detail and discussion follows:

Plasmid and Point Mutant Constructlon Plasmid hGR-SB was generated by reco~bination at the ClaI site between linker scanning mutants I532 and !; I403S (Giguere et al., Cell 46, 645 (1986)). I403S was derived from I403 by introduction of the SstI adaptor 5' GATCGAGCTCGC 3' into the BamHI site. This hGR derivative is parent to all point mutants and is indistinguishable from wild-type receptor in DNA b~nd1ng and transcriptonal activation To convert the desired codon of hGR-SB to one encoding glycine, the SstI/BamHI, 400-nucleotide fragment from plasmid hGR-SB was first introduced into M13mpl8 to yield single-stranded template. Synthetic oligonucleotides of 13-15 bases were then used to change the desired codon to GGN by standard techniques followed by re-introduction of the altered SstI/BamHI fragment into hGR-SB. Deletions ~420-451 and a450-487 were generated with longer oligonucleotides (20mers); amino acid positions given define non-deleted residues at the deletion junction. Mutant sequences were directly determined from double-stranded plasmid DNA using alkaline denaturation (Hattori ~ 31-, Anal.8iochem 152, 232 (1986)) followed by chain termination of synthetic hGR primers.
Reporter plasmid MTV-CAT and its deletion derivative ~MTV-CAT were generated as follows. The HindIII site of pB~CAT2 (Luckow y~ ucl.Acids Res.
15, 5490 (1987)) was first destroyed to generate pTXCAT-H. The HSV-thymidine-kinase promoter of pTKCAT-H was then excised by digestion with 8amHI/BglII and replaced with the BamHI, MTV-LTR fragment of pMTV-TK (Kuhnel et al., J.Mol.Biol. 190, 367 ~1986)), pLS-l90/-181 or pLS-96/-88 ~Buetti et al., J Mol.~iol. l9o, 379 (1986)) to generate MTV-CAT, -190/-181 MTV-CAT, or -96/-88 MTV-CAT, respectively. ~ MTV-CAT was constructed from -190/-181-and -90/-88 MTV-CAT by recombination between the HindIII
sites of these mutants. ~ MTV-CAT was converted to .~. ,..~_ .

MTV-GAL-cAT by introduction of the synthetic GAL4 binding site "17MX" ~Webster et al., Cell 52, 169 (1988)) ~nto the unique HindIII site. Plasmid MTV-LUC was constructed by conversion of the HindIII site of pSVOA/L-A ~5' ~de Wet et al., Mol.Cell.Biol. 7, 725 ~1987)) to XhoI and introduction of luciferase coding and SV40 polyadenylation sequences from this derivative ~generated by ~amHI digestion, Klenow polymerase I end-filling and XhoI digestion) into XhoI/SmaI-digested MTV-CAT.
GgalG was derived from pG525 (Laughon et al., Mol.Cell.Biol. 4, 260(19~4)). Primer-directed mutageneSiS was used to introduce NotI and XhoI sites at codinq-sequence nucleotides -10 to -3 and ~223 to +228, respectively. ~he GAL4 DNA binding domain was excised from this derivative by digestion with NotI and inserted into pRShGR~ (Giguere et al., Nature 330, 624(1987)) in place of the endogenous DNA binding domain. Ggal~. and 4gal ~ were produced by digestion of GgalG and ~galG, respectively, with XhoI, followed by end-filling and ligation. ~ galG was constructed by introductlon of a synthetic oligonucleotide duplex ( aAN, 5' GTACCACCATGGGGC 3') containing a consensus ribosome binding site ~Kozak, ~ucl.Acid.Res. 12, 85~ (1984)), in place of the amino-terminal-coding, Asp718/NotI fragment of GgalG.
Generation of mutants ~77-262, I515* and ~ 9-385 has been described by ~ollenberg ç_al-, ~ 49, 39~
(1987). r~ mutants were constructed using the BglII/BamHI
fragment from I262 (Giguere et al., Cell 46, 645 (1986)), encoding amino acids 77-262 of the hGR. This fragment was inserted into the ~amHI sites of hGR linker scanning mutants I262 and Isls to generate GRll and GR12, respectively. GR13 was derived from pRShGR~ by replacement of the amino terminal coding sequences with the ~ AN adaptor described above. GR17 was created by insertiDn of the amino terminal coding BamHI fragment, generated by recombination between linker scanning , ' ' ' ' , .

