Note: Descriptions are shown in the official language in which they were submitted.
W~ 96/00739 2 1 9 ~ 8 ~ C t ~ ~ [
T~TLE OF THE INVENTION
MODIFIED G-PROTEIN COUPLED RECEPTORS
CROSS RELATED TO OTHER APPLICATIONS
S This is a c~-ntinll~tion-in-part of U.S. Serial Number
0~s/267,9~i7, filed June 29, 1994, now pending, and an continuation-in-
part of U. S. Serial Number 0~/267,9~7, filed June 29, 1994, now
pending.
BACKGROUND OF THE INVENTION
G-protein coupled receptors are cell surface receptors that
mediate the responses of the cell to a variety of environmental signals.
Upon binding an agonist, the receptor interacts with one or more
specific G protein.s, which then regulate the activities of specific effector
1:~ proteins. By this means, activation of G-protein coupled receptors
amplifie.s the effects of the environmental signal and initiates a cascade
of intracellular events that ultimately leads to a defined cellular
re.sponse. The family of G-protein coupled receptor.s function as a
complex information processing network within the plasma membrane
of the cell, acting to coordinate a cell's response to multiple
environmental signals.
G-protein coupled receptors are characteri~ed by the ability
of agonists to promote the formation of a high affinity ternary complex
between the agonist, the receptor and the G-protein (Figure 1). The oc-
25 . .subunit of the G protein contain.s a guanine nucleotide binding site
which, in the high affinity ternary rG protein-receptor-agonist]
complex, is occupied by GDP. In the presence of physiological
concentrations of GTP, the GDP molecule in the guanine nucleotide
binding site of the G protein i.s displaced by a GTP molecule. The
30 binding of GTP dissociates the o~ subunit of the G protein from its 13
subunits and from the receptor, thereby activating the G-protein to
stimulate down.stream effectors (adenylyl cyclase in the case of the ,~-
adrenergic receptor (~AR)) and propagating the intracellular signal.
Thus, the ternary complex is transient in the presence of physiological
WO 9~/00739 2 i ~ 3 ~ o 9 . ~ C ~
.
GTP concentrations. Because the affinity of the agonist for the
receptor-G protein complex is higher than its affinity for the
uncomplexed receptor, one consequence of the destabilization of the
ternary complex is a reduction in the affinity of the receptor for the
5 agonist. Thus, the affinity of agonists for G-protein coupled receptor.s
i.s a function of the efficiency with which the receptor i.s coupled to the
G-protein. In contrast, antagoni.sts bind with the same affinity to the
receptor in the presence or absence of G-protein coupling.
The observation that agonist affinity can be reduced by
10 conditions under which a receptor is not optimally coupled to it.s G-
protein has important implication.s for the identification of agonists of
G-protein coupled receptors, particularly identification based on ligand
binding. If the receptor is not optimally coupled to the G-protein under
the conditions of binding a.ssays, an agonist will bind to the receptor
15 with relatively low affinity. Thus, a screen that relies on a binding
assay based on displacement of a radiolabeled ligand, although attractive
for its ease and the potential for high throughput, poses the risk that a
promising partial agoni.st might be overlooked because the agonist
would bind predominantly to the low affinity state of the receptor, and
20 thus would have low affinity in the binding assay. Consequently,
functional assays are frequently used to screen for agoni.sts of G-protein
coupled receptor.s. However, functional assays (ranging from ex vivo
muscle contraction assays to determination of second me.ssenger levels
in cells expressing exogenous cloned G-protein coupled receptors) are
25 .tediou.s and much more time-consuming than ligand binding as.says, and
hence are not readily adapted to high throughput screen.s. Because the
modified receptor.s of the pre.sent invention bind agonists with high
affinity in the presence or absence of the G-protein. tbey can be used in
high throughput radioligand binding a.ssays to screen for high affinity
30 ligands, regardless of whether the ligands are agonists or antagonists.
G-protein coupled receptors consist of seven hydrophobic
domains cr-nn~c~ing eight hydrophilic domain.s. The hydrophobicity or
hydrophilicity of the domains may be determined by standard
hydropathy profiles, such as Kyte-Doolittle analysis (Kyte, J. and
... .
2 1 a2Qn~
W0 96/00739 1'~
,. -
Doolittle, R.J.F. J. Mol. Biol. 157: 105 (19~S2)). The receptors are
thought to be oriented in the plasma membrane of the cell in such a way
that the N-terminus of the receptor faces the extracellular space and the
Cl-terminus of the receptor faces the cytoplasm, such that each of the
hydrophobic domains crosses the plasma membrane. The receptor.s
have been modeled and the putative boundaries of the extracellular,
transmembrane and intracellular domain.s are generally agreed upon
based on these models (for a review, see Baldwin, EMBO i. 12:1693,
1993). In general, the tran.smembrane domains are comprised of
.stretches of 20-25 amino acid.s in which most of the amino acid re.sidue.s
have hydrophobic side chains (including cy.steine, methionine,
phenylalanine, tyrosine, tryptophan, proline, glycine, alanine, valine,
leucine, isoleucine), wherea.s the intracellular and extracellular loops are
defined by contiguous stretche.s of several amino acids that have
hydrophilic or polar side chain.s (including a.spartate, glutamate,
asparagine, glllt~lmini~, serine, threonine, histidine, Iysine, and arginine).
Polar amino acids, especially uncharged ones (such as .serine, threonine,
asparagine, and glllt~min~) are found in both transmembrane and
extramembrane regions.
The extramembrane regions are characterized by
contiguous stretches of three or more hydrophilic residues. In contra.st,
hydrophilic residues are found only in groups of 1-2, surrounded by
hydrophobic residues, in the transmembrane domain. Thus, the
transmembrane and extramembrane regions can be identified by the
number of contiguous hydrophilic or hydrophobic amino acids in the
primary .sequence of the receptor, in addition to the constraints on the
length of the hydrophobic segments given above. The boundaries
between the transmembrane and extramembrane regions are often
defined by the presence of charged or polar residues at the beginning or
end of a stretch of hydrophobic amino acids. The locations of the
mutations in the receptors of the present invention are described on the
basis of these models and can be specifically defined by the specific
amino acid numbers of the residues being mutated.
2t938o~
W0 96/00739 r~
- 4 -
By these criteria, the third intracellular loop is defined as
the hydrophilic loop connecting the hydrophobic, putative
lla~ ,lelllbrane domains V and Vl. For example, in hamster ~2
adrenergic receptor, used to particularly exemplify the invention, the
third intracellular loop would refer to amino acids 221 through 273
(Figure 2). In accordance with the principles described above, the
beginning of this loop is defined by the presence of Arg221 (a charged
residue at the end of the hydrophobic stretch of residues 19~-220) and
Lys273 (a charged residue at the beginning of the hydrophobic stretch
of residues 274-29~).
The present invention pertains to modifed G-protein
coupled receptors having deletions in the third intracellular domain.
Methods of designing and making modified receptors are provided.
The modified receptors are uncoupled from or are poorly coupled to
their respective G-proteins. However, these modified receptors bind
agonists with high affinity in the absence of G protein coupling.
