Method of identifying protein CAMs (constitutively active mutants)

[0001] The present invention relates to a method of identifying protein CAMs and the use thereof.

[0002] The G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors with a common evolutionary origin. They include receptors which respond to environmental ligands (odorants, flavors) or radiations (light of various wavelengths) and to innumerable internal signals (hormones, bioactive amines, neuropeptides, arachidonic acid metabolites, purines, etc. . . . ). This extreme diversity contrasts with their stereotyped structure (seven transmembrane alpha helices, three extracellular loops, three intracellular loops, amino terminus outside and carboxyl terminus inside the cell) and with the limited number of downstream regulatory cascades they control.

[0003] The GPCR interacts with an intracellular heterotrimeric G protein consisting of αβγ subunits. Upon binding of the receptor's ligand, the α-subunit dissociates from the β- and γ-subunits, and hydrolyses GTP to GDP. Both Gα and Gβγ can then activate downstream transduction effectors or regulate other receptors (Zwick et al., 1999). in haploid Saccharomyces cerevisiae cells, GPCRs are regulating the mating process. The receptor (Ste2 or Ste3) detects the presence of cells of the opposite mating type (through binding of peptide mating pheromones), and activates intracellular heterotrimeric G proteins, thus initiating the mating process. Gpa1 (α subunit) dissociates from the βγ (Ste4-Ste18) complex which activates downstream elements of the pheromone response pathway which includes a well-characterized mitogen-activated protein kinase (MAP kinase) cascade. The transcription factor Ste12 can then initiate the transcription of several mating factor-inducible genes such as FUS1.

[0004] Reports of mammalian GPCRs expressed in yeast indicate that these heterologous proteins can be reliably expressed in yeast and properly inserted into yeast membranes (Tate and Grisshammer, 1996). Reports, e.g. (Price et al., 1995), demonstrate that a large number of heterologous GPCRs interact with the yeast heterotrimeric G protein with sufficient efficacy to induce a growth-promoting signal. In case the GPCR under investigation does not couple to Gpa1, it is co-expressed together with a chimera where the C-terminal part of Gpa1 is replaced by the corresponding amino acids of a given human Gα subunit (Brown et al., 2000). A reporter construct (such as pFUS1-HIS3 or pFUS1-lacZ) is then expected to produce a detectable response upon receptor activation.

[0005] Increasingly, it is being appreciated that endogenous receptors, and in particular those of the G protein-coupled receptor family, may possess some level of constitutive activity even in the absence of activating mutation. A potentially important physiological ramification of the constitutive activity of such receptors is that the ability of different receptor subtypes (for the same ligand) to spontaneously isomerize to the active state might well differ. Such receptor subtypes would vary substantially in their properties, thus best suiting them for one or another physiological context (Lefkowitz et al., 1993).

[0006] The discovery of constitutive GPCR activity presents a theoretical approach to the identification of ligands for orphan receptors. The basic premise for this idea is that different tertiary conformations (i.e. different allosteric change states) of the receptor protein will display different binding domains for ligands, or different binding affinities for the same ligand. Since the mutation of a receptor sequence can only affect the physico-chemical properties of the receptor, but not those of ligands, a change of affinity of a ligand for a receptor ought to be of a similar magnitude for all ligands and not proportional to the ligand's efficacy (Lefkowitz et al., 1993).

[0007] The notion that constitutive activation of G protein-coupled receptors could be responsible for hereditary diseases came first from the study of patients suffering of retinis pigmentosa (Robinson et al., 1992). Since then, several other human pathologies have been linked to constitutive activity or aberrant receptors (Dhanasekaran et al., 1995) (Rao and Oprian, 1996) (Duprez et al., 1997) (Jensen et al., 2000). It has now been recognized that very valuable information relevant to treatment of diseases caused by constitutively active receptors (for instance TSH and LH receptors (Spiegel, 1996)) can be directly obtained by identifying compounds which act as inverse agonists to constitutively activated forms of the receptor.

[0008] Additionally, some of the therapeutic effects of presently used receptor antagonists may be related to their inverse agonist properties. Recent results (Varma et al., 1999) show that almost all β-adrenergic antagonists (with the exception of pindolol) have inverse agonist properties in the heart of reserpine treated rats.

[0009] Down regulation and desensitization of GPCRs is elicited by agonists or as a consequence of spontaneous activity. Thus, inverse agonists upregulate heptahelical receptors by decreasing spontaneous downregulation (Daeffler and Landry, 2000), offering new approaches to tolerance and dependence to drugs. Amino acid residues can be mutated and lead to ligand-independent activation of the receptor and constitutive activation of signaling pathway (Lefkowitz et al., 1993) (Rao and Oprian, 1996) (Sommers et al., 2000) (Konopka et al., 1996) (Alewijnse et al., 2000).

[0010] Several techniques have been applied to the discovery or the study of constitutively active receptors. For instance, manipulation of the stoichiometry of receptors and G proteins (mainly over-expression of the receptor) can create a constitutive active receptor system (Chen et al., 2000) (Samama et al., 1997) or site directed mutagenesis on residues such as the highly conserved DRY motif were found to be involved in stabilizing intramolecular interactions (Alewijnse et al., 2000). To date, random mutagenesis has been used in many works to identify CAMs: in a random saturation mutagenesis of a critical region of the Calcium-sensing receptor (Jensen et al., 2000) or in a systematic screening in a mammalian cell based bioassay of a random mutant library of the angiotensin II AT1A receptor (Parnot et al., 2000).

[0011] The ease of genetic manipulation of yeast and the availability of an assay that allows detection of a signaling activity made it possible to search through large random mutational libraries to study the spectrum of mutations capable of causing constitutive activation. Ma & al. (Ma et al., 1987) described a fast and reliable method for plasmid construction (Gap Repair) that is based on the efficient repair of a linearized plasmid by recombination with a homologous DNA restriction fragment during yeast transformation.

[0012] Additionally, S. cerevisiae does not show such a rapid desensitization process comparable to the ligand-dependent phosphorylation of receptors followed by receptor interaction with arresting, disruption of the interaction receptor-G protein, and in some case sequestration (Tsao et al., 2001). This makes the identification of a constitutive activity much easier.

[0013] Previously, a random mutagenesis strategy combined with a yeast based in vivo sub-cloning/screening has been applied successfully on the amino-terminal and transmembrane regions (approximately the first 300 out of 431 residues) of the yeast Ste2 G protein-coupled receptor (Sommers et al., 2000) and on the second intracellular loop of the V2 Vasopressin receptor (Erlenbach et al., 2001). In contrast, the present method allows the systematic identification of activating mutations over the whole open reading frame, without the need of focusing on some regions. Although people usually choose to mutagenise only a part of the coding sequence because they believe that only this region is involved in the mechanism studied (Erlenbach et al., 2001), no doubt would persist and sometimes new prospects on structure-activity would appear.

[0014] WO 00/12705 discloses methods for improving the function of heterologous G protein-coupled receptors.

[0015] Random mutagenesis on human GPCRs and functionally studied in mammalian cells, were described by (Parnot et al., 2000)) (CAM discovery of Angiotensin II 1A receptor, full length) and (Jensen et al., 2000) (Functional Importance of the Ala116-Pro136 Region in the Calcium-sensing Receptor).

[0016] Random mutagenesis of a yeast GPCR and functionally studied in yeast cells, in particular CAM discovery of Ste2 (α-factor receptor), random mutagenesis of amino-terminal and transmembrane regions, including Gap Repair were described by (Sommers et al., 2000) and (Sommers and Dumont, 1997)).

[0017] Random mutagenesis on a human GPCR functionally studied in yeast cells, in particular coupling properties study of V2 vasopressin receptor, oligonucleotide-directed random mutagenesis of the intracellular loop 2 (228 bp), including Gap repair were described by (Erlenbach et al., 2001)).

[0018] CAMs and methods of using them are also disclosed in WO 00/21987. WO 00/06597 discloses endogenous constitutively activated G protein-coupled orphan receptors. WO 00/22129 and WO 00/22131 disclose non-endogenous constitutively activated human G protein-coupled orphan receptors (site directed mutagenesis of GPCRs to generate constitutively activated mutants) and WO 97/21731 an assay for and uses of peptide hormone receptor ligands.

[0019] The discovery of constitutively activated mutants (CAMs) is usually the result of a long process of genetic manipulations and assays in mammalian cell culture. Researchers usually choose site directed mutagenesis because of its more straight forward and fast principle (Egan et al., 1998; Alewijnse et al., 2000).

[0020] It was a task of the present invention to provide an easy and fast method for identifying CAMs of proteins, e.g. for GPCRs, ion-channel, enzymes.

[0021] The present invention provides a method for identifying protein CAMs (constitutively active mutants), wherein

[0022] a) a library of mutated sequences of a protein is generated,

[0023] b) yeast cells are transformed with such library and

[0024] c) the respective protein CAM is identified.

[0025] Examples for proteins for which CAMs can be identified are GPCRs, ion-channels, enzymes, e.g. kinases, proteases, transcription factors.

[0026] Preferably, protein CAMs of mammalian proteins are identified, e.g. CAMs of human proteins.

[0027] The present invention provides a method of identifying protein CAMs (constitutively active mutants) wherein

[0028] a) a library of mutated sequences of a protein is generated,

[0029] b) yeast cells are co-transformed with the library and a linearized expression vector,

[0030] c) the transformed yeast cells are selected for the repair of the plasmid, and

[0031] d) protein CAMs are identified by determining the activity of the respective protein mutant.

[0032] The present invention provides a method of identifying protein CAMs (constitutively active mutants) wherein

[0033] a) a library of mutated sequences of a protein is generated,

[0034] b) yeast cells are co-transformed with such library and a linearized expression vector,

[0035] c) the transformed yeast cells are selected for the repair of the plasmid, and

[0036] d) the previously selected yeast colonies are transferred on a second selective plate where they are screened for the activity of the respective protein mutant.

[0037] In a special embodiment of the invention low fidelity PCR is applied on a full length sequences of a particular protein, e.g. a GPCR, preferably a mammalian protein sequence. The PCR products were co-transformed with a linearized expression vector (e.g. containing at each end short sequences homologous to the end of the PCR product) into an engineered yeast strain. The transformed yeast cells were first selected for the repair of the plasmid (e.g. selection by colony forming on a selective medium). The colonies previously selected were replicated an another medium, selective for the activity of the protein, e.g. the receptor (e.g. by the use of a survival reporter gene expressed only upon receptor signaling). Preferably, three or more identical and independent experiments were done to avoid the PCR's bias. The protein CAMs (“mutants”) have an increased basal signaling activity and the same maximum of stimulation than the wild type protein.

[0038] A yeast based in vivo discovery of random active mutants can be applied to the entire coding sequence of a protein, e.g. a human receptor. This was done by screening for constitutive mutations of the human sphingosine 1-phosphate receptor EDG5 (Endothelial Differentiation Gene 5) (An et al., 2000) (Hla, 2001).

