Note: Descriptions are shown in the official language in which they were submitted.
WO90/14356 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~,
-2-
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.,
~ : .
' ` ~ , ' '
. :, -: -
. . . :: . ..
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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WO90/143~ PCT/US90/03113
3 ~9 ~
-4-
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
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W090/14356 2~57~49 PCT/US~/03113
~ 3^
--5--
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
-6-
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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WOgO/14356 z~7~49 PCT/US90/03113
j.` :~ .
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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- . . . .
, . .. , . , : . : ,
WO90/14356 PCT/US90/03113
~ 2~ o~
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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.
~ . . .: - - . . .
, . . . .. , -, ., .. ~ ........ .
W090/~4356 ~ PCT/US90/03113
Z~ ig`` ~
--10--
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
-
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,
~ ., . . ,, - . . . -
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
,
:
'
.
W O 90/14356 . P~r/US90/03113
2(~57~)49
-12-
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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: . . . . . .. . .
, :;, .
WO90/14356 PCT/US90/03113
t~
--13--
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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'. . . . ..
WOgO/14356 ~ PCT/US90/03113
! P~
-14-
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 ~
-16-
(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
': ` . ", :,
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W O 90/14356 2~7049 PC~r/US90/03113
~r ~h-',~,~
-17-
- 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 ~
-18-
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
.. .. . .. .,.. . ~-
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WO90/14356 2C~ 3 Sgo/03113
.:,,i ~. '.. ` .
--19--
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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. . : . :: . . : -
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WO90/14356 - PCT/US90/03113
2 ~ 7 ~ 3~`
-20-
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
h - - -
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W O 90/14356 ~S70 - PC~r/US90/03113
-21-
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~.. `. . ~
-22-
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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'`: " " ' ',,' ' ,'"'''''.',' ' '"' ' ;" -''
. -" ' ~ ,' '- . : .
W O 90/14356 PC~r/US90/03113
~ 2~ g~
-23-
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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' ' . ; - - .: ' ,'....... . .
- . .
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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(~,~, ,, ~
-25-
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-; ~
-26-
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
-27-
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
... . . . . . ..... . . . . . .
.. , . . ~. . .
W O 90/14356 ~ ~ PC~r/US90/03113
. ~
-2R-
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
-29-
~ ~, ;; 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
. .. .- , ~ ........................................ : . . .
- ' :; -' . ' - ~
- .. ,.~ : - . . . .. . . .: . .. - :....... .
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 '~
-31-
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 `
-32-
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
~: . .` ., . . ,, : ', :. . '
,` :' `' '~'" ':. ` ' ' ' ' ' ~ ' .' ".., , '
.~' ' ~ ' ': ' .-` . .
~; ' ' ' ' ":: . .:
W090/l4356 PCT/US90/03113
2C57~)49
-33-
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
.. . . . .......... . .
.. . .: .~. . ..
. , . .. . . ~ . , : . , . ...................... :
. , .- . . ~ - : . .
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
.. ...
. , .. - ,: ,.; . . -- . . . .. .
: ~ . , :, - . . .
- . . ,~. ~ . - ,. .
- . . , .. -
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
-37-
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`- . ~
-38-
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
, . . , : :
. .. . . .
- . . . . . . . .
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.. . . . . ..
. .
'' . '
WO90/14356 PCT/US90/03113
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
. .: . . .` ~ ' ' ' .. ~'. ., . '
WO90/14356 Z~57~4~ PCT~US90~03ll3
-41-
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
.
- . ;:, . . .: . : .: -
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