Because of their high intrinsic affinity for agonists, these modified
receptors may be used in hi~h throughput binding assays to identify
compounds that bind to the receptor with high affinity, regardless of
whether these compound.s are a~onists or anta~onists. The invention
includes the DNA encodin~ the modified receptors, the modified
receptors, assay.s employin~ the modifed receptors, cells expressing
the modified receptors, and substances identified through the use of
the modified receptors including specifc modulators of the modified
receptors. Modulators identified in this process are useful as
therapeutic a~ents. Modulator.s, as described herein, include but are
not limited to agonists, antagonists, suppressors and inducers.
SUMMARY OF THE INVENTION
.= M odified G-protein coupled receptors havin~ deletions
in the third intracellular domain are identified and methods of
makin~ the modified receptors are provided. The invention include.s
the modified receptors, assays employin~ the modified receptors,
cells expressing the modified receptors, and compounds identified
2 ~ 9~8~~i
WC~ 96/00739
.
~ through the use of the modified receptors, including modulators of
the receptor.s. Modulator.s identified in this proce.s.s are useful as
therapeutic agents.
BRIFF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic diagram of G-protein signal tran.sduction .sy.stem.
The receptor is shown as a seven-helical bundle. a, l~, and ~ indicate the
three subunits of the G protein. E indicates an effector enzyme, such a.s
adenylyl cyclase. The agonist (A) binding with high affinity to the
receptor-G protein complex and with low affinity to the receptor alone
i.s shown.
Figure 2. Schematic diagram of the hamster ~2 adrenergic receptor.
The third intracellular loop comprises re.sidues 221-273. The proximal
and distal segments of this loop are drawn in cylinders.
Figure 3. Stimulation of cAMP production as a function of
isoproterenol by the wild type j33AR (clo.sed circles) but not the
modified D(227-234) (triangles) or D(277-2~9)~3AR (.s~luares).
Figure 4: Binding of an agonist and an antagonist to the wild type (open
circle.s) and D(277-2~9) ,~3AR (closed circle.s). Binding of the agonist
isoproterenol (top panel) or the antagonist propranolol (bottom panel)
was mea.sured in competition with the radioligand 1251-cyanopindolol.
Figure 5. Inhibition of adenylyl cyclase activity. A concentration
dependent respon.se curve of the ability of 5-HT to inhibit adenylate
cyclase activity mediated by the wild type 5-HTID~ receptor is .shown.
However, in the histogram on the right of the figure, the inability of
100 mM 5-HT activating at the mutant receptor, D(231-239)5-HTlD,~
to produce an inhibition of adenylate cyclase activity is demonstrated.
The results shown are from a typical experiment and were repeated
three times and are representative of three independent mutant receptor
cell lines [D(231-239)5-HTID~ clones 1. 21 and 65]. Formation of
WO 96/00739 2 t q ~ 8 0 9 r~ o~
32P-cAMP from 32P-ATP was measured in crude membrane
preparation.s prepared from CHO cells stably expressing the ~ v,o,;ale
receptors .
Figure 6. Table 1: Binding and functional parameters of the wild type
and modified ~2AR.
Figure 7. Table 2: Binding parameters of the wild type and modified
,~3AR .
Figure ~. Table 3: Radioligand binding properties of modified 5HT-
I D~ receptor.s. Presented in the table are the specific binding value.s
(dpm) of 2 nm [3H]5-HT ob.served in the presence and absence of the
guanine nucleotide analog, GppNHp (100 mM). Also shown is the
percenta~e inhibition of adenylate cyclase activity (%AC inhibition) for
the respective cell lines. Results shown are from a typical experiment
and were repeated three times.
DETAILED DESCRIPTION OF THE INVENTION
Modified G-protein coupled receptors having deletions
in the third intracellular domain are identified and methods of
making the modified receptor.s are provided. The modified
receptors are uncoupled from or are poorly coupled to their
re.spective G-protein.s and may be used in assays to identify
25 . substance.s that bind to the receptor regardless of whether these
suhst~nr~s are agonists or antagonists. The invention includes the
modified receptors, assays employing the modified receptors, cells
expressing the modified receptors, and compound.s identified through
the use of the modified receptors, including modulators of the
30 receptors. Modulators identified in this process are u.seful as
therapeutic agent.s. Modulators, as described herein, include but are
not limited to agonists, antagonists, suppressors and inducers.
The term "G-protein coupled receptor" refer.s to any
receptor protein that mediates its endogenous signal transduction
wo s6A~0739 2 1 q ~ 8 0 ~
- 7 -
through activation of one or more guanine nucleotide bindung
regulatory proteins (G-proteins). These receptors share common
structural features, including seven hydrophobic tr~n~m~mhrane
domains. G-protein coupled receptors include receptors that bind to
5 small biogenic amines, including but not limited to beta-adrenergic
receptors (~AR), alpha-adrenergic receptors (o~AR) and muscarinic
receptor.s, as well as receptors whose endogenous ligands are peptides,
such as neurokinin and glucagon receptors. Examples of ~AR include
beta-l, beta-2, and beta-3 adrenergic receptors. Examples of ocAR
10 include alpha-la, alpha-lb, alpha- I c. alpha-2a, alpha-2b, and alpha-2c.
Examples of muscarinic receptors include M I, M2, M3, M4 and M5.
Example.s of neurokinin receptors include NKI, NK2 and NK3. Other
examples of G-protein coupled receptors include but are not limited to
adenosine 2 receptor, alpha-2 adrenergic receptor.s, type-l angiotensin
15 11 receptor, cholecytokinin B receptor, gastrin receptor, somatostatin
receptor, 5-hydroxytryptamine I beta receptor, A2 adenosine receptor,
Burkitt's Iymphoma receptor, neuropeptide Y receptor, tachykinin
receptor, .serotonin receptor, formyl peptide receptor like-l, tyramine
receptor, muscarinic acetylcholine receptor, certain endothelin
20 receptors, complement protein 5a receptor, choriogonadotropic
hormone receptor, high affinity interleukin ~ receptor, follicle
.stimulating hormone receptor, dopamine Dl receptor, C5a
anaphylotoxin receptor, hi~t~min~ H2 receptor, substance P receptor,
thyrotropin receptor and. Iuteininzing hormone receptor. G-protein
coupled receptors have been i.solated from a variety of animals,
including but not limited to humans, cows, goats, mice, pigs and rats.
Modified eceptors may include genetic variants, both
natural and induced. Induced modified receptor.s may be derived by a
variety of methods, including but not limited to, site-directed
30 mutagenesis. Techni~lue.s for nucleic acid and protein manipulation are
well-known in the art and are described generally in Methods in
Enzymology and in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory ( 19R9).
WO 96100739 2 1 9 3 8 0 9 P~l/UJ~ 5.
It is kno~,vn that there is a 5nh~t~nti~1 amount of
redundancy in the variou.s codons which code for specific amino
acids. Therefore, this invention is also directed to those DNA
sequences which contain alternative codon,s which code for the
5 eventual translation of the identical amino acid. For purposes of thi.s
speciflcation, a sequence bearing one or more replaced codons will
be defined as a degenerate variation. Al.so included within the scope
of this invention are mutations either in the DNA .sequence or the
tran.slated protein which do not sllhsl~nti~lly alter the ultimate
10 phy.sical properties of the expres.sed protein. For example,
sub.stitution of valine for leucine, arginine for Iysine, or asparagine
for glutamine may not cause a change in functionality of the
polypeptide.