[0039] To obtain a whole set of single point mutants directly (and also to avoid excessive secondary sub-cloning work to find out the activity conferred by each single point mutation), the PCR protocol was optimized to induce an average of less than one point mutation per copy of the gene. Indeed, the high throughput potential of an in vivo subcloning/screening strategy allows us to increase the size of the library without consuming more time/money.

[0040] In another embodiment the present invention relates to engineered yeast cells comprising a library of mutants (e.g. GPCR CAMs) and the use of such engineered yeast cells.

[0041] For such engineering for example Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans cells can be used.

[0042] The use of such an engineered yeast cell should bring three major improvements at the same time:

[0043] screening of a whole library of mutants generated by low fidelity polymerase chain reaction (PCR)

[0044] in vivo sub-cloning of each mutated sequences into the expression vector by homologous recombination

[0045] in vivo selection of the active mutants using reporter genes

[0046] All three in the same engineered yeast cell.

[0047] Further advantages are, that the yeast is a powerful tool for the study of mammalian GPCRs and their transduction characteristics because of the high homology between these eukaryotic cells (Price et al., 1995; Hadcock and Pausch, 1999; Botstein et al., 1997); Yeast has a high rate of homologous recombination and the genetic manipulations of yeast are easy (Ma et al., 1987; Oldenburg et al., 1997); Yeast allows in vivo selection of a receptor's activity (Chambers et al., 2000); In vivo screen allows the direct recovery of the plasmid carrying the mutant of interest (CAM) from the micro-organism. Yeast is cheaper to cultivate and engineer than mammalian cells. The technology used to sub-clone and detect the mutants' activity in mammalian cells is far more expensive and qualitative selection and quantification of the mutants' activity can both be done in the same yeast system.

[0048] The present method of identifying protein CAMs presents a low cost, fast and powerful method to systematically identify activating mutations along the whole coding sequence of a protein. In contrast to previous work (Parnot et al., 2000), the cloning step is simplified to a simple transformation in yeast and the selection of active mutants is not more than picking growing colonies.

[0049] The transposition of the method into mammalian cells confirmed very well the constitutive activity of the mutants screened and selected in yeast. This proves that the method is a suitable alternative to mutant screening in mammalian systems.

[0050] Another big advantage of the method is that it immediately discriminates between a moderately active and a highly active mutant (a too high basal activity would not be suitable for agonist discovery, but appropriate for inverse-agonist screening). The growth speed of the colonies on agar selective medium is well correlated to the different “intensities” of constitutive activity observed in a liquid reporter assay.

[0051] De-orphaning can also be achieved with this method, e.g. the method can be applied to orphan GPCRs. Therefore, a low fidelity PCR product was co-transfected with the linearized vector into a panel of yeast strains expressing different humanized Gα protein subunits. On selective medium, mutants were selected only from the yeast strain expressing the Gα specific for its coupling. β-Galactosidase detection after growth in a selective medium showed an increased basal activity of the receptor mutant (i.e. an increased expression level of lacZ, controlled by a FUS1 promoter).

[0052] The method of identifying protein CAMs, of e.g. GPCR CAMs can be used for:

[0053] Identifying agonists; such use is based on the fact that the affinity of a protein CAM, e.g. GPCR CAM for his agonist is increased (Lefkowitz et al., 1993) (MacEwan and Milligan, 1996) (Alewijnse et al., 2000);

[0054] Identifying inverse agonists (Chen et al., 2000);

[0055] Studying proteins oligomerization, e.g. GPCR oligomerization, depending on their state of activity;

[0056] Crystallizing protein, e.g. GPCRs in different tertiary conformations;

[0057] De-orphaning: modulators of protein action can be identified with no prior knowledge of the endogenous ligand or protein function.

FIGURES

[0058]
FIG. 1: Summary of the Method of Identifying GPCR CAMs.

[0059]
FIG. 1 summarizes the whole process of the method.

[0060] Random mutagenesis of the EDG5 gene was conducted using the yeast CEN expression plasmid p416GPD-Edg5 carrying an URA3 marker (FIG. 2).

[0061]
FIG. 2: Restriction Map of p416GPD-Edg5 (Nhel)

[0062] A Nhel restriction site was created at position 157 bp of the coding sequence of EDG5. Three nucleotides where exchanged by site directed mutagenesis to create the site. This was necessary to conserve, after linearization of the plasmid, only the first 157 bp and the last 101 bp of the open reading frame for homologous recombination.

[0063] To allow in vivo recombination, the p416GPD-Edg5 was linearized by double digestion Nhel-Xmal before co-transformation with the low-fidelity PCR amplification product.

[0064]
FIG. 3: Restriction Map of pcDNA3.1(+)-Edg5

[0065]
FIG. 4: Solid Phase Assay

[0066] Three colonies of each yeast transformation were tested for the growth and for the β-Galactosidease activity on selective plate:

[0067] the first plate (SC Glucose-Ura) shows the normal growth of the colonies;

[0068] the second plate (SC Glucose-Ura-His containing 2 mM 3-AT, pH 6.8) shows the growth of the colonies expressing a constitutively activated mutant;

[0069] the third plate (SC Glucose-Ura, X-Gal, pH 7) is testing the β-Galactosidase activity (its substrate X-Gal is transformed in a blue metabolite) of the mutants: the frame shows the colored colonies which correspond to the most active clones and correlates with the observations from the second plate.

[0070]
FIG. 5: Liquid Assay

[0071] After 24 hours of growth of the different mutants in selective liquid medium, in the presence of an increasing concentration of Sphingosine 1-Phosphate, β-Galactosidase activity was measured in a calorimetric assay by adding the substrate CPRG, incubating 2 hours and measuring the absorbance at 574 nm.

[0072]
FIG. 6: Cell Culture Assay

[0073] Luciferase activity measured in triplicates after 24 hours of stimulation by a serial dilution of Sphingosine 1-Phosphate.

EXAMPLES

Example 1

Synthesis of the Random Mutational Library

[0074] A modified PCR protocol (Svetlov and Cooper, 1998) was used: initial denaturation at 95° C. for 3 min, 30 cycles of denaturation at 95° C. for 5 s, annealing at 50° C. for 5 s, and primer extension at 72° C. for 5 s, and final extension at 72° C. for 5 min, performed on the Cycler PTC-200 (MJ-Research).

[0075] The reaction was carried out with 2.5 U of Taq polymerase (Promega) using standard reaction buffer (10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl) supplemented with 0.5 mM MnSO4. An equimolar mix of dNTPs (Amersham Pharmacia Biotech Inc) was used to provide 500 μM of each nucleotide triphosphate in a 100 μl reaction volume. 10 ng of the p416GPD-Edg5 plasmid were used as template. The following oligonucleotides (30 pmol of each) were used as primers for the PCR amplification: EDG5 fwd CAR (SEQ ID NO. 1: 5′-ATG GGC AGC TTG TAC TCG GAG T-3′) and EDG5 rev CAR (SEQ ID NO. 2: 5′-TCA GAA CAC CGT GTT GCC CTC-3′). They correspond exactly to the first 22 and last 21 nucleotides of the receptor's sequence, thus the PCR amplifies exactly the open reading frame.

[0076] The PCR product (about 5 μg) was purified by electrophoresis through a 1% agarose-TBE gel followed by elution into 40 μl sterile water (QIAquick Gel Extraction Kit, Qiagen). The DNA final concentration was about 0.1 μg/μl.

Example 2

In Vivo Recombination (Gap Repair)

[0077] 2 μl of the PCR product were cloned into the TA Topo Cloning Vector (Invitrogen) for further qualitative and quantitative analysis of the randomly induced mutations.

[0078] The remaining volume of purified PCR product (3-5 μg in 38 μl) was co-transformed with 1 μg of p416GPD-Edg5 (linearized by double digestion with Nhel-Xmal) into about 109 cells of the yeast strain (W303 MATa far1::hisG, sst2::ura3FOA, fus1::HIS3, ΔSte2::KanR, mfa2-fus1-lacZ::ura3FOA) according to a modified Lithium acetate method (Ito et al., 1983).

[0079] The transformed yeast cells were plated on 10 plates SC/Glucose-Ura medium to select for the cells with a “repaired plasmid” (about 108 yeast cells per plate).

[0080] After 36 hours incubation at 30° C. (when very small colonies were visible), the transformation plates were replica-plated onto selective medium: SC/Glucose-Ura-His, pH 6.8, containing 2 mM 3-Aminotriazol (3-AT).

Example 3

Selection

[0081] After 48 hours incubation at 30° C., the colonies still growing were picked and restreaked as patches on a new selective plate (SC/Glucose-Ura-His, pH 6.8, containing 2 mM of 3-AT).

[0082] To eliminate false positive mutants (plasmid independent activity), the following steps were performed:

[0083] The plasmid from each selected clone was recovered by a Zymolase/SDS treatment protocol adapted from H. Ma & al. (Ma et al., 1987).

[0084] After ethanol purification, each plasmid was transformed into E. coli DH5α electro-competent cells. Individual bacterial transformants, one for each mutant, were grown in mini culture for plasmid preparation (QIAprep Spin Miniprep Kit, Qiagen).

[0085] The purified DNA was then transformed again into the same yeast strain and each mutant assayed.

Example 4

Solid Phase Assay

[0086] From a 16 hours culture in SC/glucose-Ura medium, about 3×105 cells of each mutant (in triplicates) were spotted onto three different plates:

[0087] SC/Glucose-Ura as a control (to make sure that every spot contains roughly the same number of cells);

[0088] SC/Glucose-Ura-His, pH 6.8, containing 2 mM 3-AT;

[0089] SC/Glucose-Ura, pH 7, containing 100 μg/ml X-Gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside).

[0090] The two first plates were analyzed after 48 hours of growth at 30° C.; the third one was kept for 2 or 3 additional days at 4° C. to develop the blue coloration due to β-Galactosidase activity.

[0091] We selected the clones which grew on selective medium (SC/Glucose-Ura-His, pH 6.8, containing 2 mM 3-AT) and gave rise to a blue colored patch on X-Gal medium (SD-Ura, pH 7, containing 100 μg/ml X-Gal). These were candidates for constitutive activity (FIG. 4).

Example 5

Liquid Assay in 96 Well Format

[0092] The same 16 hours culture was diluted 200 times in selective medium (SC/Glucose-Ura-His, pH 6.8, containing 2 mM 3-AT) and 90 μl were dispensed into the 8 wells of a microtiter plate column already containing 10 μl of a serial dilution of the ligand Spingosine 1-Phosphate (Matreya) (solubilized and diluted in water from 10−3 to 10−9 M).