Z0~339 2~
mutants 19 and I384 (Giguere et al., ~eE~,) into the Baml3I site of I515. Mutant I550* was produced by recombination between the BamHI sites of mutants I550 and I696, thus shifting the reading frame after amino acid 550. This mutant incorrectly described as ~532-697 in a previous report (Hollenberg et al., sUPra). To create T2 derivatives, an exciseable r2 coding-fragment was generated by conversion of hGR nucleotides 1704-1709 and 1800-1805 (Hollenberg et al., Nature 31~, 635 (1985)) to BamHI and BglII sites, respectively, using oligonucleotide-dlrected mutagenesis. The sequence encoding r2 was then introduced into the BamHI site of I262 to produce GR20 or in place of the BglII/BamHI
fragment of I262 to generate GR21. GR23 was created by replacement of the BglII/BamHI fragment of I262 with a synthetic oligonucleotide duplex (5'GATCT GGAAT TACAA
GAGCT GCAGG AACTA CAAGC ATTGT TACAA CAGCA AGAG 3') encoding the ~'aah~ sequence ~G1niger and Ptashne, 1987).
Mutants with tandem copies of this sequence and r2 (GR24 and GR22) were generated by standard techniques (Rosenfeld and Kelly, 1986). Double mutant derivatives of constructions described above were generated by recombinatiOn at the ClaI gite: GR14 from GR12 and GR13:
GR15 from ~ 77-262 and Isl5* GR16 fro~ GRll and I515~;
GR18 from GR13 and GR17: GR25 from G~22 and I515~: GR26 from ~ 9-385 and I515~.

Immunoprecipitation DNA Binding DNA binding was measured as described previously (Hollenberg et al., ~uE~). Mutant receptor, obtained in a crude COS-l cell extract after transfection, was incubated with a mixture of radiolabeled DNA fragments, one of which contained GREs.
Receptor-DNA complexes were immunoprecipitated with receptor-specific antiserum and Staph A, freed of protein, counted Cerènkov, and then electrophoresed through a denaturlng polyacrymide gel to verify specific binding. Total immunoprecipitated counts were compared.
The presence of mutant hGR protein in each coS 1 cell extract was confirmed by Western blot analysis.
s Transfection and Luciferase Assays Transfection of CV-l and COS-l cells was as described previously (Giguere et al., and Hollenberg et al., su~ra) using 5 micrograms of each plasmid per 10 cm dish. Luciferase assays were performed as described (de Wet ~ 31-, supra).

Cell Culture and Transfection Conditions for growth and transfection of CV-l (African green mon~ey Xidney) cells were as previously described (Giguere et al., Cell 46, 645 (1986)), except that the calcium phosphate precipitate was left on the cells for 4-8 hours, at whlch time the media was changed to DMEM with 5% T~ free bovine serum minus or plus 10-7 M
T~ ~Sigma). Cells were harvested 36 hours after the additlon of T3, and CAT assays were performed as described (Gorman et al.,Mol.Cell.Biol. ~, 1044 (1982~ Hollenberg et al., ~11 49, 39 (1987)). Typically, 5 ~g reporter and 1 ~g expression vector were cotransfected, along wlth 2.5 ~g RSV-~gal as a control for transfection efficlency.
Acetylated and non-acetylated forms of tl'C]chloramphenicol were separated by thin layer chromatography, excised, and quantitated by liquid scintillation counting in Econofluor (DuPont) with 5~
DMS0. ~-galactosidase assays were performed as described ~}lerbomel et al., Cell 39, 653 ~1984)). CAT activity is expressed as percent conversion divided by ~ f -galactosidase activity.

