CA2193809A1 - Modified g-protein coupled receptors - Google Patents

Abstract

Modified G-protein coupled receptors having deletions in the third intracellular domain are identified and methods of making the modified receptors are provided. The invention includes the modified receptors, assays employing the modified receptors, cells expressing the modified receptors, and compounds identified through the use of the modified receptors, including modulators of the receptors.

Description

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~

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 ~

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 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~"~

(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 .... . _ ..... ... .. _ .. ...... ... _ _ ... _ ..... .. . . _ .. . _ _ ... . ......

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 .

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, ~ ' [ [

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~

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.

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~

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-~ 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.

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~"~ ' 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

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.. ~ ;

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.

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~

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.

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~

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.

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 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.

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

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.

Claims (21)

WHAT IS CLAIMED IS:

1. Isolated DNA encoding a modified receptor, the modified receptor being derived from a G-protein coupled receptor having seven transmembrane domains and the modified receptor having deletions in the third intracellular domain, or a functional derivative thereof.

2. The DNA of Claim 1 wherein the modified receptor is a modified .beta.3-adrenergic receptor.

3. Isolated RNA encoded by the isolated DNA of Claim 1 or its complementary sequence.

4. Isolated RNA encoded by the isolated DNA of Claim 2 or its complementary sequence.

5. An expression vector containing the isolated DNA of Claim 1.

6. A recombinant host cell containing the expression vector of Claim 5.

7. A process for the production of a modified G-protein coupled receptor, comprising:
a) transforming a host cell with the isolated DNA of Claim 1 to produce a recombinant host cell;
b) culturing the recombinant host cell under conditions which allow the production of modified G-protein coupled receptor; and c) recovering the modified G-protein coupled receptor.

8. The modified G-protein coupled receptor produced by the process of Claim 7.

9. The process of Claim 7 wherein the modified G-protein coupled receptor is a modified beta-3 adrenergic receptor.

10. An isolated and purified modified G-protein coupled receptor, the receptor having seven transmembrane domains and having amino acids deleted from the third transmembrane domain, or a functional derivative thereof.

11. The purified modified G-protein coupled receptor of Claim 10 which is a modified beta-3 adrenergic receptor.

12. A method of identifying compounds that modulate modified G-protein coupled receptor activity, comprising:
(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.

13. Compounds identified by the method of Claim 12.

14. Pharmaceutical compositions comprising the compound of Claim 13.

15. A method for identifying compounds which specifically bind to a modified G-protein coupled receptor, comprising:
(a) cloning a 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 expression vector to form a construct;
(d) introducing the construct into a cell which does not express the altered receptor in the absence of the introduced construct;
(e) incubating cells or membranes isolated from cells produced in step c with a quantifiable compound known to bind to the receptor; and (f) adding test compounds so as to compete the quantifiable compound from the receptor.

16. Compounds identified by the method of Claim 15.

17. A method of making a modified G-protein coupled receptor, comprising:
(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;
(c) isolating the altered DNA;
(d) expressing the altered DNA; and (e) recovering the modified G-protein coupled receptor.

18. The modified G-coupled protein receptors of Claim 17.

19. The method of Claim 17 wherein between six and thirteen nucleotides are deleted from DNA encoding the third intracellular domain of the G-protein coupled receptor.

20. The isolated DNA of Claim 1 wherein the modified G-protein coupled receptor is selected from the group consisting of:

(a) D(277-289) beta-3 adrenergic receptor; and (b) D(227-234) beta-3 adrenergic receptor.

21. The isolated and purified receptor of Claim 10 wherein the modified beta adrenergic receptor is selected from the group consisting of D(277-289) beta-3 adrenergic receptor and D(227-234) beta-3 adrenergic receptor.