[0093] After 18 to 24 hours of stimulation/growth in a shaking incubator (700 rpm, 30° C.), the β-Galactosidase activity was detected with the substrate Chlorophenolred-β-D-galactopyranoside (CPRG, Boehringer).

Example 6

Experimental Procedures in HEK 293

[0094] The wild type Edg5 receptor and 3 of the 22 CAMs were sub-cloned into the mammalian expression vector pcDNA3.1(+) to be tested in cell culture (FIG. 3). A HEK 293 cell line stably transfected with the reporter construct 6SRE-Luciferase was utilized for the assay.

[0095] This adherent cell line was grown under normal conditions (37° C., 5% CO2, humid atmosphere) in DMEM-Glutamax (Gibco BRL)+1% Penicillin/Streptomycin+10% Fetal Bovine Serum.

Example 6.1

Transfection

[0096] Day 1—40.000 cells/well were plated in a white 96-well plate

[0097] Day 2—the cells were rinsed with 200 μl Opti-MEM (Gibco BRL) and each well received 100 μl of a transfection mix containing: 0.5 μg receptor plasmid+0.25 μg CMV-β Gal (Promega)+1.2 μl Lipofectamine (Life Technologies) in Opti-MEM.

[0098] After 5 hours of incubation with this mix, the wells were emptied and received 180 μl of the normal culture medium (DMEM-Glutamax+1% Penicillin/Streptomycin) containing only 0.5% of Fetal Bovine Serum.

[0099] Day 3—20 μl of a 10-fold concentrated serial dilution of Sphingosine 1-Phosphate (from 10−4 to 10−10 M) was added to each well.

[0100] Day 4—the wells were emptied, rinsed with 200 μl phosphate buffer (without calcium and magnesium) and received 50 μl of Glo Lysis Buffer (Promega), after 5 minutes at room temperature, they received 50 μl of Steady-Glo Luciferase Reagent, and the measurement was achieved 5 minutes later in a Luminoskan (Labsystem), 15 seconds integration of the signal.

[0101] To normalize the results of the assay, β-Galactosidase activity was measured, from the same plate, after 5 minutes incubation with 25 μl of Gal-Screen Reagent (Tropix), 5 seconds integration of the signal. The luciferase numbers were then divided by the β-Galactosidase numbers.

Example 7

Results

Example 7.1

TA Cloning Analysis

[0102] The analysis of 38 randomly sequenced clones revealed 28 nucleotide mutations: 5 silent mutations, 1 STOP codon and 22 amino acid substitutions. These results suggest that under the experimental conditions the probability for an amino acid substitution to occur in the 354 residues of the wild-type sequence is 0.61.

Example 7.2

Analysis of Selected Mutants

[0103] The solid phase assay gives a confirmation of the first selection done after replica-plating the gap repair plates on selection plates. After being grown again on selective plate as patches (for confirmation), the plasmid DNA carrying the active mutant was purified, amplified in E. coli and re-transformed into the same yeast strain.

[0104] Three colonies of each yeast transformation were tested for growth and for β-Galactosidase activity on selective plates. The FIG. 4 illustrates the clear response from the different mutants obtained in this assay. Here, mutants 1 (contains two mutations, Ala82Val and Ile197Thr) and 7 (Ala82Val only) look the most active (i.e. fast growth on selective plate and blue coloration on X-Gal plate). Mutants 2 (Thr196Ile), 3 (Ser159Pro), 5 (Phe242Leu) and 8 (Ser159Pro and Val215Met) were also selected (at least two of the three clones grew), but appear less active (i.e. the blue coloration is not so obvious). Clone 4 had the same activating Phe242Leu mutation but only one of the three colonies grew. Clone 6 had no activating mutation.

[0105] To further characterize the mutants, their activity was tested in a liquid assay. Triplicates of each mutant (re-transformed in yeast) were grown to saturation in a pre-culture. These cell suspensions were diluted 200 times into a medium lacking histidine, permitting the growth of only activated receptors (HIS3 gene under the control of the FUS1 promoter) and distributed in a 96-well microtiterplate together with an increasing concentration of Sphingosine 1-Phosphate. After 24 hours of incubation and shaking at 30° C., the presence of β-Galactosidase activity was measured in a colorimetric assay by adding the substrate CPRG, incubating 2 hours at 30° C. and measuring the absorbance at 574 nm.

[0106]
FIG. 5 shows that even in the absence of ligand, mutant Ala82Val is hyper-active (which correlates very well with the observations made in the plate assay), while others have a basal activity intermediate between the Ala82Val mutant and the wild type receptor (Ser159Pro and Val238Glu).

[0107] Out of three independent screens, 22 mutations have been found to confer constitutive activity to the Edg5 receptor (increased basal activity and the ability to be further stimulated by Sphingosine 1-Phosphate) (Table 1). Interestingly, the mutation Ser159Pro was isolated in each of the three screens, and the mutations Ala82Val and Phe242Leu were isolated in two of the three screens.

Example 7.3

HEK 293 Assay

[0108] A Serum Responsive Element (SRE)-Luciferase reporter assay in HEK 293 was chosen to verify in mammalian cells the activity of the CAMs selected with the yeast system. A stable HEK 293 cell line carrying the 6SRE-Luciferase construct was transfected with the wild type Edg5 or the mutants Ala82Val, Ser159Pro and Val238Glu. After 24 hours of stimulation, the measurement of Luciferase reflected the receptor's activity.

[0109] This assay (FIG. 6) shows an increased basal activity (i.e. in absence of agonist) of the three mutants compared to the wild type, although the maximum response of all four receptors (wild type and mutants) was not changed.

Example 7.4

Systematic Screen

[0110] To validate a screen, the whole process must be repeated in the same conditions. Indeed, the PCR principle can create an important bias introducing an activating (i.e. the Ala 82->Val mutation found 68 times in the third screen) or inactivating mutation in an early stage of the reaction. This mutation is then present in a high percentage of clones and can mask other interesting point mutations. This has to be circumvented. The best and fastest way would be doing at least three low-fidelity PCRs at the time and all the following steps in parallel.

1TABLE 1Selected mutants analysisOut of three independent experiments, 22 mutations have been foundto confer constitutive activity to the Edg5 receptor:FirstSecondThirdscreenscreenscreen(4 active(3 active(118 activemutantsmutantsmutantsLocationLeu 70 -> Pro2Transmembranedomain 2Phe 71 -> Leu7Transmembranedomain 2Ala 82 -> Val268Transmembranedomain 2Val 87 -> Ala1Transmembranedomain 2Ser 113 -> LeTransmembranedomain 3Leu 139 -> Pro2Intracellularloop 2Ser 155 -> Pro1Transmembranedomain 4Ser 159 -> Pro1125Transmembranedomain 4Val 183 -> Ala2Extracellularloop 2Ala 187 -> Thr1Extracellularloop 2Lys 188 -> Arg1Extracellularloop 2Thr 196 -> Ile1Transmembranedomain 5Ile 205 -> Phe3Transmembranedomain 5Leu 229 -> Pro1Transmembranedomain 6Leu 232 -> Arg3Transmembranedomain 6Thr 234 -> Ala4Transmembranedomain 6Val 235 -> Ile4Transmembranedomain 6Thr 236 -> Ile1Transmembranedomain 6Val 238 -> Glu2Transmembranedomain 6Val 238 -> Ala3Transmembranedomain 6Phe 242 -> Leu21Transmembranedomain 6Phe 250 -> Tyr2Transmembranedomain 6

[0111]

2

TABLE 2

Nucleotide Sequence of p416 GPD-Edg5

(SEQ ID NO. 3)

