WO1995001430A1

WO1995001430A1 - Insect diuretic hormone receptor, preferably isolated from manduca sexta

Info

Publication number: WO1995001430A1
Application number: PCT/EP1994/002159
Authority: WO
Inventors: Jeff Dale Reagan
Original assignee: Sandoz Ltd.; Sandoz-Patent-Gmbh; Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H.
Priority date: 1993-07-02
Filing date: 1994-07-01
Publication date: 1995-01-12
Also published as: EP0710284A1; AU7490694A

Abstract

Insect diuretic hormone receptors useful in controlling insects, identifying insect control agents and controlling gene expression.

Description

INSECT DIURETIC HORMONE RECEPTOR, PREFERABLY ISOLATED FROM MANDUCA SEXTA.

The present invention concerns insect hormone receptor proteins, genes encoding said proteins and uses of said receptor proteins and genes in the identification of insect control agents and in the regulation of gene expression. In particular the hormone receptor is a diuretic hormone (DH) receptor. The following abbreviations are used in this application: Mt, Malpighian tubule; Mas-DH, Manduca sexta diuretic hormone; Mas-DH-R, Manduca sexta diuretic hormone receptor, Lorn DH, Locusta migratoria diuretic hormone; Acd DH, Acheta domesticus diuretic hormone; BSA, bovine serum albumin; DMF dimethylformamide; TFA, trifluoroacetic acid; BOC, t-butoxycarbonyl; and SF9, Spodoptera frugiperda.

The control of fluid secretion in insects is modulated at least in part by diuretic hormones and antidiuretic hormones. The primary target site for diuretic hormones is the Malpighian tubules (Mt) which are the main organs for fluid and ion secretion in insects. In addition diuretic hormones may act by decreasing fluid resorption by the rectum. Thus far diuretic hormones from Locusta migratoria, Manduca sexta. Achete domesticus and Periplenata americana have been identified and sequenced and these peptides share between 34% and 50% sequence identity with the 41 amino acid peptide isolated from Manduca sexta. The 41 amino acid diuretic hormone isolated from Manduca sexta is an amidated peptide which has been shown to stimulate fluid secretion in a number of insects such as Manduca sexta, Pieiris rapae and Achete domesticus. The high degree of sequence homology with the other diuretic hormones referred to above suggests that these hormones belong to a single family of corticotropin releasing factor (CFR)-related peptides.

Manduca sexta diuretic hormone, its isolation, characterization, preparation and use are described in US Patent Application Serial No. 07/774,391 the contents of which are incorporated herein by reference.

In connection with the present invention it was postulated that diuretic hormones exert their effects on fluid secretion by binding to a membrane bound receptor on Malpighian tubule cells initiating a cascade of events such as secondary messenger formation followed by opening of a specific ion channel. It would thus be desirable for a number of reasons such as in the control of insect pests and for the regulation of gene expression to obtain information on such receptors and the genes controlling them. A detailed discussion of these uses of insect hormone receptors based on the example of steroid receptors can be found in US Patent Application Serial No. 07/485,749 pp 4 to 8 the contents of which are incorporated herein by reference.

The present invention therefore provides a polypeptide comprising an insect diuretic hormone receptor or analog or fragment thereof which is substantially free from associated insect polypeptides and exhibits biological activity characteristic of a native insect diuretic hormone receptor.

More particularly the invention concerns an insect diuretic hormone receptor isolated from Manduca sexta having the amino acid sequence shown in SEQ ID NO. 2 and analogs or fragments thereof. The receptor consists of 395 amino acids and contains seven putative membrane spanning regions. Analogs and fragments of an insect diuretic hormone receptor as used herein refers to modified amino acid sequences resulting from truncation of, deletion of, substitution of, addition of or extension by one or more amino acids which retain the biological activity characteristic of a native insect diuretic hormone receptor.

Preferred such analogs and fragments are preferably at least 70% homologous, more preferably 80% homologous even more preferably 85% homologous with a native insect diuretic hormone receptor. The invention also provides DNA sequences encoding an insect diuretic hormone receptor and analogs or fragments thereof. These DNA sequences are preferably combined with heterologous DNA sequences such as promoter or other operator and regulator sequences to form systems for the expression, production and/or delivery of polypeptides having the biological activity characteristic of a native insect receptor hormone receptor. Such systems may also contain other DNA sequences encoding other functional polypeptides whereby the DNA sequence encoding an insect diuretic hormone receptor or an analog or fragment thereof can be used to regulate expression.

By way of illustration SEQ ID No. 1 shows a DNA sequence encoding a native insect diuretic hormone receptor isolated from Manduca sexta. It will be appreciated that this sequence is but one of a variety which may be constructed to encode an insect diuretic hormone receptor or an analog or fragment thereof. Different codons encoding the same amino acids may be readily substituted as allowed by the genetic code and any DNA sequence encoding a particular amino acid sequence according to the invention is included within the invention.