It is known that DNA sequences coding for a peptide
11; may be altered so as to code for a peptide having properties that are
different than those of the naturally-occurring peptide. Methods of
altering the DNA sequences include, but are not limited to site
directed mutagenesis. Examples of altered properties include but are
not limited to changes in the affinity of an enzyme for a substrate or
20 a receptor for a ligand.
A.s used herein, a "functional derivative" of a modified
receptor is a compound that posses.se.s a biological activity (either
functional or structural) that is substantially similar to the biological
activity of the modified receptor. The term "functional derivative"
2~ is intended to include the "fragments," "variants," "degenerate
variants," "analogs" and "homologues" or to "chemical derivatives"
of modified receptors. The term "fragment" is meant to refer to any
polypeptide subset of modified receptors. The term "variant" is
meant to refer to a molecule substantially similar in structure and
30 function to either the entire modified receptor molecule or to a
fragment thereof. A molecule is "substantially similar" to a
modified receptor if both molecules have substantially similar
structures or if both molecules po.s.se.ss similar biological activity.
Therefore, if the two molecules possess substantially similar activity,
..... ..... ... . . . ... . . ....
2 ~ q3~i~q
W096/00739 - rc.,.
they are considered to be variants even if the structure of one of the
molecule.s is not found in the other or even if the two amino acid
sequences are not identical.
The term "analog" refers to a molecule suh.st~nti~lly
~; similar in function to either the entire modified receptor molecule or
to a fragment thereof.
"Sub,stantial homology" or "substantial similarity", when
referring to nucleic acids means that the segments or their
complementary .strand.s, when optimally aligned and compared, are
identical with appropriate nucleotide insertions or deletion.s~ in at least
75% of the nucleotides. Alternatively, .substantial homology exists when
the segments will hybridize to a .strand or its complement.
The nucleic acids claimed herein may be present in whole
cells or in cell Iysates or in a partially purified or .sllhst~nli~lly purified
1:; form. A nucleic acid is considered substantially purified when it is
purified away from envilul.,~ l cnnt:~min~ts. Thus, a nucleic acid
.sequence isolated from cells is considered to be s~lh.~t~nti~lly purified
when purified from cellular components by standard method.s while a
chemically synthesized nucleic acid sequence is considered to be
sub.stantially purified when purified from it.s chemical precursons.
Nucleic acid composition.s of this invention may be derived
from genomic DNA or cDNA, prepared by synthesis or by a
combination of techni4ues.
The natural or synthetic nucleic acids encoding the
modified G-coupled protein receptors of the present invention may be
incorporated into expression vectors. Usually the expression vector.s
incorporating the modified receptor.s will be suitable for replication in a
ho.st. Examples of acceptable hosts include, but are not limited to,
prokaryotic and eukaryotic cells.
The phrase "recombinant expression system" as used
herein means a substantially homogenous culture of .suitable host
organism.s that stably carry a recombinant expression vector. Example.s
of suitable hosts include, but are not limited to, bacteria, yeast, fungi,
insect cells, plant cell.s and m~mm~ n cells. Generally, cells of the
W0 96/00739 2 1 q ~ 8 (~ 9 r~ r
- 10 -
expression system are the progeny of a single ancestral transformed
cell.
The cloned modified receptor DNA obtained through
the methods described herein may be recombinantly expressed by
5 molecular cloning into an expression vector containing a suitable
promoter and other appropriate transcription regulatory elements7
and transferred into prokaryotic or eukaryotic host cells to produce
recombinant modified receptor. Techni4ue.s for .such manipulations
are fully described in Sambrook, J., et al., supra, and are well known
10 in the art.
Expres.sion vectors are defined herein as DNA se~luences
that are re~luired for the transcription of cloned copies of genes and
the translation of their mRNAs in an dp~ iat~ host. Such vectors
can be used to express eukaryotic genes in a variety of hosts such as
15 bacteria, bluegreen algae, plant cells, insect cells, fungal cells and
animal cells.
Specifically designed vectors allow the shuttling of DNA
between hosts such as bacteria-yeast or bacteria-animal cells or
bacteria-fungal cells or bacteria-invertebrate cells. An appropriately
20 con.structed expression vector should contain: an origin of replication
for autonomous replication in host cells, selectable markers, a
limited number of useful restriction enzyme sites, a potential for
high copy number, and active promoters. A promoter is defined as a
DNA se4uence that directs RNA polymera.se to bind to DNA and
25 .initiate RNA synthesis. A .strong promoter is one which causes
mRNAs to be initiated at high fre~luency. Expression vectors may
include, but are not limited to, cloning vector.s, modified cloning
vectors, specifically designed plasmids or viruses.
A variety of m~mm~ n expression vectors may be used
30 to express recombinant modified receptor in m:~mm~ n cells.
Commercially available m:lmm~ n expression vectors which may
be suitable for recombinant modified receptor expression, include
but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene),
pXTI (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC
_ _ _ _ _ . _ _ . _ , ~ . .. .
WG96100739 2~ q~Q~ rc~
37593) pBPV~ -2) (ATCC 37110), pdBPV-MMTneo(342-12)
(ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 3719~),
~ pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and ~ZD35
(ATCC 37565).
S A variety of bacterial expression vectors may be u.sed to
express recombinant modified receptor in bacterial cells.
Commercially available bacterial expression vectors which may be
suitable for recombinant modified receptor expression include, but
are not limited to pETI la (Novagen), lambda gtl l (Invitrogen),
pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
A variety of fungal cell expre.s.sion vectors may be u.sed
to express recombinant modified receptor in fungal cell.s.
Commercially available fungal cell expression vectors which may be
.suitable for recombinant modified receptor expression include but
are not limited to pYES2 (lnvitrogen), Pich~l expression vector
(Invitrogen).
A variety of insect cell expression vectors may be used
to expre.ss recombinant receptor in insect cells. Commercially
available insect cell expre.ssion vector.s which may be suitable for
recombinant expression of modified receptor include but are not
Iimited to pBlue Bac III (Invitrogen).
An expression vector containing DNA encoding
modified receptor may be used for expre.ssion of modified receptor
in a recombinant host cell. Recombinant host cells may be
prokaryotic or eukaryotic, including but not limited to bacteria such
as E. coli, fungal cells such as yeast, m~mm~ n cells including but
not limited to cell lines of human, bovine, porcine, monkey and
rodent origin, and insect cells including but not limited to Drosophila
and .silkworm derived cell lines. Cell lines derived from m~mm~ n
species which may be suitable and which are commercially available,
include but are not limited to, L cells L-M(TK-)(ATCC CCL 1.3),
L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC
CCL ~6), CV-I (ATCC CCL 70), COS-I (ATCC CRL 1650), COS-7
(ATCC CRL 1651), CHO-KI (ATCC CCL 61~, 3T3 (ATCC CCL
WO 96100739 , 1 q ~ 8 a 9 r~
92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271
(ATCC CRL 1616), BS-C-I (ATCC CCL 26) and MRC-5 (ATCC
CCL 171).