1
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG

AGCGCGCAAA GCCACTACTG CCACTTTTGG AGACTGTGTA CGTCGAGGGC

51
GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG

CTCTGCCAGT GTCGAACAGA CATTCGCCTA CGGCCCTCGT CTGTTCGGGC

101
TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG GTTAACTATG

AGTCCCGCGC AGTCGCCCAC AACCGCCCAC AGCCCCGACC GAATTGATAC

151
CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATACCAC AGCTTTTCAA

GCCGTAGTCT CGTCTAACAT GACTCTCACG TCGTATGGTG TCCAAAAGTT

201
TTCAATTCAT CATTTTTTTT TTATTCTTTT TTTTGATTTC GGTTTCTTTG

AAGTTAAGTA GTAAAAAAAA AATAAGAAAA AAAACTAAAG CCAAAGAAAC

251
AAATTTTTTT GATTCGGTAA TCTCCGAACA CAAGGAAGAA CGAAGGAAGG

TTTAAAAAAA CTAAGCCATT AGAGGCTTGT CTTCCTTCTT CCTTCCTTCC

301
AGCACACACT TAGATTGGTA TATATACGCA TATGTAGTGT TGAAGAAACA

TCGTGTCTGA ATCTAACCAT ATATATGCGT ATACATCACA ACTTCTTTGT

PstI

˜˜˜˜˜˜

351
TGAAATTCCC CAGTATTCTT AACCCAACTG CACAGAACAA AAACCTGCAG

ACTTTAACGG GTCATAAGAA TTGGGTTGAC GTGTCTTGTT TTTGGACGTC

401
GAAACGAAGA TAAATCATGT CGAAAGCTAC ATATAAGGAA CGTGCTGCTA

CTTTGCTTCT ATTTAGTACA GCTTTCGATG TATATTCCTT GCACGACGAT

451
CTCATCCTAG TCCTGTTGCT GCCAAGCTAT TTAATATCAT GCACGAAAAG

GAGTAGGATC AGGACAACGA CGGTTCGATA AATTATAGTA CGTGCTTTTC

501
CAAACAAACT TGTGTGCTTC ATTGGATGTT CGTACCACCA AGGAATTACT

GTTTGTTTGA ACACACGAAG TAACCTACAA GCATGGTGGT TCCTTAATGA

551
GGAGTTAGTT GAAGCATTAG GTCCCAAAAT TTGTTTACTA AAAACACATG

CCTCAATCAA CTTCGTAATC CAGGGTTTTA AACAAATGAT TTTTGTGTAC

EcoRV NcoI

˜˜˜˜˜˜ ˜˜˜˜˜˜

601
TGGATATCTT GACTGATTTT TCCATGGAGG GCACAGTTAA GCCGCTAAAG

ACCTATAGAA CTGACTAAAA AGGTACCTCC CGTGTCAATT CGGCGATTTC

BstBI

˜˜˜˜˜˜

651
GCATTATCCG CCAAGTACAA TTTTTTACTC TTCGAAGACA GAAAATTTGC

CGTAATAGGC GGTTCATGTT AAAAAATGAG AAGCTTCTGT CTTTTAAACG

701
TGACATTGGT AATACAGTCA AATTGCAGTA CTCTGCGGGT GTATACAGAA

ACTGTAACCA TTATGTCAGT TTAACGTCAT GAGACGCCCA CATATGTCTT

751
TAGCAGAATG GGCAGACATT ACGAATGCAC ACGGTGTGGT GGGCCCAGGT

ATCGTCTTAC CCGTCTGTAA TGCTTACGTG TGCCACACCA CCCGGGTCCA

801
ATTGTTAGCG GTTTGAAGCA GGCGGCAGAA GAAGTAACAA AGGAACCTAG

TAACAATCGC CAAACTTCGT CCGCCGTCTT CTTCATTGTT TCCTTGGATC

851
AGGCCTTTTG ATGTTAGCAG AATTGTCATG CAAGGGCTCC CTATCTACTG

TCCGGAAAAC TACAATCGTC TTAACAGTAC GTTCCCGAGG GATAGATGAC

901
GAGAATATAC TAAGGGTACT GTTGACATTG CGAAGAGCGA CAAAGATTTT

CTCTTATATG ATTCCCATGA CAACTGTAAC GCTTCTCGCT GTTTCTAAAA

951
GTTATCGGCT TTATTGCTCA AAGAGACATG GGTGGAAGAG ATGAAGGTTA

CAATAGCCGA AATAACGAGT TTCTCTGTAC CCACCTTCTC TACTTCCAAT

1001
CGATTGGTTG ATTATGACAC CCGGTGTGGG TTTAGATGAC AAGGGAGACG

GCTAACCAAC TAATACTGTG GGCCACACCC AAATCTACTG TTCCCTCTGC

1051
CATTGGGTCA ACAGTATAGA ACCGTGGATG ATGTGGTCTC TACAGGATCT

GTAACCCAGT TGTCATATCT TGGCACCTAC TACACCAGAG ATGTCCTAGA

1101
GACATTATTA TTGTTGGAAG AGGACTATTT GCAAAGGGAA GGGATGCTAA

CTGTAATAAT AACAACCTTC TCCTGATAAA CGTTTCCCTT CCCTACGATT

1151
GGTAGAGGGT GAACGTTACA GAAAAGCAGG CTGGGAAGCA TATTTGAGAA

CCATCTCCCA CTTGCAATGT CTTTTCGTCC GACCCTTCGT ATAAACTCTT

1201
GATGCGGCCA GCAAAACTAA AAAACTGTAT TATAAGTAAA TGCATGTATA

CTACGCCGGT CGTTTTGATT TTTTGACATA ATATTCATTT ACGTACATAT

1251
CTAAACTCAC AAATTAGAGC TTCAATTTAA TTATATCAGT TATTACCCTA

GATTTGAGTG TTTAATCTCG AAGTTAAATT AATATAGTCA ATAATGGGAT

1301
TGCGGTGTGA AATACCGCAC AGATGCGTAA GGAGAAAATA CCGCATCAGG

ACGCCACACT TTATGGCGTG TCTACGCATT CCTCTTTTAT GGCGTAGTCC

1351
AAATTGTAAA CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA

TTTAACATTT GCAATTATAA AACAATTTTA AGCGCAATTT AAAAACAATT

1401
ATCAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA

TAGTCGAGTA AAAAATTGGT TATCCGGCTT TAGCCGTTTT AGGGAATATT

1451
ATCAAAAGAA TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTGGAACA

TAGTTTTCTT ATCTGGCTCT ATCCCAACTC ACAACAAGGT CAAACCTTGT

1501
AGAGTCCACT ATTAAAGAAC GTGGACTCCA ACGTCAAAGG GCGAAAAACC

TCTCAGGTGA TAATTTCTTG CACCTGAGGT TGCAGTTTCC CGCTTTTTGG

1551
GTCTATCAGG GCGATGGCCC ACTACGTGAA CCATCACCCT AATCAAGTTT

CAGATAGTCC CGCTACCGGG TGATGCACTT GGTAGTGGGA TTAGTTCAAA

1601
TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT AAAGGGAGCC

AAACCCCAGC TCCACGGCAT TTCGTCATTT AGCCTTGGCA TTTCCCTCGG

1651
CCCGATTTAG AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA

GGGCTAAATC TCGAACTGCC CCTTTCGGCC GCTTGCACCG CTCTTTCCTT

1701
GGGAAGAAAG CGAAAGGAGC GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT

CCCTTCTTTC GCTTTCCTCG CCCGCGATCC CGCGACCCTT CACATCGCCA

1751
CACGCTGCGC GTAACCACCA CACCCGCCGC GCTTAATGCG CCGCTACAGG

GTGCGACGCG CATTGGTGGT GTGGGCGGCG CGAATTACGC GGCGATGTCC

˜˜˜

1801
GCGCGTCGCG CCATTCGCCA TTCAGGCTGC GCAACTGTTC GGAAGGCCGA

CGCGCAGCGC GGTAAGCGGT AAGTCCGACG CGTTGACAAC CCTTCCCGCT

PvuI PvuII

˜˜˜ ˜˜˜˜˜˜

1851
TCGGTGCGGG CCTCTTCGCT ATTACGCCAG CTGGCGAAAG GGCGATGTGC

AGCCACGCCC GGAGAAGCGA TAATGCGGTC GACCGCTTTC CCCCTACACG

1901
TGCAAGGCGA TTAAGTTGGG TAACGCCAGG GTTTTCCCAG TCACGACGTT

ACGTTCCGCT AATTCAACCC ATTGCGGTCC CAAAAGGGTC AGTGCTGCAA

BssHII

˜˜˜˜˜˜

1951
GTAAAACGAC GGCCAGTGAG CGCGCGTAAT ACGACTCACT ATAGGGCGAA

CATTTTGCTG CCGGTCACTC GCGCGCATTA TGCTGAGTGA TATCCCGCTT

KpnI

˜˜˜˜˜˜

Asp718

˜˜˜˜˜˜

2001
TTGGGTACCG GCCGCAAATT AAAGCCTTCG AGCGTCCCAA AACCTTCTCA

AACCCATGGC CGGCGTTTAA TTTCGGAAGC TCGCAGGGTT TTGGAAGAGT

2051
AGCAAGGTTT TCAGTATAAT GTTACATGCG TACACGCGTC TGTACAGAAA

TCGTTCCAAA AGTCATATTA CAATGTACGC ATGTGCGCAG ACATGTCTTT

2101
AAAAAGAAAA ATTTGAAATA TAAATAACGT TCTTAATACT AACATAACTA

TTTTTCTTTT TAAACTTTAT ATTTATTGCA AGAATTATGA TTGTATTGAT

2151
TAAAAAAATA AATACCGACC TAGACTTCAG GTTGTCTAAC TCCTTCCTTT

ATTTTTTTAT TTATCCCTGG ATCTGAAGTC CAACAGATTG AGGAAGGAAA

2201
TCGGTTAGAG CGGATGTGGG GGGAGGGCGT GAATGTAAGC GTGACATAAC

AGCCAATCTC GCCTACACCC CCCTCCCGCA CTTACATTCG CACTGTATTG

SalI

˜˜˜˜˜˜˜

XhoI

˜˜˜˜˜˜

2251
TAATTACATG ACTCGAGGTC GACTCAGAAC ACCGTGTTGC CCTCCAGAAA

ATTAATGTAC TGAGCTCCAG CTGAGTCTTG TGGCACAACG GGAGGTCTTT