Preferred DNA sequences according to the invention are those which will hybridize under stringent conditions to the DNA sequence shown in SEQ ID No. 1. Stringent hybridization conditions as contemplated herein are those in which hybridization is effected in a standard manner at 60°C in 2.5 X saline citrate buffer (a.k.a. SSC buffer) followed by merely washing at 37°C at a reduced buffer concentration, which will not affect true hybrids which have formed.

The invention also includes procaryotes and eukaryotes transformed with DNA according to the invention. Examples include viruses and bacterial, insect, yeast, mammalian and plant cells.

Transformation can be effected in conventional manner with naked DNA or with the DNA cloned into suitable vectors such as plasmids such as described e.g. in Chu, G,; Hayakawa, H.; Berg, P. Electroporation for the efficient transfection of mammalian cells with DNA. Nuc. Acid. Res..15, 1311-1326 (1987); Trevors, J.T. Electrotransformation of Bacteria Met. in Mol. and Cell. Biol. 2:247-253 (1991). Transformed cells can themselves be employed to transform other cells of the same or different type e.g. plant cells by breeding or insect cells by transformed baculoviruses. Other aspects of the invention include: rDNA comprising a regulating element which is responsive to a ligand which binds to an insect diuretic hormone receptor or analog or fragment thereof according to the invention and cells transformed therewith;

Antibodies both poly- and mono-clonal and binding fragments thereof with binding specificity for an epitope characteristic for an insect diuretic hormone receptor or analog or fragment thereof according to the invention;

Methods for selecting ligands with binding specificity for a diuretic hormone binding domain of an insect diuretic hormone receptor or analog or fragment thereof according to the invention; Methods for selecting DNA sequences which are capable of being bound specifically by an insect diuretic hormone receptor or analog or fragment thereof according to the invention;

Chimeric polypeptides comprising a diuretic hormone binding domain of an insect diuretic hormone receptor or analog or fragment thereof according to the invention and at least one further polypeptide and DNA encoding same;

Methods of producing a polypeptide by introducing into a cell which is insensitive to ligands which bind to an insect diuretic hormone receptor or analog or fragment thereof according to the invention a receptor for that ligand and gene encoding the polypeptide which is operably linked to a regulating element which is responsive to said ligand and exposing the cell thus transformed to said ligand.

Methods for isolation and characterization of insect diuretic hormone receptors and for their use as outlined above are conventional and are analogous e.g. to methods and techniques described in US Patent Applications Serial No. 07/485,749 (pp 8 to 47) and Serial No. 07 774,391 (pp 3 to 13), the contents of which are incorporated herein by reference.

Detergents useful in solubilization include Triton^® X 100, CHAPS digitonin and deoxycholate, Haga et. al. in Receptor Biochemistry: A Practical Approach Hulme E.C. ed. pp 1-50 OUP, 1990 describes other techniques and the contents thereof are incorporated herein by reference. Colonies of Manduca sexta are raised on an artificial diet (Troetschler etal. J. Econ. Entomol. 78, 1521-1523 (1985)). Peptides are prepared by solid phase synthesis on a Biosearch 9600 peptide synthesizer using a t-butoxycarbonyl protocol and p-methylbenzylhydrylamine resin (Kataoka et. al. Proc. Nat. Acad. Sci. 86, pp 2976-80 (1989)).

The invention will be further apparent from the following description, taken together with drawings and sequence listings. Of the drawings:

Figure 1 shows a saturation isotherm of Nα-[2,3-³H-propanoyl]Mas-DH binding to CHAP solubilized Mt membrane from 5th stage larvae Manduca sexta;

Figure 2 shows a time course of Nα-[2,3-³H-propanoyl]Mas-DH binding to CHAP solubilized Mt membrane from 5th stage larvae Manduca sexta:

Figure 3 shows displacement of Nα-[2,3-³H-propanoyl]Mas-DH binding by Mas-DH and biotinylated Mas-DH;

Figure 4 shows ligand bound solubilized Mas-DH receptor,

Figure 5 shows the peak corresponding to the receptor-hormone complex when CHAPS solubilized Mt membranes are treated with Nα-[2,3-³H-propanoyl]Mas-DH binding by Mas-DH and separated;

Figure 6 shows the affinity of the receptor for Mas-DH depicted as saturation kinetics;

Figure 7 shows the affinity of the receptor for Mas-DH depicted as the reciprocal of Figure 6 kinetics;

Figure 8 shows the affinity of DH structurally related proteins for the recombinant Mas- DH-R;

Figure 9 shows the plasmid pKSl;

Figure 10 shows the expression of Mas-DH-R in recombinant baculovirus infected Sf9 cells;

Figure 11 shows the stimulation of cAMP synthesis by Mas-DH and N-terminal truncated analogs.

Of the Sequences:

SEQ ID No. 1 shows the cDNA sequence of the Mas-DH-R;

SEQ ID No. 2 shows a 395 amino acid sequence having 7 putative membrane spanning regions and corresponding to the cDNA open reading frame in SEQ ID No. 1;

SEQ ID No. 3 shows the nucleotide sequences of pKSl;

SEQ ID Nos. 4 and 5 show respectively forward and reverse PCR primers used in the modification of the 5' and 3' non-coding regions of the cDNA encoding the Mas-DH-R.