The expres.sion vector may be introduced into host cell.s
5 via any one of a number of techniques including but not limited to
transformation, transfection, lipofection, protoplast fusion, and
electroporation. The expre.ssion vector-containing cell.s are clonally
propagated and individually analy~ed to determine whether they
produce modified receptor protein. Identification of modified
10 receptor expressing host cell clones may be done by several means,
including but not limited to immunological reactivity with anti-
modified receptor antibodies.
Expression of modified receptor DNA may also be
performed using iM l~i~l O produced synthetic mRNA or native
15 mRNA. Synthetic mRNA or mRNA isolated from modified receptor
producing cells can be efficiently translated in various cell-free
systems, including but not limited to wheat germ extracts and
reticulocyte extracts, as well as efficiently translated in cell based
sy.stems, including but not limited to microinjection into frog
20 oocyte.s, with microinjection into frog oocytes being preferred.
The term "substantial homology", when referring to
polypeptides, indicates that the polypeptide or protein in question
exhibits at lea.st about 30% homology with the naturally occurring
protein in question, usually at least about 65~o homology.
25 . The modified receptors may be expressed in an
appropriate host cell and used to discover compounds that affect the
modified receptor. Preferably, the modified receptors are expressed in
a m~mm~ n cell line, including but not limited to, COS-7, CHO or L
cells, or an in.sect cell line, including but not limited to Sf9 and Sf21,
30 and may be used to di.scover ligands that bind to the receptor and alter
or stimulate its function. The modified receptors may al.so be produced
in bacterial, fungal or yeast expression systems.
The expression of the modified receptor mày be detected
by use of a radiolabeled ligand specific for the receptor. For the ~2
wog61oa739 21 93809 r ~ r
,.. ..
adrenergic receptor used herein to exemplify the invention, such a
ligand may be 1251-iodocyanopindolol (1251-CYP).
The specificity of binding of compounds showing affinity
for the modified receptors is shown by measuring the affinity of the
5 compounds for cells transfected with the cloned modified receptor or
for membranes from these cells. Expression of the cloned modified
receptor and screening for compounds that inhibit the binding of
radiolabeled ligand to these cells provides a rational way for selection of
compound.s with high affinity for the receptor. The.se compounds may
10 be agonist.s or antagonists of the receptor. Because the modified
receptor does not couple well to G proteins, the agonist activity of these
compounds is best as.sessed by using the wild-type receptor, either
natively expres.sed in tissues or cloned and exogenously expressed.
Once the modified receptor is cloned and expressed in a
15 mammalian cell line, such as COS-7 cells or CHO cells, the recombinant
modified receptor is in a well-characterized environment. The
membranes from the recombinant cells expres.sing the modified
receptor are then isolated according to methods known in the art. The
i.solated membranes may be u.sed in a variety of membrane-based
20 receptor binding assays. Becau.se the modified receptor has a high
affinity for agonists, ligand.s (either agonists or antagonists) may be
identified by standard radioligand binding assays. These assays will
mea.sure the intrinsic affinity of the ligand for the receptor.
The present invention provides methods of generating
25 .modified G-protein coupled receptors. Such methods generally
comprise the deletion of at least one nucleotide from the third
intracellular domain of the receptor. Additional methods include, but
are not limited to, enzymatic or chemical removal of amino acids from
- the third intracellular domain of the receptor. One method of
30 generating modified G-protein receptors comprises:
~ (a) isolating DNA encoding a G-protein coupled receptor~
(b) altering the DNA of step (a) by deleting at least one
nucleotide from DNA encoding the third intracellular domain of the G-
protein coupled receptor;
WO 96/00~39 2 1 9 ~ ~ ~ 9 r~"~
- 14 -
(c) isolating the altered DNA;
(d) expressing the altered DNA; and
(e) recovering the modified G-protein coupled receptor.
The third intracellular domain of a G-protein coupled receptor is
5 located between the fifth and sixth hydrophobic transmembrane domains
of the receptor (Figure 2).
The present invention provides methods of identifying
compounds that bind to modified G-protein coupled receptors. Methods
of identifying compound.s are exemplified by an assay, comprising:
a) cloning the G-protein coupled receptor;
b) altering the DNA .sequence encoding the third
intracellular domain of the cloned G-protein coupled receptor;
c) .splicing the altered receptor into an expre.ssion vector to
produce a construct such that the altered receptor is operably linked to
15 transcription and translation signals .sufficient to induce expression of
the receptor upon introduction of the construct into a prokaryotic or
eukaryotic cell;
d) introducing the construct into a prokaryotic or
eukaryotic cell which does not express the altered receptor in the
20 absence of the introduced construct; and
e) incubating cells or membranes i.solated from cells
produced in step c with a quantifiable compound known to bind to the
receptors, and subsequently adding test compounds at a range of
concentrations so as to compete the quantifiable compound from the
25 receptor, such that an IC50 for the test compound is obtained as the
concentration of test compound at which 50% of the quantifiable
compound becomes displaced from the receptor.
The present invention i.s also directed to methods for
.screening for compounds which modulate the expression of DNA or
30 RNA encoding modified receptors or which modulate the function of
modifed receptor protein. Compounds which modulate these
activities may be DNA,RNA, peptides, proteins, or non-
proteinaceous organic molecules. Compounds may modulate by
increa.sing or ~tt~n--~ting the expression of DNA or RNA encoding
.... . _ ..... ... .. _ .. ...... ... _ _ ... _ ..... .. . . _ .. . _ _ ... . ......
21 93809
wal 961011739 r "~1 ~ f~ ~ -
.
modified receptor, or the function of modihed receptor protein.
Compounds that modulate the expression of DNA or RNA encoding
~ modified receptor or the function of modified receptor protein may
be detected by a variety of assays. The assay may be a simple
5 "yeslno" a.ssay to determine whether there is a change in expression
or function. The as.say may be made quantitative by comparing the
expre.ssion or function of a test sample with the levels of expression
or function in a standard sample.
Kits containing modified receptor DNA, antibodies to
10 modified receptor, or modified receptor protein may be prepared.
Such kit.s are used to detect DNA which hybridizes to modified
receptor DNA or to detect the presence of modified receptor protein
or peptide fragment.s in a sample. Such characterization is useful for
a variety of purpose.s including but not limited to forensic,
1:~ taxonomic or epidemiological studies.
The DNA molecules, RNA molecule.s, recombinant
protein and antibodies of the present invention may be used to .screen
and measure leveh; of modified receptor DNA, modified receptor
RNA or modified receptor protein. The recombinant proteins, DNA
20 molecules, RNA molecules and antibodies lend themselves to the
formulation of kits .suitable for the detection and typing of modified
receptor. Such a kit would compri.se a compartmentalized carrier
suitable to hold in close c~ le.ll~ at least one container. The
carrier would further comprise reagents such as recombinant
~~ modihed receptor protein or anti-modified receptor antibodies
suitable for detecting modified receptor. The carrier may also
contain a means for detection .such a.s labeled antigen or enzyme
substrates or the like.