~.

Construction of Reporter and Expression Plasmids Synthetic oligonucleotides corresponding to -169 to -200 of the rat growth hormone gene or a palindromic TRE (TCAGGTCATGACCTGA) (Glass et al., Cell 54, 313 (1988)) were insertçd into a linXer scanning mutant of MTV-CAT that has a Hind III site at position -190/-181 (Buetti et al., J.Mol.Biol. l90, 379 (1986)) or -lso/-88 MTV-CAT, which has a Hind III site replacing the lo nucleotides between -88 and -190. Expression vectors were constructed for the thyroid hormone receptors by inserting the full-length cDNAs of pheA12 (Weinberger et al., Nature 324, 641 (1986)) and rbeA12 (Thompson et al., Science 237, 1610 (1987)) between the KpnI and BamHI
sites of the pRS vector (Giguere et al., Cell 46, 645 (1986) and Nature 330, 124 (1987)).

Construction of Chimeric Receptors.

The construction of hGR~ has been described (Giguere, Nature ~Ye~). To construct hTR~, the cDNA
insert of phe A12 tWeinberger, Nature, su~ra) was subcloned between the XpnI and BamHI sites of M13mpl9 and mutagenized by the method of Kunkel, PNAS ~, 488 (1985).
The oligonucleotide used to create the NotI site changed three amino acids: Asp97 to Arg, Lys98 to Pro, Asp99 to Pro. The oligonucleotide used to create the XhoI site changed two amino acids: Thrl71 to Leu, Aspl72 to Gly.
The mutant receptor cDNA was then transferred to the expression vector pRS (Giguere, Cell, Nature ~Ye~);
hybrids were constructed by exchanging RpnI-NotI, XpnI-XhoI or NotI-XhoI restriction fragments between RShGR~
and RShTR~. RShGR~ has about 75% of wild-type activity, and RShTR ~ has about 60% of wild-type activity. For the addition of rl to rTR~, the unique BstEII site at amino acid 21 was changed to a BamHI site by inserting an oligonucleotide adaptor that encoded a ~aml~I site flanked by BstEII ends. This allowed the in frame lnsertion of a BamllI-BglII fragment encoding amino acids 77-262 of the hGR into this site. ~TT and ~GG
were constructed by deleting the Asp718-NotI fragment of RShTR ~ and RShGR~ respectively and replacing it with an oligonucleotide adaptor of a consensus ribosome binding site (Kozak Nucl.Acids Res. 12, 857 (19~4)).
Amino acids 77 to 262 of the hGR, called r1~
were inserted in frame after amino acid 21 of rTR~ in one or multiple copies and the resulting hybrid receptors assayed for trans-activation. Table 1 shows that addition of one Tl domain increased activity by at least four-fold, while the presence of multiple such domains further increased acti~ity. This is consistent with the modular nature of this domain, and demonstrates that the activity of thyroid hormone receptors can be augmented by the addition of a trans-activation domain from a different receptor.

Table 1. Activity of thyroid hormone receptor~rl hybrids.
Receptor # of ~1domains- Relative CAT Activity-RShGR 1 0 RSrTR~-Bm+ o 100 RSrTR-Tl~l 1 1430 , RSrTR-Tl-2 2 ~ 1140 RSrTR-Tl3 3 1960 RSrTR-Tl, 4 820 'CAT activity is relative to the induced activity of RSrTR-Bm+, which is the rat alpha thyroid hormone receptor with the BstEII site at amino acid 21 changed to a BamHI site.
In certain experiments where the amount of receptor expression vector is increased from l~g to 5~g, relative CAT activity was shown to increase several-fold.

, . . .
' 200~339 The foregoing description details specific ~ethods that can be employed to practice the present invention. 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 sequences thereof, the art skilled will well enough know how to devise alternative reliable methods for arriving at the same information and for extending this information to other intraspecies and interspecies related receptors. Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope hereof; rather, the ambit of the present invention is to be governed only by the lawful construction of the appended claims.