2301
CGTGGGTGAC GTGGGCATGT GCATGCCCCT CTCCAGGGAG CTGGAGCTGC

GCACCCACTG CACCCGTACA CGTACGGGGA GAGGTCCCTC GACCTCGACG

XmaI

˜˜˜˜˜˜

SmaI

˜˜˜˜˜˜

2351
GGAGTGGCAG GAGGTGGTGG CCCGGGGTCC CGCCCCGCCT CCGTCCTTGC

CCTCACCGTC CTCCACCACC GGGCCCCAGG GCGGGGCGGA GGCAGGAACG

PstI

˜˜˜˜˜˜˜

2401
ACCCCCACCC CCGGCCGCCA GCACTGCAGC GGCCGAAGCA CCTCCCGCCG

TGGGGGTGGG GGCCGGCGGT CGTGACGTCG CCGGCTTCGT GGAGGGCGGC

EcoRI

˜˜˜˜˜˜

2451
CAGGTCCCGG CTGCGCCACG TGTAGATGAC GGGGTTGAGC AGGGAATTCA

GTCCAGGGCC GACGCGGTGC ACATCTACTG CCCCAACTCG TCCCTTAAGT

2501
GGGTGGAGAC GGCGAAAAAG TAGTGGGCTT TGTAGAGGAT CGGGCAGGAG

CCCACCTCTG CCGCTTTTTC ATCACCCGAA ACATCTCCTA GCCCGTCCTC

2551
TGGACGGGAC AGGCATAGTC CAGAAGGAGG ATGCTGAAGG CGGGCAGCCA

ACCTGCCCTG TCCGTATCAG GTCTTCCTCC TACGACTTCC GCCCGTCGGT

2601
GCAGACGATA AAGACGCCTA GCACGATGGT GACCGTCTTG AGCAGGGCTA

CGTCTGCTAT TTCTGCGGAT CGTGCTACCA CTGGCAGAAC TCGTCCCGAT

NheI

˜˜

2651
GCGTCTGCGG GGCGGCCATG TCAGCGTGGC TTGAGCGGAC CACGCAGTAG

CGCAGACGCC CCGCCGGTAC AGTCGCACCG AACTCGCCTG GTGCGTCATC

MscI

˜˜˜˜˜˜

2701
ATGCGCACGT ACAGGGCCAC GATGGCCAAC AGGATGATGG AGAAGATGGT

TACGCGTGCA TGTCCCGGTG CTACCGGTTG TCCTACTACC TCTTCTACCA

2751
CACCACGCAC AGCACATAAT GCTTGGCGTA GAGAGGCAGG ACAGTGGAGC

GTGGTGCGTG TCGTGTATTA CGAACCGCAT CTCTCCGTCC TGTCACCTCG

XhoI

˜˜˜˜˜˜˜

2801
AGGCCTCGAG GTGGCCCAGG CAGTTCCAGC CAAGGATGGG CAGGCCACCG

TCCGGAGCTC CACCGGGTCC GTCAAGGTCG GTTCCTACCC GTCCGGTGGC

2851
AGGACCAGCG AGATGAGCCA CGAGGCCCCG ATGAGCAGAA GCATGCGGCA

TCCTGGTCGC TCTACTCGGT GCTCCGGGGC TACTCGTCTT CGTACGCCGT

MscI

˜˜˜˜˜˜˜

2901
GCTCTTGTCG CTGCCATACA GCTTGACCTT GGCAATGGCC ACGTGGCGCT

CGAGAACAGC GACGGTATGT CGAACTGGAA CCGTTACCGG TGCACCGCGA

MscI

˜˜˜˜˜˜˜

2951
CAATGGCGAT GGCCAGGAGG CTGAAGACAG AGGCCGAGAG CGTGATGAAG

GTTACCGCTA CCGGTCCTCC GACTTCTGTC TCCGGCTCTC GCACTACTTC

XmaI

˜˜˜˜˜˜

SmaI

˜˜˜˜˜˜

3001
GCAGAGCCCT CCCGGGCAAA CCACTGCACA GGCGTCAGCC TCAGCGTGAC

CGTCTCGGGA GGGCCCGTTT GGTGACGTGT CCGCAGTCGG AGTCGCACTG

3051
AGAGCCAGAG AGCAAGGTAT TGGCTACGAA GGCCACGCCT GCCAGTAGAT

TCTCGGTCTC TCGTTCCATA ACCGATGCTT CCGGTGCGGA CGGTCATCTA

3101
CGGAGGCGGC CAGGTTGCCC AGAAACAGGT ACATTGCCGA GTGGAACTTG

GCCTCCGCCG GTCCAACGGG TCTTTGTCCA TGTAACGGCT CACCTTGAAC

NheI

˜˜˜˜˜˜˜

3151
CTGTTTCGGG CCACCGCAAT GAGCGCTAGC AGGTTTTCCA CCACAATGGC

GACAAAGCCC GGTGGCGTTA CTCGCGATCG TCCAAAAGGT GGTGTTACCG

3201
GCAACAGAGG ATGACGATGA AGGCCGAGGC CACCTGGCGG GAGGTCGTCT

CGTTGTCTCC TACTGCTACT TCCGGCTCCG GTGGACCGCC CTCCAGCAGA

3251
CCTGCGTTTC CAGCGTCTCC TTGGTATAAT TATAGTGTTC CTGGACCTTG

GGACGCAAAG GTCGCAGAGG AACCATATTA ATATCACAAG GACCTGGAAC

HindIII

˜˜˜˜˜˜

ClaI

˜˜˜˜˜˜

3301
TTGGGGTTCA GGTACTCCGA GTACAAGCTG CCCATTTTAT CGATAAGCTT

AACCCCAAGT CCATGAGGCT CATGTTCGAC GGGTAAAATA GCTATTCGAA

EcoRV PstI

˜˜˜˜˜˜ ˜˜˜˜˜˜

EcoRI XbaI

˜˜˜˜˜˜˜ ˜˜˜˜˜˜

3351
GATATCGAAT TCCTGCAGCC CGGCTAGTTC TAGAATCCGT CGAAACTAAG

CTATAGCTTA AGGACGTCGG GCCGATCAAG ATCTTAGGCA GCTTTGATTC

3401
TTCTGGTGTT TTAAAACTAA AAAAAAGACT AACTATAAAA GTAGAATTTA

AAGACCACAA AATTTTGATT TTTTTTCTGA TTGATATTTT CATCTTAAAT

3451
AGAAGTTTAA GAAATAGATT TACAGAATTA CAATCAATAC CTACCGTCTT

TCTTCAAATT CTTTATCTAA ATGTCTTAAT GTTAGTTATG GATGCCAGAA

3501
TATATACTTA TTAGTCAAGT AGGGGAATAA TTTCAGGGAA CTGGTTTCAA

ATATATGAAT AATCAGTTCA TCCCCTTATT AAAGTCCCTT GACCAAAGTT

3551
CCTTTTTTTT CAGCTTTTTC CAAATCAGAG AGAGCAGAAG GTAATAGAAG

GCAAAAAAAA GTCGAAAAAG GTTTAGTCTC TCTCGTCTTC CATTATCTTC

3601
GTGTAAGAAA ATGAGATAGA TACATGCGTG GGTCAATTGC CTTGTGTCAT

CACATTCTTT TACTCTATCT ATGTACGCAC CCAGTTAACG CAACACAGTA

3651
CATTTACTCC AGGCAGGTTG CATCACTCCA TTGAGGTTGT GCCCGTTTTT

GTAAATGAGG TCCGTCCAAC GTAGTGAGGT AACTCCAACA CGGGCAAAAA

3701
TGCCTGTTTG TGCCCCTGTT CTCTGTAGTT GCGCTAAGAG AATGGACCTA

ACGGACAAAC ACGGGGACAA GAGACATCAA CGCGATTCTC TTACCTGGAT

3751
TGAACTGATG GTTGGTGAAG AAAACAATAT TTTGGTGCTG GGATTCTTTT

ACTTGACTAC CAACCACTTC TTTTGTTATA AAACCACGAC CCTAAGAAAA

3801
TTTTTCTGGA TGCCAGCTTA AAAAGCGGGC TCCATTATAT TTAGTGGATG

AAAAAGACCT ACGGTCGAAT TTTTCGCCCG AGGTAATATA AATCACCTAC

3851
CCAGGAATAA ACTGTTCACC CAGACACCTA CGATGTTATA TATTCTGTGT

GGTCCTTATT TGACAAGTGG GTCTGTGGAT GCTACAATAT ATAAGACACA

3901
AACCCGCCCC CTATTTTGGG CATGTACGGG TTACAGCAGA ATTAAAAGGC

TTGGGCGGGG GATAAAACCC GTACATGCCC AATGTCGTCT TAATTTTCCG

3951
TAATTTTTTG ACTAAATAAA GTTAGGAAAA TCACTACTAT TAATTATTTA

ATTAAAAAAC TGATTTATTT CAATCCTTTT AGTGATGATA ATTAATAAAT

SacI

˜˜˜˜˜˜

4001
CGTATTCTTT GAAATGGCAG TATTGATAAT GATAAACTGA GCTCCAGCTT

GCATAAGAAA CTTTACCGTC ATAACTATTA CTATTTGACT CGAGGTCGAA

BssHII

˜˜˜˜˜˜˜

4051
TTGTTCCCTT TAGTGACGGT TAATTGCGCG CTTGGCGTAA TCATGGTCAT

AACAAGGGAA ATCACTCCCA ATTAACGCGC GAACCGCATT AGTACCAGTA

4101
AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA

TCGACAAAGG ACACACTTTA ACAATAGGCG AGTGTTAAGG TGTGTTGTAT

4151
GGAGCCGGAA GCATAAAGTG TAAAGCCTCC CCTCCCTAAT GAGTGAGGTA

CCTCGGCCTT CGTATTTCAC ATTTCGGACC CCACGGATTA CTCACTCCAT

4201
ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG TCGGGAAACC

TGAGTGTAAT TAACGCAACG CGAGTGACGG GCGAAAGGTC AGCCCTTTGG

PvuII

˜˜˜˜˜˜˜

4251
TGTCGTGCCA GCTGCATTAA TGAATCGGCC AACGCGCGGG GAGAGGCGGT

ACAGCACGGT CGACGTAATT ACTTAGCCGG TTGCGCGCCC CTCTCCGCCA

4301
TTGCGTATTG GGCGCTCTTC CGCTTCCTCG CTCACTGACT CGCTGCGCTC

AACGCATAAC CCGCGAGAAG GCGAAGGAGC GAGTGACTGA GCGACGCGAG

4351
GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG GCGGTAATAC

CCAGCAAGCC GACGCCGCTC GCCATAGTCG AGTGAGTTTC CGCCATTATG

4401
GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA

CCAATAGGTG TCTTAGTCCC CTATTGCGTC CTTTCTTGTA CACTCGTTTT

4451
GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT

CCGGTCGTTT TCCGGTCCTT GGCATTTTTC CGGCGCAACG ACCGCAAAAA

4501
CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC

GGTATCCGAG GCGGGGGGAC TGCTCGTAGT GTTTTTAGCT GCGAGTTCAG

4551
AGAGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT

TCTCCACCGC TTTGGGCTGT CCTGATATTT CTATGGTCCG CAAAGGGGGA

4601
GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG ACCCTGCCGC TTACCGGATA

CCTTCGAGGG AGCACGCGAG AGGACAAGGC TGGGACGGCG AATGGCCTAT

4651
CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT CATAGCTCAC

GGACAGGCGG AAAGAGGGAA GCCCTTCGCA CCGCGAAAGA GTATCGAGTG

4701
GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT

CGACATCCAT AGAGTCAAGC CACATCCAGC AAGCGAGGTT CGACCCGACA

4751
GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA

CACGTGCTTG GGGGGCAAGT CGGGCTGGCG ACGCGGAATA GGCCATTGAT

4801
TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA CTCGCAGCAG

AGCAGAACTC AGGTTGGGCC ATTCTGTGCT GAATAGCGGT GACCGTCGTC

4851
CCACTGGTAA CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG

GGTGACCATT GTCCTAATCG TCTCGCTCCA TACATCCGCC ACGATGTCTC

4901
TTCTTGAAGT GGTGGCCTAA CTACGGCTAC ACTAGAAGGA CAGTATTTCC

AAGAACTTCA CCACCGGATT GATGCCGATG TGATCTTCCT GTCATAAACC

4951
TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA GTTGGTAGCT

ATAGACGCGA GACGACTTCG GTCAATGGAA GCCTTTTTCT CAACCATCGA

5001
CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC

GAACTAGGCC GTTTGTTTGG TGGCGACCAT CGCCACCAAA AAAACAAACG

5051
AAGCAGCAGA TTACGCGCAG AAAAAAAGCA TCTCAAGAAG ATCCTTTGAT

TTCGTCGTCT AATGCGCGTC TTTTTTTCCT AGAGTTCTTC TAGGAAACTA

5101
CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA

GAAAAGATGC CCCAGACTGC GAGTCACCTT GCTTTTGAGT GCAATTCCCT

5151
TTTTGGTCAT GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT

AAAACCAGTA CTCTAATAGT TTTTCCTAGA AGTGGATCTA GGAAAATTTA

5201
TAAAAATGAA GTTTTAAATC AATCTAAAGT ATATATGAGT AAACTTGGTC

ATTTTTACTT CAAAATTTAG TTAGATTTCA TATATACTCA TTTGAACCAG

5251
TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA GCGATCTGTC

ACTGTCAATG GTTACGAATT AGTCACTCCG TGGATAGAGT CGCTAGACAG

5301
TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG

ATAAAGCAAG TAGGTATCAA CGGACTGAGG GGCAGCACAT CTATTGATGC

5351
ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA

TATGCCCTCC CGAATGGTAG ACCGGGGTCA CGACGTTACT ATGGCGCTCT

5401
CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA

GGGTGCGAGT GGCCGAGGTC TAAATAGTCG TTATTTGGTC GGTCGGCCTT

5451
GGGCCGAGCG CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT

CCCGGCTCGC GTCTTCACCA GGACGTTGAA ATAGGCGGAG GTAGGTCAGA

5501
ATTAATTGTT GCCGGGAAGC TAGAGTAAGT AGTTCGCCAG TTAATAGTTT

TAATTAACAA CGGCCCTTCG ATCTCATTCA TCAAGCGGTC AATTATCAAA

5551
GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA CGCTCGTCGT

CGCGTTGCAA CAACGGTAAC GATGTCCGTA GCACCACAGT GCGAGCAGCA

5601
TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA

AACCATACCG AAGTAAGTCG AGGCCAAGGG TTGCTAGTTC CGCTCAATGT

PvuI

˜˜˜˜

5651
TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT

ACTAGGGGGT ACAACACGTT TTTTCGCCAA TCGAGGAAGC CAGGAGGCTA

PvuI

˜˜

5701
CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG

GCAACAGTCT TCATTCAACC GGCGTCACAA TAGTGAGTAC CAATACCGTC

5751
CACTGCATAA TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG

GTGACGTATT AAGAGAATGA CAGTACGGTA GGCATTCTAC GAAAAGACAC

5801
ACTGGTGAGT ACTCAACCAA GTCATTCTGA GAATAGTGTA TGCGGCGACC

TGACCACTCA TGAGTTGGTT CAGTAAGACT CTTATCACAT ACGCCGCTGG

5851
GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG CCACATAGCA

CTCAACGAGA ACGGGCCGCA GTTATGCCCT ATTATGGCGC GGTGTATCGT

5901
GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC

CTTGAAATTT TCACGAGTAG TAACCTTTTG CAAGAAGCCC CGCTTTTGAG

5951
TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC

AGTTCCTAGA ATGGCGACAA CTCTAGGTCA AGCTACATTG GGTGAGCACG

6001
ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG

TGGGTTGACT AGAAGTCGTA GAAAATGAAA GTGGTCGCAA AGACCCACTC

6051
CAAAAACAGG AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG

GTTTTTGTCC TTCCGTTTTA CGGCGTTTTT TCCCTTATTC CCGCTGTGCC

6101
AAATGTTGAA TACTCATACT CTTCCTTTTT CAATATTATT GAAGCATTTA

TTTACAACTT ATGAGTATGA GAAGGAAAAA GTTATAATAA CTTCGTAAAT

6151
TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT ATTTAGAAAA

AGTCCCAATA ACAGAGTACT CGCCTATGTA TAAACTTACA TAAATCTTTT

6201
ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGGG

TATTTGTTTA TCCCCAAGGC GCGTGTAAAG GGGCTTTTCA CGGTGGACCC

6251
TCCTTTTCAT CACGTGCTAT AAAAATAATT ATAATTTAAA TTTTTTAATA

AGGAAAAGTA GTGCACGATA TTTTTATTAA TATTAAATTT AAAAAATTAT

6301
TAAATATATA AATTAAAAAT AGAAAGTAAA AAAAGAAATT AAAGAAAAAA

ATTTATATAT TTAATTTTTA TCTTTCATTT TTTTCTTTAA TTTCTTTTTT

6351
TAGTTTTTGT TTTCCGAAGA TGTAAAAGAC TCTAGGGGGA TCGCCAACAA

ATCAAAAACA AAAGGCTTCT ACATTTTCTG AGATCCCCCT AGCGGTTGTT

6401
ATACTACCTT TTATCTTGCT CTTCCTGCTC TCAGGTATTA ATGCCGAATT

TATGATGGAA AATAGAACGA GAAGGACGAG AGTCCATAAT TACGGCTTAA

6451
GTTTCATCTT GTCTGTGTAG AAGACCACAC ACGAAAATCC TGTGATTTTA

CAAAGTAGAA CAGACACATC TTCTGGTGTG TGCTTTTAGG ACACTAAAAT

6501
CATTTTACTT ATCGTTAATC GAATGTATAT CTATTTAATC TGCTTTTCTT

GTAAAATGAA TAGCAATTAG CTTACATATA GATAAATTAG ACGAAAAGAA

6551
GTCTAATAAA TATATATGTA AAGTACGCTT TTTGTTGAAA TTTTTTAAAC

CAGATTATTT ATATATACAT TTCATGCGATA AAACAACTTT AAAAAATTTG

6601
CTTTGTTTAT TTTTTTTTCT TCATTCCGTA ACTCTTCTAC CTTCTTTATT

GAAACAAATA AAAAAAAAGA AGTAAGGCAT TGAGAAGATG GAAGAAATAA

6651
TACTTTCTAA AATCCAAATA CAAAACATAA AAATAAATAA ACACAGAGTA

ATGAAAGATT TTAGGTTTAT GTTTTGTATT TTTATTTATT TGTGTCTCAT

6701
AATTCCCAAA TTATTCCATC ATTAAAAGAT ACGAGGCGCG TGTAAGTTAC

TTAAGGGTTT AATAAGGTAG TAATTTTCTA TGCTCCGCGC ACATTCAATG

6751
AGGCAAGCGA TCCGTCCTAA GAAACCATTA TTATCATGAC ATTAACCTAT

TCCGTTCGCT AGGCAGGATT CTTTGGTAAT AATAGTACTG TAATTGGATA

6801
AAAAATAGGC GTATCACGAG GCCCTTTCGT C

TTTTTATCCG CATAGTGCTC CGGGAAAGCA G

[0112]

3

TABLE 3

Nucleotide Sequence of pcDNA3.1 (+)-Edg 5

(SEQ ID NO. 4)