Pharmaceutically useful compositiom; comprising
30 modulators of modified receptor activity, may be formulated
according to known methods such as by the admixture of a
pharm~relltic~lly acceptable carrier. E~xamples of such carriers and
method.s of formulation may be found in Remington's
Ph~ reutir~l Sciences. To form a pharmaceutically acceptable
, .. . . .
W0 96~073g 2 1 9 ~ 8 ~ 5
.
- 16 -
compo~sition suitable for effective administration, such compositions
will contain an effective amount of the protein, DNA, RNA, or
modulator.
Therapeutic or diagno.stic compositions of the invention
are admini.stered to an individual in amounts sufficient to treat or
diagnose disorders. The effective amount may vary according to a
variety of factors such as the individual'.s condition, weight, .sex and
age. Other factors include the mode of administration.
The pharmaceutical compositions may be provided to
the individual by a variety of routes such a.s subuu~ eous, topical,
oral and intramuscular.
The term "chemical derivative" describes a molecule
that contains additional chemical moieties which are not normally a
part of the base molecule. Such moieties may improve the solubility,
half-life, absorption, etc. of the base molecule. Alternatively the
moieties may attenuate unde.sirable side effects of the base molecule
or decrease the toxicity of the base molecule. Examples of such
moieties are described in a variety of texts, .such as Remington'.s
Pharmaceutical Sciences.
Compounds identified according to the methods disclosed
herein may be u.sed alone at appropriate dosages. Alternatively, co-
administration or se4uential administration of other agents may be
desirable .
The present invention also has the objective of providing
~~ suitable topical, oral, systemic and parenteral pharmaceutical
formulations for use in the novel methods of treatment of the present
invention. The compositions containing compounds identified
according to this invention as the active ingredient can be
administered in a wide variety of therapeutic dosage forms in
conventional vehicles for admini.stration. For example, the
compound.s can be administered in such oral dosage forms as tablet.s,
capsules (each including timed release and sustained release
~ formulations), pills, powders, granules, elixirs, tinctures, solutions,
suspensions, syrups and emulsions, or by injection. Likewise, they
W0 96/00739 2 1 9 3 ~ 0 9 ~ , C, ~ ' [ [
- 17 -
may also be administered in intravenous (both bolus and infusion),
intraperitoneal, subcutaneous, topical with or without occlusion, or
intramuscular form, all using form.s well known to tho.se of ordinary
skill in the pharmaceutical arts.
Advantageously, compound.s of the present invention
may be administered in a single daily do.se, or the total daily dosage
may be administered in divided do.ses of two, three or four times
daily. Furthermore, compounds for the present invention can be
administered in intranasal forrn via topical use of .suitable intrana.sal
vehicles, or via tran.sdermal routes, using tho.se forms of transdermal
skin patche.s well known to those of ordinary skill in that art. To be
administered in the form of a transdermal delivery system, the
dosage administration will, of course, be continuous rather than
intermittent throughout the dosage regimen.
lS For combination treatment with more than one active
agent, where the active agents are in separate dosage forrnulations,
the active agent.s can be ~dministl~red concurrently, or they each can
be a~lrnini~stered at separately .staggered times.
The dosage regimen utilizing the compounds of the
present invention i.s selected in accordance with a variety of factors
including type, species, age, weight, sex and medical condition of the
patient; the severity of the condition to be treated; the route of
administration, the renal and hepatic function of the patient; and the
particular compound thereof employed. A physician or veterinarian
of ordinary skill can readily determine and prescribe the effective
amount of the drug required to prevent, counter or arrest the
progre.ss of the condition. Optimal precision in achieving
concentrations of drug within the range that yields efficacy without
toxicity re~luires a regimen based on the kinetics of the drug's
availability to target sites. This involves a consideration of the
distribution, equilibrium, and elimination of a drug.
The modified G-protein coupled receptors of the present
invention are exemplified herein by the hamster beta-2 (,~2) adrenergic
receptor, the human ~3 receptor and the human SHT-lD,~ receptor.
.. . . .. . . . . .....
WO 96/00739 2 1 ~ 3 8 ~ 9 ~ --
Deletion mutagenesis of the ,~2-adrenergic receptor has
shown that none of the hydrophobic clusters of amino acids (the putative
tran.smembrane helices) could be deleted without substantial loss of
binding. In contrast, most of the connecting loops could be deleted
5 without affecting the ]igand binding properties of the receptor. This
indicates that these hydrophilic loop.s are not required for ligand
binding to the receptor, .suggesting that the ligand binding pocket is
located predominantly within the tran.smembrane domain of the protein
(Strader, et al FASEB J .3: 1~2-1~3 (19~9)). Deletion.s in the
10 connecting loops that were large enough to encompass the entire loop
led to .steric problems, re.sulting in incorrect processing of the protein
(Dixon, et al. EMBO J. 6: 3269-3~75 (19~7)). Certain connecting loop
deletion mutations, however, led to loss of functional activation of
adenylyl cyclase by the receptor. For example, deletion of the carboxy
15 terminal region of the third intracellular loop attenuated the ability of
the receptor to activate adenylyl cyclase, and deletion of the amino
terminal portion of this loop abolished adenylyl cyclase activation
(Strader, et al J. Bi~l. Chem. 262: 16439-16443 (19~7)). Moreover, the
agonist binding isotherms for the.se modifed receptor.s displayed a
20 .single affinity site, suggesting altered G protein interaction.s. Since
these modified receptors also retain their functional activation of Na+-
H+ exchange, which is mediated through a different G protein (Barber,
et al. M~l. Pha~-m. 41: 1056-1060 (1992)), the deletions appear not to
result in gross structural perturbations of the receptor, suggesting that
25 .the changes seen in adenylyl cyclase activation are due to alteration of a
specific G protein interaction. Subsequent amino acid repl~PmP.nt.s in
the third intracellular loop confirmed the role of this region in G
protein interaction (Cheung, et al. Mol. Pharm. 41: 1061-1065 (1992)).
The following examples are provided to further define the
30 invention without, however, limiting the invention to the particular.s of
the example.s.
WO 96/00739 21 9 ~ 8 ~ 9 r~
- 19 -
EXAMPLE I
Deletion of 6-12 amino acids at the N-terminal portion of the third
- jntracellular loop of the hamster ~ adrener~ic receptor
Modified receptor D(222-229)~2AR wa.s de.scribed in
5 Strader et al. (J. Biol. Chem. 262: 16349, 1987). A modified cDNA
encoding the ham.ster ,~2AR in which residues 222-229 (Val-Phe-Gln-
Val-Ala-Lys-Arg-Gln) are deleted was constructed by .standard
oligonucleotide-directed mutagenesis procedures.
The modified receptor is designed so as to disrupt the
10 proximal portion of the third intracellular loop, without affecting the
adjacent fifth tran~smembrane helix. Thu.s, the charged amino acid
(Arg221) that delineate.s the bottom of helix 5 is left intact in the D(222-
229) modified receptor, while the following eight arnino acids are
deleted. The size of the deletion in the present invention may vary from
15 six to 13 amino acid.s in the.se regions, beginning immediately after the
charged residue at the end of transmembrane helix 5.