The following Examples are intended to illustrate the present invention without in any way restricting its scope.

EXAMPLE A: Preparation and solubilization of Mt membranes

Malpighian tubules (Mt) are isolated from 60 pre-wandering fifth instar Manduca sexta larvae weighing between 7 and 11 g. The Mt are washed in modified Manduca saline (4 mM NaCl, 40 mM KCl, 18 mM CaCl₂, 3 mM CaCl₂, 5 mM HEPES, pH 6.6) homogenized on ice, and centrifuged at 1200 x g for 10 minutes at 4°C. The supernatant is removed and the pellet washed further with Manduca saline. The pooled supernatants are ultracentrifuged in a Beckman SW28 rotor at 112,700 x g f or 1 hr at 4°C and the resulting pellet resuspended in 6 mL of 45% sucrose/25 mM HEPES, pH 6.6 in an SW28 polyallomer centrifuge tube. Ten percent sucrose/25 mM HEPES pH 6.6 is carefully layered upon the 45% sucrose solution and the resulting step gradient is centrifuged as above. The membranes located at the interface of the sucrose gradient are removed, diluted with 25 mM HEPES pH 6.6 and pelleted at 112,700 x g. The pellet is resuspended in 1 mL of 25 mM HEPES pH 6.6 and stored at -70°C. Protein is determined using a BCA protein assay reagent (Pierce, Rockford, IL). Mt membranes are solubilized at a protein concentration of 2.25 mg/ml in 0.6% CHAPS, 25 mM HEPES, pH 7.0 for 2 h at 4°C. The membranes are then centrifuged in a Beckman, TL-100 ultracentrifuge at 125,000 x g for 45 minutes at 4°C. The supernatant is removed and may be used for solubilized receptor studies. Protein concentrations are determined by use of a BioRad protein assay kit (Bio-Rad, Richmond, CA).

EXAMPLE B: Biotinylation of Mas-DH

Biotin is incorporated into the α-amino group of Mas-DH (cf Kataoka et.al. ibid) via an amidation reaction using sulfosuccinimidyl 2-(biotinamido) ethyl- 1,3-dithiopropionate (NHS- SS-Biotin, Pierce, Rockford, IL). Fully protected Mas-DH is assembled on MBHA resin by the Boc/DIC method on a Biosearch/Millgen 9600 Peptide Synthesizer as described previously (Kataoka et. al. ibid). The Ncc-Boc group of the peptidyl resin (1.64 g, 0.157 mmol) is deprotected with 50% TFA in DCM and the DNP group on His²⁷ is removed by treating with 17.5% thiophenol in DMF in the usual manner. After deprotonating with 10% DIE A in DCM according to the solid phase peptide synthesis protocol, the selectively deprotected peptidyl resin is treated two times with NHS-SS-biotin reagent (100 mg, 0.165 mmol) and diisopropyl ethylamine (DIEA) 55 μl, 0.314 mmol) in 10 ml DMF for 45 min, with DMF wash between biotinylation reactions. Kaiser test for free amino function is carried out. The biotinylated peptide (crude yield 0.82 g) is completely deprotected and cleaved from the solid support by treating 1.81 g biotinylated peptidyl resin with 15 ml HF in the presence of 1.5 ml anisole and 1 mL ethyl sulfϊde at 0°C for 1 hr. The crude peptide is purified by reversed-phase liquid chromatography (RPLC) using a Vydac C₁₈ column, 0.46 x 15 cm, particle size 5 μm, flow rate at 1 ml/min linear gradient elution form 28% to 31.5% acetonitrile in 0.1% trifluoroacetic acid (TFA) over 21 min, monitoring at 220nm and or 280nm. The purified peptide is a white powder and has the correct amino acid composition with a 43% peptide content. The structure is confirmed by electrospray ionization mass spectrometry on a Nermag Quadrupole Mass Spectronometer, Model R30-10, with an electrospray ion source from Analytica, Branford, CT (conducted under contract by TexMS Analytical Services, Houston): calculated for C₂₂.H₃₇₇N₆s.S₅ 5121, found 5122 +-2.

EXAMPLE C: Preparation of Nα-r2.3-³H-Propanoyll Mas-DH

To a solution of Mas-DH (10 μg, 2 nmol) in dry DMF was added 0.057 M diisopropylethylamine in DMF (1.6 μl, 91.2nmol). The resulting solution was added to solvent-free 100 Ci/mmol N-succinimidyl 2,3-³H-propionate (lOOμCi, 1 nmol) and mixed well. After 3 hours at room temperature the mixture was chromatographed by reverse phase HPLC, using a Vydac C18 column (0.46 x 25 cm), a Spectro-Physics 8700 pumping system, and the following gradient elution: flow-rate at 1 ml/min, 0-60% CH₃CN/0.1%. TFA over 60 min. Nα-[2,3-³H-propanoyl] Mas-DH collected at a retention time of 41 min. The yield was 0.68 μg of Nα-[2,3-³H-propanoyl] Mas-DH with a specific activity of 25 Ci mmol. The label was believed to attach to the N-terminal amino function on the basis that this modified Mas-DH resisted Edman degradation.