SalI
BglII

˜˜˜ ˜˜˜˜˜˜

1
GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC

CTGCCTAGCC CTCTAGAGGG CTAGGGGATA CCACGTGAGA GTCATGTTAG

51
TGCTCTGATG CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT

ACGAGACTAC GGCGTATCAA TTCGGTCATA GACGAGGGAC GAACACACAA

101
GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG

CCTCCAGCGA CTCATCACGC GCTCGTTTTA AATTCGATGT TGTTCCGTTC

151
GCTTGACCGA CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG

CGAACTGGCT GTTAACGTAC TTCTTAGACG AATCCCAATC CGCAAAACGC

SpeI

˜˜˜˜

201
CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT GATTATTGAC

GACGAAGCGC TACATGCCCG GTCTATATGC GCAACTGTAA CTAATAACTG

SpeI

˜˜˜˜

251
TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA

ATCAATAATT ATCATTAGTT AATGCCCCAG TAATCAAGTA TCGGGTATAT

301
TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG

ACCTCAAGGC GCAATGTATT GAATGCCATT TACCGGGCGG ACCGACTGGC

351
CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT

GGGTTGCTGG GGGCGGGTAA CTGCAGTTAT TACTGCATAC AAGGGTATCA

401
AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT

TTGCGGTTAT CCCTGAAAGG TAACTGCAGT TACCcACCTC ATAAATGCCA

451
AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC

TTTGACGGGT GAACCGTCAT GTAGTTCACA TAGTATACGG TTCATGCGGG

501
CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA

GGATAACTGC AGTTACTGCC ATTTACCGGG CGGACCGTAA TACGGGTCAT

551
CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA

GTACTGGAAT ACCCTGAAAG GATGAACCGT CATGTAGATG CATAATCAGT

NcoI

˜˜˜˜˜˜˜

601
TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA

AGCGATAATG GTACCACTAC GCCAAAACCG TCATGTAGTT ACCCGCACCT

651
TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA

ATCGCCAAAC TGAGTGCCCC TAAAGGTTCA GAGGTGGGGT AACTGCAGTT

701
TGGGAGTTTG TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA

ACCCTCAAAC AAAACCGTGG TTTTAGTTGC CCTGAAAGGT TTTACAGCAT

751
ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG

TGTTGAGGCG GGGTAACTGC GTTTACCCGC CATCCGCACA TGCCACCCTC

SacI

˜˜˜˜˜˜˜

801
GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG

CAGATATATT CGTCTCGAGA GACCGATTGA TCTCTTGGGT GACGAATGAC

NheI

˜˜˜˜˜˜

851
GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC

CGAATAGCTT TAATTATGCT GAGTGATATC CCTCTGGGTT CGACCGATCG

HindIII

˜˜˜˜˜˜

PmeI ClaI

˜˜˜˜˜˜˜˜ ˜˜˜˜˜˜˜

901
GTTTAAACTT AAGCTTATCG ATAAAATGGG CAGCTTGTAC TCGGAGTACC

CAAATTTGAA TTCGAATAGC TATTTTACCC GTCGAACATG AGCCTCATGG

951
TGAACCCCAA CAAGGTCCAG GAACACTATA ATTATACCAA GGAGACGCTG

ACTTGGGGTT GTTCCAGGTC CTTGTGATAT TAATATGGTT CCTCTGCGAC

1001
GAAACGCAGG AGACGACCTC CCGCCAGGTG GCCTCGGCCT TCATCGTCAT

CTTTGCGTCC TCTGCTGGAG GGCGGTCCAC CGGAGCCGGA AGTAGCAGTA

1051
CCTCTGTTGC GCCATTGTGG TGGAAAACCT TCTGGTGCTC ATTGCGGTGG

GGAGACAACG CGGTAACACC ACCTTTTGGA AGACCACGAG TAACGCCACC

1101
CCCGAAACAG CAAGTTCCAC TCGGCAATGT ACCTGTTTCT GGGCAACCTG

GGGCTTTGTC GTTCAAGGTG AGCCGTTACA TGGACAAAGA CCCGTTGGAC

1151
GCCGCCTCCG ATCTACTGGC AGGCGTGGCC TTCGTAGCCA ATACCTTGCT

CGGCGGAGGC TAGATGACCG TCCGCACCGG AAGCATCGGT TATGGAACGA

XmaI

˜˜˜˜˜˜SmaI

1201
CTCTGGCTCT GTCACGCTGA GGCTGACGCC TGTGCAGTGG TTTGCCCGGG

GAGACCGAGA CAGTGCGACT CCGACTGCGG ACACGTCACC AAACGGGCCC

MscI

˜˜˜˜˜

1251
AGGGCTCTGC CTTCATCACG CTCTCGGCCT CTGTCTTCAG CCTCCTGGCC

TCCCGAGACC GAAGTAGTGC GAGAGCCGGA GACAGAAGTC GGAGGACCGG

MscI MscI

˜ ˜˜˜˜˜˜

1301
ATCGCCATTG AGCGCCACGT GGCCATTGCC AAGGTCAAGC TGTATGGCAG

TAGCGGTAAC TCGCGGTGCA CCGGTAACGG TTCCAGTTCG ACATACCGTC

1351
CGACAAGAGC TGCCGCATGC TTCTGCTCAT CGGGGCCTCG TGGCTCATCT

2351
AGCGTGACCG CTACACTTGC CAGCGCCCTA GCGCCCGCTC CTTTCGCTTT

TCGCACTGGC GATGTGAACG GTCGCGGGAT CGCGCGCGAG GAAAGCGAAA

2401
CTTCCCTTCC TTTCTCGCCA CGTTCGCCOG CTTTCCCCGT CAAGCTCTAA

GAAGGGAAGG AAAGAGCGGT GCAAGCGGCC GAAAGGGGCA GTTCGAGATT

2451
ATCGGGGGCT CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC

TAGCCCCCGA GGGAAATCCC AAGGCTAAAT CACGAAATGC CGTGGAGCTG

2501
CCCAAAAAAC TTGATTAGGG TGATGGTTCA CGTAGTGGGC CATCGCCCTG

GGGTTTTTTG AACTAATCCC ACTACCAAGT GCATCACCCG GTAGCGGGAC

2551
ATAGACGGTT TTTCGCCCTT TGACGTTGGA GTCCACGTTC TTTAATAGTG

TATCTGCCAA AAAGCGGGAA ACTGCAACCT CAGGTGCAAG AAATTATCAC

2601
GACTCTTCTT CCAAACTGGA ACAACACTCA ACCCTATCTC GGTCTATTCT

CTGAGAACAA GGTTTGACCT TGTTGTGAGT TGGGATAGAG CCAGATAAGA

2651
TTTGATTTAT AAGGGATTTT GCCGATTTCG GCCTATTGGT TAAAAAATGA

AAACTAAATA TTCCCTAAAA CGGCTAAAGC CGGATAACCA ATTTTTTACT

2701
GCTGATTTAA CAAAAATTTA ACGCGAATTA ATTCTGTGGA ATGTGTGTCA

CGACTAAATT GTTTTTAAAT TGCGCTTAAT TAAGACACCT TACACACAGT

2751
GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA

CAATCCCACA CCTTTCAGGG GTCCGAGGGG TCGTCCGTCT TCATACGTTT

2801
GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC

CGTACGTAGA GTTAATCAGT CGTTGGTCCA CACCTTTCAG GGGTCCGACG

2851
CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT

GGTCGTCCGT CTTCATACGT TTCGTACGTA GAGTTAATCA GTCGTTGGTA

2901
AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG CCCAGTTCCG

TCAGGGCGGG GATTGAGGCG GGTAGGGCGG GGATTGAGGC GGGTCAAGGC

NcoI

˜˜˜˜˜˜˜

2951
CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC

GGGTAAGAGG CGGGGTACCG ACTGATTAAA AAAAATAAAT ACGTCTCCGG

3001
GAGGCCGCCT CTGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT

CTCCGGCGGA GACGGAGACT CGATAAGGTC TTCATCACTC CTCCGAAAAA

XmaI

˜˜˜˜˜˜˜

SmaI

˜˜˜˜˜˜˜

3051
TGGAGGCCTA GGCTTTTGCA AAAAGCTCCC GGGAGCTTCT ATATCCATTT

ACCTCCGGAT CCGAAAACGT TTTTCGAGGG CCCTCGAACA TATAGGTAAA

BclI

3101
TCGGATCTGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT GATTGAACAA

AGCCTAGACT AGTTCTCTGT CCTACTCCTA GCAAAGCGTA CTAACTTGTT

3151
GATGGATTGC ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG

CTACCTAACG TGCGTCCAAG AGGCCGGCGA ACCCACCTCT CCGATAAGCC

3201
CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC

GATACTGACC CGTGTTGTCT GTTAGCCGAC GAGACTACGG CGGCACAAGG

3251
GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT TTCTCAAGAC CGACCTGTCC

CCGACAGTCG CGTCCCCGCG GGCCAAGAAA AACAGTTCTG GCTGGACAGG

PstI MscI

˜˜˜˜˜˜˜ ˜˜˜˜

3301
GGTGCCCTGA ATGAACTGCA GGACGAGGCA GCGCGGCTAT CGTGGCTGGC

CCACGGGACT TACTTGACGT CCTGCTCCGT CGCGCCGATA GCACCGACCG

MscI PvuII

˜˜ ˜˜˜˜˜˜

3351
CACGACGGGC GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG

GTGCTGCCCG CAAGGAACGC GTCGACACGA GCTGCAACAG TGACTTCGCC

3401
GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA

CTTCCCTGAC CGACGATAAC CCGCTTCACG GCCCCGTCCT AGAGGACAGT

3451
TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG

AGAGTGGAAC GAGGACGGCT CTTTCATAGG TAGTACCGAC TACGTTACGC

3501
GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA

CGCCGACQTA TGCGAACTAG GCCGATGGAC GGGTAAGCTG GTGGTTCGCT

3551
AACATCGCAT CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT

TTGTAGCGTA GCTCGCTCGT GCATGAGCCT ACCTTCGGCC AGAACAGCTA

3601
CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT

GTCCTACTAG ACCTGCTTCT CGTAGTCCCC GAGCGCGGTC GGCTTGACAA

BssHII NcoI

˜˜˜˜˜˜ ˜˜

3651
CGCCAGGCTC AAGGCGCGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC

GCGGTCCGAG TTCCGCGCGT ACGGGCTGCC GCTCCTAGAG CAGCACTGGG

NcoI

˜˜˜˜

3701
ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG CCGCTTTTCT

TACCGCTACG GACGAACGGC TTATAGTACC ACCTTTTACC GGCGAAAAGA

3751
GGATTCATCG ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT

CCTAAGTAGC TCACACCGGC CGACCCACAC CGCCTGGCGA TAGTCCTGTA

3801
AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG

TCGCAACCGA TGGQCACTAT AACGACTTCT CCAACCGCCG CTTACCCGAC

3851
ACCGCTTCCT CGTGCTTTAC GGTATCGCCC CTCCCGATTC GCAGCGCATC

TGGCGAAGGA GCACGAAATG CCATAGCGGC GAGGGCTAAG CGTCGCGTAG

BstBI

˜˜˜

3901
GCCTTCTATC GCCTTCTTGA CCAGTTCTTC TGAGCGGGAC TCTGGGGTTC

CGGAAGATAG CGGAAGAACT GCTCAAGAAG ACTCGCCCTG AGACCCCAAG

BstBI

˜˜˜

3951
GAAATGACCG ACCAAGCGAC GCCCAACCTG CCATCACGAG ATTTCGATTC

CTTTACTGGC TGGTTCGCTG CGGGTTGGAC GGTAGTGCTC TAAAGCTAAG

4001
CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT TTCCGGGACG

GTGGCGGCGG AAGATACTTT CCAACCCGAA GCCTTAGCAA AAGGCCCTGC

4051
CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC

GGCCGACCTA CTAGGAGGTC GCGCCCCTAG AGTACGACCT CAAGAAGCGG

4101
CACCCCAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG

GTGGGGTTGA ACAAATAACG TCGAATATTA CCAATGTTTA TTTCGTTATC

4151
CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG

GTAGTGTTTA AAGTGTTTAT TTCGTAAAAA AAGTGACGTA AGATCAACAC

SalI

˜˜˜˜˜˜

4201
GTTTGTCCAA ACTCATCAAT GTATCTTATC ATGTCTGTAT ACCGTCGACC

CAAACAGGTT TGAGTAGTTA CATAGAATAG TACAGACATA TGGCAGCTGG

4251
TCTAGCTAGA GCTTGGCGTA ATCATGGTCA TAGCTGTTTC CTGTGTGAAA

AGATCGATCT CGAACCGCAT TAGTACCAGT ATCGACAAAG GACACACTTT

4301
TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGA AGCATAAAGT

AACAATAGGC GAGTGTTAAG GTGTGTTGTA TGCTCGGCCT TCGTATTTCA

4351
GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATT AATTGCGTTG

CATTTCGGAC CCCACGGATT ACTCACTCGA TTGAGTGTAA TTAACGCAAC

PvuII

˜˜˜˜˜˜

4401
CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC AGCTGCATTA

GCGAGTGACG GGCGAAAGGT CAGCCCTTTG GACAGCACGG TCGACGTAAT

4451
ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT

TACTTAGCCG GTTGCGCGCC CCTCTCCGCC AAACGCATAA CCCGCGAGAA

4501
CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA

GGCGAAGGAG CGAGTGACTG AGCGACGCGA GCCAGCAAGC CGACGCCGCT

4551
GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG

CGCCATAGTC GAGTGAGTTT CCGCCATTAT GCCAATAGGT GTCTTAGTCC

4601
GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA

CCTATTGCGT CCTTTCTTGT ACACTCGTTT TCCGGTCGTT TTCCGGTCCT

4651
ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT

TGGCATTTTT CCGGCGCAAC GACCGCAAAA AGGTATCCGA GGCGGGGGGA

4701
GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC

CTGCTCGTAG TGTTTTTAGC TGCGAGTTCA GTCTCCACCG CTTTGGGCTG

4751
AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT

TCCTGATATT TCTATGGTCC GCAAAGGGGG ACCTTCGAGG GAGCACGCGA

4801
CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT

GAGGACAAGG CTGGGACGGC GAATGGCCTA TGGACAGGCG GAAAGAGGCA

4851
TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC

AGCCCTTCGC ACCGCGAAAG AGTATCGAGT GCGACATCCA TAGAGTCAAG

4901
GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC

CCACATCCAG CAAGCGAGGT TCOACCCGAC ACACGTGCTT GGGGGGCAAG

4951
AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG

TCGGGCTGGC GACGCGGAAT AGGCCATTGA TAGCAGAACT CAGGTTGGGC

5001
GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG

CATTCTGTGC TGAATAGCGG TGACCGTCGT CGGTGACCAT TGTCCTAATC