EXAMPI F 2
Deletion of amino acid.s at the C-terminal pollion of the third
20 intraçellular loop of the ham~ster ,~ adrener~ic receptor
Modified receptor D(258-270)~2AR was described in
Strader et al. (J. Biol. Chem. 262:16349, 1987~. A modified cDNA
encoding the hamster ~2AR in which residues 258-270 (Leu-Arg-Arg-
Ser-Ser-Ly.s-Phe-Cys-Leu-Lys-Glu-His-Lys) were deleted was
25 constructed by standard oligonucleotide-directed mutagenesis
procedures.
The modified receptor is designed so as to disrupt the distal
portion of the third intracellular loop, without affecting the adjacent
sixth transmembrane helix. Thus, the charged amino acid (Lys273) that
30 delineates the bottom of helix 6 is left intact in the D(258-270) modified
receptor, while the nearby proximal residues 258-270 are deleted. The
size of the deletion in the pre.sent invention may vary from six to 13
amino acids in these regions, ending 1-3 residues before the charged
residue at the beginning of helix 6.
W0 96100739 ~ & ~ ~ P~
1~
- 20 -
EXAMPLE 3 ~
Expres.sion and characterization of the altered ,~2 adrener~ic receptor.
COS-7 cells are transfected with the modified receptor
5 cDNA subcloned into a eukaryotic expression vector such as the
eukaryotic expression vector pcDNA l/neo (Invitrogen). Cells are
harvested after incubation for about 60-72 h. Membranes containing
the expressed receptor protein are prepared as described (C. D. Strader
et al., Pro(. Natl. Aca(l. Sci. U.S.A. 84, 4324-4322 (1927).
Binding reaction.s are performed in a final volume of 250
111 of TME buffer (75 mM Tris; 12.5 mM MgC12; 1.5 mM EDTA, pH
7.5) as described (Strader, et al J. Biol. Chem. 262: 16439 (1927)).
Adenylyl cyclase activity is measured as described (Strader, et al J. Biol.
Chem. 262: 16439 (1927)), with cAMP determined by the method of
15 Salomon (Anal. Biochem. 52: 541-542 (1974)).
Membrane.s prepared from the COS-7 cells transfected with
a vector containing either the wild type or the modified receptor cDNA
specifically bind the ~ receptor antagonist 1251-CYP. However, the
modified receptor is characterized by an absence of coupling to G.s, an
20 inability to mediate the activation of adenylyl cyclase, and an increased
affinity for agonists.
As shown in Table 1, the modified D(222-22~)~2AR,
- when expressed in L cells, does not stimulate adenylyl cyclase activation
in response to the agonist isoproterenol. In contrast, when the wild type
25 receptor i.s expressed in the same cell line. adenylyl cyclase activity is
stimulated by 3.2 fold, with an EC50 of 15 nM. The modified receptor
retains its ability to stimulate Na+-H+ exchange, indicating that .some
level of coupling to a G-protein other than Gs is retained (Barber et al.
Mol. Pharm. 41, 1056, 1992). Similarly, D(252-270),BAR show.s
30 impaired cAMP stimulation compared to the wild type receptor, with
only a small (1.3 fold) .stimulation over ba.sal levels.
These modified receptors have increased affinity for
agonists when compared to the wild type receptor. This is shown in
Table I, where the modified D(222-229) receptor binds the agonist
- 2~3809
WC~ 96/00739 1 ~II IJ~,5,'~C~ C-
- 21 -
~ isoproterenol with a single high affinity of 6 nM. The high affinity of
the agonist for the modified receptor is not affected by agents that
~ uncouple the receptor from the G protein; such agents include the nonhydrolyzable GTP analog GppNHp, sodium fluoride, and the
5 detergent digitonin. In contrast, the wild type receptor binds
isoproterenol with two affinity ~state.s: a high affinity state (Kd = 3 nM)
indicative of binding to the receptor-G protein complex, and a low
affnity .state (Kd = 200 nM) reflecting binding to the uncoupled
receptor alone (Table 1). In the presence of agents that interfere with
10 C protein coupling (GppNHp is such an agent shown in Table 1), the
agonist binds to the wild type receptor with a single low affinity state
(Kd = 200 nM).
The data in Table I demonstrate that when the receptor is
not optimally coupled to the G protein, a binding as.say using the
15 modified receptor will detect agonist.s with more sensitivity than will the
identical binding assay u.sing the wild type receptor. Similarly, D(25~-
270)1~AR binds to the agoni.st isoproterenol with a single high affinity of
~ nM, which is not significantly affected by the addition of Gpp(NH)p.
EXAMPLE 4
Screening A.ssay u.sing D(222-229) ~AR or D(2~-270)~AR
Tran.sfected cells expressing recombinant modified receptor
may be u.sed to identify compounds that bind to the receptor with high
affinity. Thi.s may be accomplished in a variety of way.s, such as by
.incubating the test compound in a final volume of 0.25 ml of TME
buffer with membranes containing 5-7 pM of the modified ~2AR and
35 pM 1251-CYP for I hour at 25~. The reaction is .stopped by
filtration over GF/C glass fiber filter.s, washing with 3 x 5 ml of cold
TME buffer, and counting the filters in a gamma counter to measure
bound radioactivity. This assay will detect a compound that has a high
intrinsic affinity for the receptor. Such compounds may be either
agoni.sts or antagonists.
W096,0073g 21 9~809 1~"~ '
- 22 -
EXAMPLE 5
Construction of Modified D(227-234) Beta-3 Adrenergic Receptor
Modified receptor D(227-234) ~3AR was constructed by
digesting the wild-type human ,~3 receptor cDNA (Granneman, et al.
5 Mol. Pharm. 42: 964-970 (1992))) with Accl and PvuII, followed by
re-ligation with a linker adaptor. The sequence of the linker adaptor is:
5'CTACGCGC~G3'/3'TGCGCGCC5' (SEQ IP NO:I).
10 The modified DNA sequence encodes a ~3AR lacking ~ amino acid
re.sidues (VFVVATRQ) at the N-terminal portion of the third
intracellular loop. The nucleotide sequence of the modified receptor
was confirmed by DNA se~luencing. As was the case for the modified
,~2 receptors, this modified ~3 receptor is designed so as to di.srupt the
15 proximal portion of the third intracellular loop, without affecting the
adjacent fifth ~ elllbrane helix. Thus, the charged amino acid
(Arg226) that delineates the bottom of helix 5 is left intact in the
D(227-234) modified receptor, while the eight amino acids which
follow it are deleted. The size of the deletion in the present invention
20 may vary from six to 13 amino acids in this region, beginning
immt~Ai~l~ly after the charged residue at the bottom of transmembrane
helix 5.
E~XAMPLE 6
25 .Co~struction of Modified D(277-2~9) Beta-3 Adrenergic ReceDtor
Modified D(277-2~9), lacking 13 residues at the C-terminal
portion of the third intracellular loop, was prepared by standard PCR-
based mutagenesis procedures. The nucleotide sequences of the
modified receptor.s were confirmed by DNA sequencing. As was the
30 case for the modified ~2 receptors, this modified ~3 receptor is
designed so as to disrupt the distal portion of the third intracellular
loop, without affecting the adjacent ~sixth transmembrane helix. Thu.,
the polar amino acids (C292,T293) that define the bottom of helix 6 are
left intact, while the nearby proximal residues 277-2~9 are deleted. The
W1~ 96/00739 2 1 9 3 ~ O q ~ C
- 23 -
size of the deletion in the present invention may vary from six to 13
amino acids in this region, ending immediately before the polar re.sidues
at the bottom of helix 6.