EXAMPLE D: Saturation and Displacement Binding Experiments

Solubilized membranes are dissolved in 250 μl of ice cold binding buffer (0.2% CHAPS, 25 mM HEPES, pH 7.0, 100 mM NaCl, 10 mg/ml BSA, 2.5 mg/ml bacitracin, 1 mg/ml amastatin) and various concentrations of Nα-[2,3-³H-propanoyl] Mas-DH are added (Nα-[2,3-³H- propanoyl] Mas-DH is dissolved in 20% propanol 0.1% TFA). To tubes used to determine ^' nonspecific binding, 0.1 μg of unlabelled Mas-DH is added. The tubes are incubated on ice for 2 hr. After the incubation period the samples are filtered through a GF/B filter (pretreated for 1 hour with 0.1% PEI). The filters are washed with ice cold 25 mM HEPES, pH 7.0 containing 14% isopropanol and dried under a heat lamp. The dried filters are placed in 5 ml of Ready Protein scintillation cocktail (Beckman, Palo Alto, CA) and counted in a Beckman LS5000 scintillation counter- Data are analyzed by the computer program LIGAND (Munson et.al. 1980).

Figure 1 shows a saturation isotherm of Nα-[2,3-³H-propanoyl] Mas-DH binding to CHAPS solubilized Mt membranes from 5th stadium Manduca sexta larvae. The binding is saturable and specific. Analysis of the binding data by LIGAND indicated a single binding site with a Kd=l.l nM (S.D. = 0.46, n = 7) and a Bmax= 5.8 pmol/mg detergent solubilized protein (S.D. = 3.3 n = 7).

The time course of Nα-[2,3-³H-propanoyl] Mas-DH binding to CHAPS solubilized Mt membranes is shown in Figure 2. Maximum binding occurred approximately 2 hours after the addition of tritiated peptide at 4°C. Addition of an excess of unlabelled Mas-DH to the tritiated Mas-DH bound receptor preparation leads to an exchange of the tritiated peptide for unlabelled peptide (Fig. 2). Complete exchange occurs approximately 1 hour after addition of unlabelled peptide.

Displacement of Nα-[2,3-³H-propanoyl] Mas-DH by Mas-DH and biotinylated Mas-DH is shown in Figure 3. Both Mas-DH and biotinylated Mas DH show similar affinity for the solubilized receptor.

EXAMPLE E: Sucrose Gradient and Gel Filtration

Solubilized Mt membranes are incubated with Nα-[2,3-³H-propanoyl] Mas-DH (unlabelled Mas-DH is included for determination of nonspecific binding) for 2 hours on ice and then layered upon a 5% to 20% linear sucrose gradient and centrifuged in a VTi65.1 rotor (Beckman, Palo Alto, CA) at 269,000xg for 1.5 hours using a slow acceleration and no brake profile. Fractions (1 ml) are collected and the radioactivity in each sample is determined by liquid scintillation counting as described above. For gel filtration studies, solubilized Mt membranes are incubated with Nα-[2,3-³H-propanoyl] Mas-DH (unlabelled Mas-DH is included for determination of nonspecific binding) for 2 hours on ice. The solubilized membranes are then loaded on a 26 cm x 1 cm Sepharose CL-6B column and eluted a buffer containing 0.2% CHAPS, 25 mM HEPES, pH 7.0, 100 mM NaCl at a flow rate of 0.5 ml/min. Fractions (1 ml) are collected and the radioactivity in each sample is determined as above.

When the CHAPS solubilized MT membranes are treated with Ncc-[2,3-³H-propanoyl] Mas-DH and centrifuged in a 5%-20% linear sucrose gradient as described, a radiolabelled peak corresponding to the ligand bound solubilized Mas-DH receptor appears (Fig. 4). When an excess of unlabelled Mas-DH is included in the binding mixture, the radiolabelled peak is absent indicating specific binding of tritium labelled Mas-DH to the solubilized receptor. When the CHAPS solubilized MT membranes are treated with Nα-[2,3-³H-propanoyl] Mas-DH and separated on a sepharose CL-6B column as described a peak corresponding to the receptor- hormone complex elutes prior to unbound tritiated peptide (Fig. 5). This radioactive peak is absent when an excess of unlabelled Mas-DH is present indicating binding of tritium labelled Mas-DH to the solubilized receptor-

EXAMPLE F: Isolation of Mas DH Receptor (Mas-DH-R)