5051
CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA

GTCTCGCTCC ATACATCCGC CACGATGTCT CAAGAACTTC ACCACCGGAT

5101
ACTACGGCTA CACTAGAAGA ACAGTATTTG GTATCTGCGC TCTGCTGAAG

TGATGCCGAT GTGATCTTCT TGTCATAAAC CATAGACGCG AGACGACTTC

5151
CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC

GGTCAATGGA AGCCTTTTTC TCAACCATCG AGAACTAGGC CGTTTGTTTG

5201
CACCGCTGGT AGCGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA

GTGGCGACCA TCGCCAAAAA AACAAACGTT CGTCGTCTAA TGCGCGTCTT

5251
AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT

TTTTTCCTAG AGTTCTTCTA GGAAACTAGA AAAGATGCCC CAGACTGCGA

5301
CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA

GTCACCTTGC TTTTGAGTGC AATTCCCTAA AACCAGTACT CTAATAGTTT

5351
AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA

TTCCTAGAAG TGGATCTAGG AAAATTTAAT TTTTACTTCA AAATTTAGTT

5401
TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC

AGATTTCATA TATACTCATT TGAACCAGAC TGTCAATGGT TACGAATTAG

5451
AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC

TCACTCCGTG GATAGAGTCG CTAGACAGAT AAAGCAAGTA GGTATCAACG

5501
CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG

GACTGAGGGG CAGCACATCT ATTGATGCTA TGCCCTCCCG AATGGTAGAC

5551
GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT

CGGGGTCACG ACGTTACTAT GGCGCTCTGG GTGCGAGTGG CCGAGGTCTA

5601
TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC

AATAGTCGTT ATTTGGTCGG TCGGCCTTCC CGGCTCGCGT CTTCACCAGG

5651
TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA

ACGTTGAAAT AGGCGGAGGT AGGTCAGATA ATTAACAACG GCCCTTCGAT

5701
GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT

CTCATTCATC AAGCGGTCAA TTATCAAACG CGTTGCAACA ACGGTAACGA

5751
ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC

TGTCCGTAGC ACCACAGTGC GAGCAGCAAA CCATACCGAA GTAAGTCGAG

5801
CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA

GCCAAGGGTT GCTAGTTCCG CTCAATGTAC TAGGGGGTAC AACACGTTTT

PvuI

˜˜˜˜˜˜

5851
AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC

TTCGCCAATC GAGGAAGCCA GGAGGCTAGC AACAGTCTTC ATTCAACCGG

5901
GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT

CGTCACAATA GTGAGTACCA ATACCGTCGT GACGTATTAA GAGAATGACA

5951
CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT

GTACGGTAGG CATTCTACGA AAAGACACTG ACCACTCATG AGTTGGTTCA

6001
CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA

GTAAGACTCT TATCACATAC GCCGCTGGCT CAACGAGAAC GGGCCGCAGT

6051
ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT

TATGCCCTAT TATGGCGCGG TGTATCGTCT TGAAATTTTC ACGAGTAGTA

6101
TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA

ACCTTTTGCA AGAAGCCCCG CTTTTGAGAG TTCCTAGAAT GGCGACAACT

6151
GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT

CTAGGTCAAG CTACATTGGG TGAGCACGTG GGTTGACTAG AAGTCGTAGA

6201
TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC

AAATGAAAGT GGTCGCAAAG ACCCACTCGT TTTTGTCCTT CCGTTTTACG

6251
CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT

GCGTTTTTTC CCTTATTCCC GCTGTGCCTT TACAACTTAT GAGTATGAGA

6301
TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC

AGGAAAAAGT TATAATAACT TCGTAAATAG TCCCAATAAC AGAGTACTCG

6351
GOATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG

CCTATGTATA AACTTACATA AATCTTTTTA TTTGTTTATC CCCAAGGCGC

SalI

˜˜˜˜

6401
CACATTTCCC CGAAAAGTGC CACCTGACGT C

GTGTAAAGGG GCTTTTCACG GTGGACTGCA G

REFERENCE LIST

[0113] Alewijnse A E, Timmerman H, Jacobs E H, Smit M J, Roovers E, Cotecchia S and Leurs R (2000) The Effect of Mutations in the DRY Motif on the Constitutive Activity and Structural Instability of the Histamine H2 Receptor. Mol Pharmacol 57: pp 890-898.

[0114] An S, Zheng Y and Bleu T (2000) Sphingosine 1-Phosphate-Induced Cell Proliferation, Survival, and Related Signaling Events Mediated by G Protein-Coupled Receptors Edg3 and Edg5. J Biol Chem 275: pp 288-296.

[0115] Botstein D, Chervitz S A and Cherry J M (1997) Yeast As a Model Organism. Science 277: pp 1259-1260.

[0116] Brown A J, Dyos S L, Whiteway M S, White J H, Watson M A, Marzioch M, Clare J J, Cousens D J, Paddon C, Plumpton C, Romanos M A and Dowell S J (2000) Functional Coupling of Mammalian Receptors to the Yeast Mating Pathway Using Novel Yeast/Mammalian G Protein α-Subunit Chimeras. Yeast 16: pp 11-22.

[0117] Chambers J K, Macdonald L E, Sarau H M, Ames R S, Freeman K, Foley J J, Zhu Y, McLaughlin M M, Murdock P, McMillan L, Trill J, Swift A, Aiyar N, Taylor P, Vawter L, Naheed S, Szekeres P, Hervieu G, Scott C, Watson J M, Murphy A J, Duzic E, Klein C, Bergsma D J, Wilson S and Livi G P (2000) A G Protein-Coupled Receptor for UDP-Glucose. J Biol Chem 275: pp 10767-10771.

[0118] Chen G, Way J, Armour S, Watson C, Queen K, Jayawickreme C K, Chen W J and Kenakin T (2000) Use of Constitutive G Protein-Coupled Receptor Activity for Drug Discovery. Mol Pharmacol 57: pp 125-134.

[0119] Daeffler L and Landry Y (2000) Inverse Agonism at Heptahelical Receptors: Concept, Experimental Approach and Therapeutic Potential. Fundam Clin Pharmacol 14: pp 73-87.

[0120] Dhanasekaran N, Heasley L E and Johnson G L (1995) G Protein-Coupled Receptor Systems Involved in Cell Growth and Oncogenesis. Endocr Rev 16: pp 259-270.

[0121] Duprez L, Parma J, Costagliola S, Hermans J, Van Sande J, Dumont J E and Vassart G (1997) Constitutive Activation of the TSH Receptor by Spontaneous Mutations Affecting the N-Terminal Extracellular Domain. FEBS Lett 409: pp 469-474.

[0122] Egan C T, Herrick-Davis K and Teitler M (1998) Creation of a Constitutively Activated State of the 5-Hydroxytryptamine2A Receptor by Site-Directed Mutagenesis: Inverse Agonist Activity of Antipsychotic Drugs. J Pharmacol Exp Ther 286: pp 85-90.

[0123] Erlenbach I, Kostenis E, Schmidt C, Serradeil-Le Gal C, Raufaste D, Dumont M E, Pausch M H and Wess J (2001) Single Amino Acid Substitutions and Deletions That Alter the G Protein Coupling Properties of the V2 Vasopressin Receptor Identified In Yeast by Receptor Random Mutagenesis. J Biol Chem pp M103203200.

[0124] Hadcock J R and Pausch M (1999) Ligand screening of G protein-coupled receptors in yeast, in G Protein-Coupled Receptors (Haga T and Berstein G eds) pp 49-69, CRC Press LLC, Boca Raton, Fla.

[0125] Hla T (2001) Sphingosine 1-Phosphate Receptors. Prostaglandins & Other Lipid Mediators 64: pp 135-142.

[0126] Ito H, Fukuda Y, Murata K and Kimura A (1983) Transformation of Intact Yeast Cells Treated With Alkali Cations. J Bacteriol 153: pp 163-168.

[0127] Jensen A A, Spalding T A, Burstein E S, Sheppard P O, O'Hara P J, Brann M R, Krogsgaard-Larsen P and Bräuner-Osborne H (2000) Functional Importance of the Ala116-Pro136 Region in the Calcium-Sensing Receptor. J Biol Chem 275: pp 29547-29555.

[0128] Konopka J B, Margarit S M and Dube P (1996) Mutation of Pro-258 in Transmembrane Domain 6 Constitutively Activates the G Protein-Coupled α-Factor Receptor. Proc Natl Acad Sci USA 93: pp 6764-6769.

[0129] Lefkowitz R J, Cotecchia S, Samama P and Costa T (1993) Constitutive Activity of Receptors Coupled to Guanine Nucleotide Regulatory Proteins. Trends Pharmacol Sci 14: pp 303-307.

[0130] Ma H, Kunes S, Schatz P J and Botstein D (1987) Plasmid Construction by Homologous Recombination in Yeast. Gene 58: pp 201-216.

[0131] MacEwan D J and Milligan G (1996) Inverse Agonist-Induced Up-Regulation of the Human B2-Adrenoceptor in Transfected Neuroblastoma X Glioma Hybrid Cells. Mol Pharmacol 50: pp 1479-1486.

[0132] Oldenburg K R, Vo K T, Michaelis S and Paddon C (1997) Recombination-Mediated PCR-Directed Plasmid Construction in Vivo in Yeast. Nucleic Acids Res 25: pp 451-452.

[0133] Parnot C, Bardin S, Miserey-Lenkei S, Guedin D, Corvol P and Clauser E (2000) Systematic Identification of Mutations That Constitutively Activate the Angiotensin II Type 1A Receptor by Screening a Randomly Mutated CDNA Library With an Original Pharmacological Bioassay. Proc Natl Acad Sci USA 97: pp 7615-7620.

[0134] Price L A, Kajkowski E M, Hadcock J R, Ozenberger B A and Pausch M H (1995) Functional Coupling of a Mammalian Somatostatin Receptor to the Yeast Pheromone Response Pathway. Mol Cell Biol 15: pp 6188-6195.

[0135] Rao V R and Oprian D D (1996) Activating Mutations of Rhodopsin and Other G Protein-Coupled Receptors. Annu Rev Biophys Biomol Struct 25: pp 287-314.

[0136] Robinson P R, Cohen G B, Zhukovsky E A and Oprian D D (1992) Constitutively Active Mutants of Rhodopsin. Neuron 9: pp 719-725.

[0137] Samama P, Bond R A, Rockman H A, Milano C A and Lefkowitz R J (1997) Ligand-Induced Overexpression of a Constitutively Active B2-Adrenergic Receptor: Pharmacological Creation of a Phenotype in Transgenic Mice. Proc Natl Acad Sci USA 94: pp 137-141.

[0138] Sommers C M and Dumont M E (1997) Genetic Interactions Among the Transmembrane Segments of the G Protein Coupled Receptor Encoded by the Yeast STE2 Gene. J Mol Biol 266: pp 559-575.

[0139] Sommers C M, Martin N P, Akal-Strader A, Becker J M, Naider F and Dumont M E (2000) A Limited Spectrum of Mutations Causes Constitutive Activation of the Yeast Alpha-Factor Receptor [In Process Citation]. Biochemistry 39: pp 6898-6909.

[0140] Spiegel A M (1996) Mutations in G Proteins and G Protein-Coupled Receptors in Endocrine Disease. J Clin Endocrinol Metab 81: pp 2434-2442.

[0141] Svetlov V and Cooper T G (1998) Efficient PCR-Based Random Mutagenesis of Sub-Genic (100 Bp) DNA Fragments. Yeast 14: pp 89-91.

[0142] Tate C G and Grisshammer R (1996) Heterologous Expression of G-Protein-Coupled Receptors. Trends Biotechnol 14: pp 426-430.

[0143] Tsao P, Cao T and von Zastrow M (2001) Role of Endocytosis in Mediating Downregulation of G-Protein-Coupled Receptors. Trends Pharmacol Sci 22: pp 91-96.

[0144] Varma D R, Shen H, Deng X F, Peri K G, Chemtob S and Mulay S (1999) Inverse Agonist Activities of B-Adrenoceptor Antagonists in Rat Myocardium. Br J Pharmacol 127: pp 895-902.

[0145] Zwick E, Hackel P O, Prenzel N and Ullrich A (1999) The EGF Receptor As Central Transducer of Heterologous Signalling Systems. Trends Pharmacol Sci 20: pp 408-412.

Method of identifying protein CAMs (constitutively active mutants)

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Priority Claims (1)

Provisional Applications (1)