EXAMPLE 7 =
Expre.ssion and characterization of the modified ,~AR
The modified receptor was subcloned into the expression
vector pRC/CMV (Invitrogen, San Diego, CA) and expressed in mouse
L cells by DEAE-Dextran tran.sfection. 72 hours after transfection,
cells were harve.sted for radioligand binding or adenylyl cyclase assay.s.
For binding assay.s, the membrane.s were prepared by
harve.sting the cell.s in ice-cold Iysi.s buffer (5 mg Tri.s, pH 7.4; 2 mM
EDTA), followed by 15 min centrifugation at 38,000 x g. The
membrane pellet was then resu.spended in TME buffer. Equilibrium
binding to the wild type or modifed ~3AR was performed in a final
volume of 0.25 ml containing membranes, 240 pM 1251-CYP, and
serial dilution of the cr-mpeting ligands. Binding reactions were
incubated for 90 min at 23~C, and terrninated by rapid filtration over
GF/C filter.s pre-soaked in 0.1% polyethyen~mine The radioactivity
was quantifed with a Packard gamma counter.
For adenylyl cyclase activity, cells are harvested in PBS
with 5 mM EDTA, pe]leted and, then resuspended in ACC buffer (75
mM Tri.s, pH 7.4; 250 mM .sucrose; 12.5 mM MgC12; 1.5 mM EDTA; I
IlM ascorbic acid; 0.6 mM 3-i.sobutyl-1-1 - methylxanthine). The cell.s
.are incubated with various concentrations of test compound (usually
agoni.st compound) for 45 min at room temperature, and the reaction
tenminated by boiling for 3 min. The concentration of cAMP in the
Iy.sate wa.s determined via protein kinase A (PKA) binding assay
(Barton, A.C., Black, L.E., Sibley, D.R., Mol. Pha~macy. 39:650-658,
1991) or an automated cAMP IRA assay (At In.struments, MD). For the
PKA binding assay, the Iy.sate was incubated with 3.6 nM 3H-cAMP
and 5 ~g of PKA in a final volume of 1~5 ~I for 2 to 24 hour.s at 4~ C,
followed by rapid filtration over GF/C filters with cold washing buffer
(20 mM potas.sium phosphate, pH 6.0). The radioactivity on the filter
... .... . . . . . .. . . .
W0 96/00739 2 1 q 3 8 ~ ~ r~l"J.. ~ ;
- 24 -
was then ~uantified on a beta counter. The final concentration of cAMP
wa.s determined according to the standard curve of cAMP. The data for
both binding and cycla.se assays were analyzed by using graphed
software (San Diego, CA).
Figure 3 shows that, when stimulated with the beta agonist
isoproterenol, there is a four-fold increase in the production of cAMP
in L cell.s transfected with the wild type human ~3AR, with a ECso ~f
2.7 + 0.5 x 10-~ M (n=4). By contrast, the ~3AR-mediated production
of cAMP is essentially abolished in cell.s transfected with modified
receptor D(227-234)~3AR and strongly attenuated in cells expressing
the D(277-289) modified receptor.
Radioligand binding with 1251-CYP indicates that the wild
type ~3AR displays two affinity sites for isupluL~I~nol binding: a high
affinity site (2~%, IC50=5 x 10-~ M), and a low affinity site (72%,
IC50=2.6 x 10-6 M). Deletion of residues 227-234 or residues 277-289
from the ~3AR results in a single high affinity binding state (Table 2
and Figure 4). No increase in binding affinity is observed for the ~AR
antagoni.st propranolol for either modified receptor (Figure 4).
These modified ~3 receptor.s can therefore be u.sed in a
screening assay to detect compounds that bind with high affinity to the
~3 adrenergic receptor, regardless of whether these compounds are
agonists or antagonists.
EXAMPLE 8
CDnstructiQn of Modified D(231-238)5HT-ID~ Receptor
Modified receptor D(231-238)5HT-ID,~ receptor was
constructed from the wild-type human 5HT-lD~ receptor cDNA (Jin, et
al J. Biol. Chem. 267: 5735 (1992)) by standard mutagenesis techniques.
The modified 5HT-ID~ receptor lack.s 8 amino acid residues
(IYVEARSR) at the N-terminal portion of the third intracellular loop.
The nucleotide se~luences of the modified receptors were conflrmed by
DNA se~uencing. As was the case for the modified ~2 and ~3
receptors, this modified 5HT-ID~ receptor is designed so as to disrupt
the proximal portion of the third intracellular loop without affecting the
wo96/C0739 2 1 9 3 8 0 9 r~ c~
- 25 -
adjacent fifth transmembrane helix. Thus, the charged amino acid
(Arg230) that delineates the bottom of helix 5 is left intact in the
~ modifed receptor, while the following eight amino acids are deleted.
The size of the deletion in the present invention may vary from six to
5 13 amino acid.s in this region, beginning immediately after the charged
residue at the end of transmembrane helix 5.
EXAMPLE 9
Expres.sion ~nd Characteri7~tion of Modified D(231-238)5HT-ID,~
10 Receptor
The modified receptor was subcloned into a m~mm~ n
expre~sion vector and expres.sed in CHO cell.s using standard
transfection method.s. Stable cell lines were selected by G-418
resistance and u.sed for radioligand binding or adenylyl cyclase assays.
For binding assays, the membranes were prepared by
harvesting the cell.s in ice-cold Iy.si.s buffer (5 mg Tris, pH 7.4; 2 mM
EDTA), followed by 15 min centrifugation at 38,000 x g. The
membrane pellet was then resuspended in buffer A. E4uilibrium
binding to the wild type or modified 5HT-ID~ was performed in a
mixture containing membranes, 5 nM 3H 5-hydroxytryptamine, and
.serial dilutions of the competing ligands. Binding reactions were
incubated for x min at 23~C, and terminated by rapid filtration over
GF/C filter.s. The bound radioactivity was 4uantified with a gamma
counter.
Adenylyl cyclase activity was measured essentially as
de.scribed by McAllister et al. (McAllister, G., Charle.sworth, A.,
Snodin, C., Beer, M. S., Noble, A. J., Middlemiss, D. N., Iversen, L.
L., and Whiting, P., 199~, PNAS 89:5517-5521), with the addition of
forskolin. Inhibition of the forskolin-stimulated response by receptor
agonists, including 5-hydroxytryptamine (serotonin), was determined.
Figure 5 shows that, when stimulated ~ith the agonist
serotonin, there is a 50~o inhibition in the forskolin-stimulated
production of cAMP in cells expressing with the wild type human 5HT-
ID13 receptor. with a EC50 of 30 nM. By contrast, the agonist-
, .. . .. _ . . . _ , .
WO96100739 2 ~ ~3 809 ~ s~
- 26 -
mediated inhibition of cAMP production i.s essentially abolished in cells
transfected with modified receptor D(231-239)5HT-ID~.