Mas-DH-R is isolated via an expression cloning procedure developed by Aruffo, A. Seed, B. Proc. (1987) Molecular Cloning of CD28 cDNA by a High Efficiency COS Cell Expression System. Proc. Natl. Acad. Sci 84, 8573-8577. A directional cDNA library is prepared from the Malpighian tubules of 5th instar M. sexta and cloned into the EcoRl/Xhol site of the vector pKSl (obtainable from Dr. Peter Mclntyre, Sandoz Institute for Medical Research, London, U.K.). The library is divided into pools of 2200 individuals. Plasmid DNA is prepared from each pool, transfected into Cos7 cells and after three days the cells are screened for Mas-DHR expression using a 125-1 labelled Mas-DH followed by emulsion autoradiography. Positive pools are subdivided until single clones are obtained which is then sequenced. The cDNA sequence of the complete Mas-DH-R is shown in SEQ ID No. 1. The cDNA contains an open reading frame which codes for a protein 395 amino acids in length and has 7 putative membrane spanning domains (SEQ ID No. 2).

The recombinant receptor has a high affinity for Mas-DH as shown in Figures 6 and 7. The Kd value of 56 pM is close to that of the native receptor (Kd = 79 pM). Since the identification of Mas-DH, four other structurally related DH's have been identified. M. sexta diuretic peptide II (Mas-DPϋ), A. domesticus DH (Ach-DH), P. americana DH (Pea-DH) and L. migratoήa DH (Lom-DH). These peptides have a high degree of sequence identity with Mas-DH. Furthermore, as shown in Figure 8, they have a high affinity for the recombinant Mas-DH receptor. Pea-DH and Ach-DH are as effective as Mas-DH at displacing tritiated Mas-DH from the receptor while Lom-DH and Mas-DPII are about 5-10 fold less active.

EXAMPLE G: Expression of Mas-DH-R in recombinant Baculovirus Infected Insect Cells (a). Cell culture and virus infection: Spodoptera frugiperda (Sf9) cells are grown as monolayers in TMN-FH medium (Sigma, St. Louis, MO) supplemented with 10% heat inactivated fetal calf serum, gentamicin and fungizone (2.5 μg/ml). Sf9 cells are treated with either wild type virus or recombinant virus at the indicated multiplicity of infection (MOI) in cell medium at room temperature. After one hour, the medium is replaced with fresh medium and the cells are incubated at 27°C.

(b). Insertion of cDNA encoding Mas-DH-R into the plasmid pVL1393: The 5' and 3' non- coding regions are deleted from the cDNA encoding the Mas-DH-R by polymerase chain reaction (PCR). A Smal site is introduced 6 base pairs upstream of the ATG initiation codon and an Xbal site 39 base pairs downstream of the stop codon by PCR using the sequence given in SEQ ID No. 4 as forward primer and the sequence given in SEQ ID No. 5 as reverse primer. The PCR product is cut with Xbal and Smal and ligated into the Xbal/Smal sites of the baculovirus transfer vector pVL1393 (Pharmingen, San Diego, CA). The PCR product is also ligated into the Sbal and Smal sites of pBluescript (Stratagene, San Diego, CA) and sequenced by the dideoxy nucleotide chain termination method using Sequenase version 2.0 (U.S. Biochemical Corp., Cleveland, OH) to ensure that no mutations are introduced by Taq polymerase.

(c). Construction and isolation of recombinant baculovirus: pVL 1393/Mas-DH-R and linearized AcRP23.1acZ baculovirus DNA (Pharmingen, San Diego, CA) are co-transfected into Sf9 cells by CaPO₄ precipitation and the recombinant baculovirus (BN/Mas-DH-R) is plaque purified using standard procedures. A high titer stock (4.5 x 10⁷ pfu/ml) is prepared by infecting Sf9 cells at a MOI of 0.1 and collecting the supernatant 4 days after infection.

(d). Expression of Mas-DH-R and binding assays: Approximately 5 x 10⁶ Sf9 cells are infected with BV/Mas-DH-R at a MOI of 1 in 75 cm² tissue culture flasks. Three days after infection, cells are harvested, centrifuged at 300 x g, and resuspended in 25 mM potassium phosphate (pH 6.6), 240 mM sucrose, 1 mM EDTA. Membranes are prepared as indicated above. Binding assays are performed as described hereinabove using 220 nanograms of membrane protein/assay tube. Binding data are analyzed by the computer program LIGAND (Munson, P.J. and Rodbard, D. (1980) Anal. Biochem. 107:220-229). Figure 10 shows a time course of Mas-DH-R expression in Sf9 cells. A time dependent increase in the level of Mas- DH-R expression which reached a maximum at two to three days post-infection is observed. A saturation isotherm of ³H-labelled Mas-DH- binding to membranes of BV/Mas-DH-R infected Sf9 cells (data not shown) reveals a single binding site displaying a Kd=80pM (S.D.=20, n=6) and a Bmax=77 pmol/mg protein (S.D=7, n=6). The Kd value is in close agreement with the Kd values of Mt-membranes and COS-7 cells transfected with the Mas-DH-R (Table 1). However, the level of Mas-DH-R expression is considerably higher.