Claims (19)

1. An assay for screening and identifying materials having a putative potential of binding to a hormone or hormone-like receptor comprising the steps comprising:
(a) providing a hormone or hormone-like receptor species in a form having improved trans-activation transcription activity, (b) challenging said form of receptor species with one or more of a battery of test materials having putative potential of binding to a hormone or hormone-like receptor, (c) monitoring the effect of said test material by measuring the amount of transcription induced by said receptor species, and (d) selecting candidates from the battery of test materials capable of having an effect on said trans-activation transcription activity of said receptor species.

2. An assay according to Claim 1 further comprising the additional step following step (d), comprising:
(e) employing said candidate in the preparation of a composition containing said candidate as an essential component, said composition being useful to impart its biofunction properties on a corresponding receptor when it is contacted in vivo with said receptor.

3. An assay according to Claim 2 further comprising the additional step following step (e), comprising:
(f) contacting said composition with a human subject.

4. A hormone or hormone-like receptor as a polypeptide having increased trans-activation transcription activity of a promoter with which it is associated, by virtue of its intrinsic ability to bind to a DNA sequence response element of said promoter or by its ability to associate with other polypeptide that bind to said DNA sequence response element, wherein said receptor contains a plurality of trans-activation transcription domains thereof, of a functional fragment thereof, located within the molecule in a location outside of its DNA-binding and ligand-binding domains.

5. The receptor according to Claim 4 wherein said receptor is based on the human glucocorticoid receptor.

6. The receptor according to Claim 4 wherein said trans-activation transcription domain is the ?1 domain.

7. The receptor according to Claim 6 wherein said receptor contains two said ?1 domains.

8. The receptor according to Claim 7 wherein the second of said ?1 domains is located adjacent to the first.

9. The receptor according to Claim 7 wherein the second of said ?1 domains is located in a C-terminal distal location from the first ?1 sequence.

10. The receptor according to any one of the preceding claims lacking a ligand-binding domain.

11. As a species of a receptor according to Claim 1, human G442 glucocorticoid receptor.

12. A hormone or hormone-like receptor having trans-activation transcription domains additional to that of parent receptor, autonomous with both the DNA-binding and ligand-binding domains of parent receptor, said additional trans-activation transcription domains being located in a position outside of each of said DNA-binding and ligand-binding domains, if present, and having a DNA-binding biofunctionality.

13. A DNA molecule that is a recombinant DNA
molecule or a cDNA molecule encoding a receptor according to Claim 12.

14. An expression vector operatively harboring DNA encoding a receptor according to Claim 13.

15. A recombinant host cell transfected with an expression vector according to Claim 14.

16. A cell culture comprising cells according to claim 15 and an extrinsic support medium assuring the viability of said cells.

17. A process of preparing a human receptor according to Claim 12 which comprises expressing in a recombinant host cell transfecting DNA encoding said receptor.

18. A process which comprises recovering and purifying a receptor according to Claim 12 to a form having quality and quantity sufficient to enable its use in assays that enable measurement of extrinsically induced biofunctionality of said receptor.

19. In a bioassay for determining the functionality of a hormone or hormone-like receptor, said bioassay comprising:
(a) transfecting into receptor negative cells one or more expression vectors harboring an operative hormone responsive promoter/enhancer element functionally linked to an operative reporter or other desired DNA and an operative DNA sequence encoding said receptor, (b) culturing said transfected cells from step (a) in the presence or absence of extrinsic material having the putative ability to activate said hormone responsive promoter/enhancer element, (c) monitoring in said cells induction of the product of said reporter or desired DNA
sequence, and (d) measuring in said cells the expression of reporter or other desired DNA, the improvement wherein said hormone or hormone-like receptor is one having increased trans-activation transcription activity compared with parent receptor, said activity additional to the parent receptor by virtue of domains, or functional fragments thereof, located within the molecule at a position outside of each of a DNA-binding and ligand-binding domain of said receptor.