Radioligand binding studies at the wild type 5-HTID,~
receptor indicate that when the guanine nucleotide analogue, GppNHp
5 (guanylylimidodiphosphate) is present (100 mM), agonist binding (2 nM
3H-5-HT) is reduced by approximately 50-60% (Table 3). This is
thought to be a re.sult of the guanine nucleotide converting the receptor
to the low affinity state. However, in three independent clones
expressing the modified receptor, D(231-239)5-HTID~ (clones 1, 21
10 and 65), no significant inhibition of agonist binding is observed,
suggesting that the modified receptor is permanently in the high affinity
state.
This modified 5HT-ID~ receptor can therefore be used in a
screening assay to detect compounds that bind with high affinity to the
15 5HT- I D~ receptor, regardless of whether these compounds are agonists
or antagoni.sts.
EXAMPLE 10
Clonin~ and Expression of Modified Receptor cDNA into Bacterial
20 F.xpression Vectors
Recombinant modified receptor is produced in a
bacterial expression system such as E. coli. The rnodified receptor
expression cassette is transferred into an E. coli expression vector,
expression vectors include but are not limited to, the pET series
25 .(Novagen). The pET vectors place modified receptor expression
under control of the tightly regulated bacteriophage T7 promoter.
Following transfer of this construct into an E. coli hcst wh~ ich
contains a chromosomal copy of the T7 RNA polymerase gene
driven by the inducible lac promoter, expression of modified
30 receptor i.s induced by addition of an appropriate lac .substrate
(IPTG) is added to the culture. The levels of expressed modified
receptor are determined by the assays described herein.
wo 96100739 2 1 ~ 3 8 a ~ r~
.
EXAMPLE I I
Cloning and Expression of Modified Receptor cDNA into a Vector
for Expression in Insect Cells
Baculovirus vectors derived from the genome of the
5 AcNPV virus are designed to provide high level expression of cDNA
in the Sf9 line of insect cells (ATCC CRL# 1711). Recombinant
baculovirus expressing modified receptor cDNA is produced by the
following .standard methods (InVitrogen Maxbac Manual): the
modified receptor cDNA constructs are ligated into the polyhedrin
10 gene in a variety of baculovirus transfer vectors, including the
pAC360 and the BlueBac vector (InVitrogen). Recombinant
baculoviruses are generated by homologous recombination following
co-transfection of the baculovirus transfer vector and linearized
AcNPV genomic DNA [Kitt.s, P.A., Nu~. Acicl. R~s. 1~, 5667
15 (1990)] into Sf9 cell.s. Recombinant pAC360 viruses are identified
by the absence of inclusion bodie.s in infected cells and recombinant
pBlueBac viruses are identified on the ba.sis of U-galactosidase
expression (Summers, M. D. and Smith, G. E., Texas Agriculture
Exp. Station Bulletin No. 1555). Following plaque purification,
20 modified receptor expression is measured.
Authentic modified receptor is found in association with
the infected cells. Active modified receptor is extracted from
infected cells by hypotonic or detergent lysis.
Alternatively, the modified receptor is expressed in the
25 .D~ C~SOPhi/~l Schneider 2 cell line by cotransfection of the Schneider 2
cells with a vector containing the modified receptor DNA
downstream and under control of an inducible metallothionin
promoter, and a vector encoding the G41~ resistant neomycin gene.
Following growth in the presence of G41~, resistant cells are
30 obtained and induced to express modified receptor by the addition of
CuSO4. Identification of modulator.s of the modified receptor is
accomplished by assay.s using either whole cells or membrane
preparations.
W0 96/00739 ' r~ J.. ot r
2 1 q3~09
- 28 -
EXAMPLE 12
Cloning of Modif1ed Receptor cDNA into a yea.st expression vector
Recombinant modified receptor is produced in the yeast
S. cerev;si~ following the insertion of the modified receptor cDNA
5 cistron into expression vectors designed to direct the intracellular or
extracellular expression of heterologous proteins. In the case of
intracellular expression, vectors such a.s EmBLyex4 or the like are
ligated to the modified receptor cistron [Rinas, U. et al.,
Bi~tech~ok~ y 8, 543-545 (1990); Horowitz B. et al., J. Biul. Chelqn
10 265, 4189-4192 (1989)]. For extracellular expression, the modified
receptor cistron is ligated into yeast expression vectors which fuse a
.secretion .signal. The levels of expressed modified receptor are
determined by the assays described herein.
EXAMPLE 13
Purification of Recombinant Modified Receptor
Recombinantly produced modified receptor may be
purified by a variety of procedures, including but not limited to
antibody affinity chromatography.
Modified receptor antibody affinity column.s are made
by adding the anti-modified receptor antibodies to Affigel-10
(Biorad), a gel support which is pre-activated with N-
hydroxysuccinimide ester.s such that the antibodies form covalent
linkages with the agarose gel bead ,support. The antibodies are then
coupled to the gel via amide bonds with the spacer arm. The
remaining activated esters are then Lluenched with I M ethanolamine
HCI (pH ~s). The column i.s washed with water followed by 0.23 M
glycine HCI (pH 2.6) to remove any non-conjugated antibody or
extraneous protein. The column is then e~luilibrated in phosphate
buffered saline (pH 7.3) together with appropriate membrane
solubilizing agents such as detergents, and the cell culture
supernatants or cell extracts containing solubilized modified receptor
or modified receptor .subunits are slowly passed through the column.
The column is then washed with phosphate-buffered saline (PBS)
.. . . .. . . .. ..
WCI 96/00739 2 1 9 3 8 0 9 r~ r ,r C
- 29 -
supplemented with d~t~ until the optical density (A280) falls to
background; then the protein is eluted with 0.23 M glycine-HCI (pH
2.6) supplemented with detergents. The purified modified receptor
protein is then dialy~ed again,st PBS.
s
EXAMPL.~, 14
Clonin~ and Expression of Modified Receptor in ~ mmalian Cell
System
A modified receptor is cloned into a m~mm~ n expression
vector. The m~mm~ n expre.ssion vector is used to tran.sform a
m~mm~ n cell line to produce a recombinant m~mm~ n cell line.
The recombinant m~mm~ n cell line is cultivated under conditions that
permit expression of the modified receptor. The recombinant
m~mm~ n cell line or membranes isolated from the recombinant
m~mm~ l cell line are used in assays to idemtify compounds that bind
to the modified receptor.
EXAMPLE 15 ~ I
Screenin~ Assay
Recombinant cells c--m~ining DNA encoding a modified
receptor, membranes derived from the recombinant cells, or
recombinant modified receptor preparations derived from the cells or
l"e"lb,d.les may be used to identify compoun.ds that modulate modified
G-protein coupled receptor activity. Modulation of such activity may
occur at the level of DNA, RNA, protein or combinations thereof. One
method of identifying compounds that modulate modified G-protein
coupled receptor, comprises:
(a) mixing a test compound with a solution
containing modified G-protein coupled receptor to form a mixture;
~b) measuring modified G-protein coupled receptor
activity in the mixture; and
(c) comparing the modified G-protein coupled
receptor activity of the mixture to a standard.