Table 1; Comparison of Kd and Bmax values for the M. sexta diuretic hormone receptor in Sf9 cells infected with BV/Mas-DH-R, COS-7 cells transfected with Mas-DH-R, and Malpighian tubule membranes.

Source Kd (pM) Bmax (pmol/mg protein) Sf9 cells 80 77

COS-7 cells 56 1.1

Malpighian tubes 79 3.1

(e). Chemical cross-linking of ^{1 5}I-labeled Mas-DH to the expressed receptor: Approximately 5 x 10¹⁶ Sf9 cells are infected with BV/Mas-DH-R at a MOI of 1 in 75 cm² tissue culture flasks. Three days after infection, cells are harvested, centrifuged at 300 x g, and resuspended in fresh cell medium. Cells are treated with 100 pM ^12sl-labeled Mas-DH [8] in the presence or absence of 1 uM unlabeled Mas-DH for 1 hour on ice. The cells are washed 3 times in phosphate buffered for 30 min on ice. Membranes are prepared as previously described and separated on a 12% SDS-PAGE gel. The gel is dried and exposed to X-ray film (Kodak, Rochester, NY) for 3 days. The results indicated a broad band of 48-52 kDa which is in close agreement with the predicted size of the Mas-DH-R based on the deduced amino acid sequence (Figure 7, SEQ ID NO.2).

(f). cAMP assay: Approximately 1 x 10⁶ Sf9 cells are infected with BV/Mas-DH-R at a MOI of 1 in 6 well tissue culture plates. Three days after infection, cells are incubated in fresh cell media containing 1 mM isobutylmethyxanthine and various concentrations of peptide hormones for 20 min at 27°C. The reaction is terminated by aspiration of the cell media and addition of 70% ice-cold ethanol. cAMP is measured by radioimmunoassay (Amersham, Arlington Heights, IL).

Figure 11 depicts the stimulation of cAMP synthesis by Mas-DH and N-terminal truncated analogs- In previous studies, it was shown that the N-terminal truncated analog [3-41] Mas- DH and [13-41] Mas-DH displays high affinity for Mas-DH-R- However, despite the [13-41] Mas-DH high affinity for the receptor, it is unable to stimulate cAMP synthesis.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Sandoz Ltd

(B) STREET: Lichtstrasse 35

(C) CITY: Basel

(D) STATE: BS

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP) : CH-4002

(G) TELEPHONE: 061-324-2327 (H) TELEFAX: 061-322-7532 (I) TELEX: 965-050-55

(A) NAME: Sandoz Patent GMBH

(B) STREET: Humboltstrasse 3

(C) CITY: Loerrach

(E) COUNTRY: Germany

(F) POSTAL CODE (ZIP) : D-7850

(A) NAME: Sandoz Erfindungen Verwaltungsgesellschaft mbH

(B) STREET: Brunner Strasse 59

(C) CITY: Vienna

(E) COUNTRY: Austria

(F) POSTAL CODE (ZIP) : A-1235

(ii) TITLE OF INVENTION: Novel Receptors (iii) NUMBER OF SEQUENCES: 5

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1626 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 254..1438

(D) OTHER INFORMATION: /codon_start= 254

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

GAATTCGGCA CGAGCACAGG CGTGCCACTC AAAACACCTG AAAATCGCGA TCGAGCCTTG 60

GAATGGAAAA GCTAACGTTC ATCGAAACCA GATAGACTGT GACAGTGTTG TGTATAGTGT 120

AATTATAACC AAAGAACAAA ACCAAAGCTG ATCGAAGCTC ATTTCTAGTG GATACGAGGT 180

AGACTTTAAC GCCAAACTGG AGGAATTACA GACGTTGGAC ACCGCAGAGA GCGTCATCGA 240

AAATGTCAGC GCG ATG GCG GAG GAG TGT CTG GCG AGG AAG TTC AAC TTG 289 Met Ala Glu Glu Cys Leu Ala Arg Lys Phe Asn Leu 1 5 10 TCG GAC AAC TAC TGC CCG GCG TAC TTC GAT GGA CTC CTG TGC TGG GAC 337 Ser Asp Asn Tyr Cys Pro Ala Tyr Phe Asp Gly Leu Leu Cys Trp Asp 15 20 25

CCG ACG CCT TGG AAC ACC TTG GCG GTG CAG AAG TGC TTT AAA GAA CTG 385 Pro Thr Pro Trp Asn Thr Leu Ala Val Gin Lys Cys Phe Lys Glu Leu 30 35 40

TAC GGG ATA CAA TAC GAT GAC ACA CAA AAT GCC TCA CGG CTG TGC TTG 433 Tyr Gly He Gin Tyr Asp Asp Thr Gin Asn Ala Ser Arg Leu Cys Leu 45 50 55 60

GAC GGA GTT TGG CAC AAC TAC ACC AAC TAT ACA AAT TGC ACG GAG AGG 481 Asp Gly Val Trp His Asn Tyr Thr Asn Tyr Thr Asn Cys Thr Glu Arg 65 70 75

ATC GCA AAC GGG TCA CCA ACA GAT GTC GCT AGT CTC ATA TAC TTG GCG 529 He Ala Asn Gly Ser Pro Thr Asp Val Ala Ser Leu He Tyr Leu Ala 80 85 90

GGC TAC TCC CTC AGC CTT GCT GTT CTA TCG TTA GCT GTC TTC GTA TTC 577 Gly Tyr Ser Leu Ser Leu Ala Val Leu Ser Leu Ala Val Phe Val Phe 95 100 105

TTG TAT TTT AAG GAT CTG CGA TGT TTA CGA AAC ACA ATT CAT ACT AAT 625 Leu Tyr Phe Lys Asp Leu Arg Cys Leu Arg Asn Thr He His Thr Asn 110 115 120

TTA ATG TCT ACA TAC ATA TTA TCA GCT TGT AGT TGG ATT TTA AAT TTA 673 Leu Met Ser Thr Tyr He Leu Ser Ala Cys Ser Trp He Leu Asn Leu 125 130 135 140

GTT TTA CAA AAC TGG TCG GAC GAA TCT CAG CAA GAC CAG ACG TCG TGT 721 Val Leu Gin Asn Trp Ser Asp Glu Ser Gin Gin Asp Gin Thr Ser Cys 145 150 155

ATG ATA CTC GTC ATT TGT ATG AAC TAT TTC TAT TTA ACT AAT TTC TTC 769 Met He Leu Val He Cys Met Asn Tyr Phe Tyr Leu Thr Asn Phe Phe 160 165 170

TGG ATG TTG GTG GAA GGT TTA TAT CTC TAC ATG CTA GTC GTC GAA ACT 817 Trp Met Leu Val Glu Gly Leu Tyr Leu Tyr Met Leu Val Val Glu Thr 175 180 185

TTT ACA GCG GAG AAT ATT AAA TTA AAA GTA TAT ACC ACT ATT GGC TGG 865 Phe Thr Ala Glu Asn He Lys Leu Lys Val Tyr Thr Thr He Gly Trp 190 195 200

GGT GCT CCA GCT GTA TTT ATT ACG ATA TGG GTG ATA TCA AGG TGT TTT 913 Gly Ala Pro Ala Val Phe He Thr He Trp Val He Ser Arg Cys Phe 205 210 215 220

GTT AAC GTT TTG CCG TCA ACG GGG CCT GAT GGA TTG GCG ATG TTC CCT 961 Val Asn Val Leu Pro Ser Thr Gly Pro Asp Gly Leu Ala Met Phe Pro 225 230 235

GAA GCA AAG ATG TGC ATA TGG ATG CAT GAA CAC CAA GTG GAC TGG ATA 1009 Glu Ala Lys Met Cys He Trp Met His Glu His Gin Val Asp Trp He 240 245 250

CAC AAA GCG CCA GCA CTT GTT GGT CTC GCT CTT AAT CTG TTC TTC CTC 1057 His Lys Ala Pro Ala Leu Val Gly Leu Ala Leu Asn Leu Phe Phe Leu 255 260 265

ATT AGG ATT ATG TGG GTG CTG ATC ACG AAA CTC CGT TCC GCC AAC ACG 1105 He Arg He Met Trp Val Leu He Thr Lys Leu Arg Ser Ala Asn Thr 270 275 280 CTA GAG ACT GAG CAA TAC CGG AAG GCT ACC AAA GCG CTT CTA GTC CTC 1153 Leu Glu Thr Glu Gin Tyr Arg Lys Ala Thr Lys Ala Leu Leu Val Leu 285 290 295 300

ATA CCC TTG CTG GGT ATA ACC AAC CTC CTG GTC CTC TGC GGC CCT AGT 1201 He Pro Leu Leu Gly He Thr Asn Leu Leu Val Leu Cys Gly Pro Ser 305 310 315

GAC GAC TCC TGG TTC GCA TAC GCA TTT GAT TAC ACT AGG GCG CTT ATG 1249 Asp Asp Ser Trp Phe Ala Tyr Ala Phe Asp Tyr Thr Arg Ala Leu Met 320 325 330

TTA TCG ACA CAG GGC TTC ACG GTA GCT CTG TTC TAC TGC TTC ATG AAC 1297 Leu Ser Thr Gin Gly Phe Thr Val Ala Leu Phe Tyr Cys Phe Met Asn 335 340 345

ACT GAG GTC CGG CAC GCG ATC AGG TAC CAC GTG GAA AGA TGG AAG ACT 1345 Thr Glu Val Arg His Ala He Arg Tyr His Val Glu Arg Trp Lys Thr 350 355 360

GGT CGG ACC ATC GGC GGG GGG AGG CGG CGA GGC GCC TCT TAC TCT AAG 1393 Gly Arg Thr He Gly Gly Gly Arg Arg Arg Gly Ala Ser Tyr Ser Lys 365 370 375 380

GAC TGG TCT CCG AGA TCG CGG ACC GAA AGC ATA AGG CTC ACA GTA 1438

Asp Trp Ser Pro Arg Ser Arg Thr Glu Ser He Arg Leu Thr Val 385 390 395

TGAAGAAGCC TGGATTTGGT ACCGGATATA TCCAACGAAT TTACTAACTG GGATGGACTT 1498

TTTACATTTA TGAAGACATT TATAGGAATA CGATAAGATG TGGATTTGGA GAGATTTTTT 1558

TAATTATATT TTTTATTTTG TTCATTTTTA ATAAATATGT ATCCAGCTTT AAAAAAAAAA 1618

AAAAAAAA 1626

(2) INFORMATION FOR SEQ ID Nθ:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 395 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

Met Ala Glu Glu Cys Leu Ala Arg Lys Phe Asn Leu Ser Asp Asn Tyr 1 5 10 15

Cys Pro Ala Tyr Phe Asp Gly Leu Leu Cys Trp Asp Pro Thr Pro Trp 20 25 30

Asn Thr Leu Ala Val Gin Lys Cys Phe Lys Glu Leu Tyr Gly He Gin 35 40 45

Tyr Asp Asp Thr Gin Asn Ala Ser Arg Leu Cys Leu Asp Gly Val Trp 50 55 60

His Asn Tyr Thr Asn Tyr Thr Asn Cys Thr Glu Arg He Ala Asn Gly 65 70 75 80

Ser Pro Thr Asp Val Ala Ser Leu He Tyr Leu Ala Gly Tyr Ser Leu 85 90 95

Ser Leu Ala Val Leu Ser Leu Ala Val Phe Val Phe Leu Tyr Phe Lys 100 105 110

Asp Leu Arg Cys Leu Arg Asn Thr He His Thr Asn Leu Met Ser Thr 115 120 125

Tyr He Leu Ser Ala Cys Ser Trp He Leu Asn Leu Val Leu Gin Asn 130 135 140

Trp Ser Asp Glu Ser Gin Gin Asp Gin Thr Ser Cys Met He Leu Val 145 150 155 160

He Cys Met Asn Tyr Phe Tyr Leu Thr Asn Phe Phe Trp Met Leu Val 165 170 175

Glu Gly Leu Tyr Leu Tyr Met Leu Val Val Glu Thr Phe Thr Ala Glu 180 185 190

Asn He Lys Leu Lys Val Tyr Thr Thr He Gly Trp Gly Ala Pro Ala 195 200 205

Val Phe He Thr He Trp Val He Ser Arg Cys Phe Val Asn Val Leu 210 215 220

Pro Ser Thr Gly Pro Asp Gly Leu Ala Met Phe Pro Glu Ala Lys Met 225 230 235 240

Cys He Trp Met His Glu His Gin Val Asp Trp He His Lys Ala Pro 245 250 255

Ala Leu Val Gly Leu Ala Leu Asn Leu Phe Phe Leu He Arg He Met 260 265 270

Trp Val Leu He Thr Lys Leu Arg Ser Ala Asn Thr Leu Glu Thr Glu 275 280 285

Gin Tyr Arg Lys Ala Thr Lys Ala Leu Leu Val Leu He Pro Leu Leu 290 295 300

Gly He Thr Asn Leu Leu Val Leu Cys Gly Pro Ser Asp Asp Ser Trp 305 310 315 320

Phe Ala Tyr Ala Phe Asp Tyr Thr Arg Ala Leu Met Leu Ser Thr Gin 325 330 335

Gly Phe Thr Val Ala Leu Phe Tyr Cys Phe Met Asn Thr Glu Val Arg 340 345 350

His Ala He Arg Tyr His Val Glu Arg Trp Lys Thr Gly Arg Thr He 355 360 365

Gly Gly Gly Arg Arg Arg Gly Ala Ser Tyr Ser Lys Asp Trp Ser Pro 370 375 380

Arg Ser Arg Thr Glu Ser He Arg Leu Thr Val 385 390 395

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4893 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: circular

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA 60

TCAGCTCACT CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG 120

AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG 180

TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG 240

TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG 300

CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA 360

AGCGTGGCGC TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC 420

TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT 480

AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT 540

GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG 600

CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT 660

ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT 720

GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT 780

TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG 840

GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT 900

AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT 960

GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC 1020

GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG 1080

CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC 1140

GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG 1200

GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA 1260

GGCATCGTGG TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA 1320

TCAAGGCGAG TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT 1380

CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG 1440

CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA 1500

ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA 1560

CGGGATAATA CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT 1620

TCGGGGCGAA AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT 1680

CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA 1740

ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC 1800

ATACTCTTCC TTTTTCAATC GCGTTGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA 1860

ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA ACTTACGGTA 1920

AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT 1980

GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA CTATTTACGG 2040 TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC 2100

GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT 2160

CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGTGAT GCGGTTTTGG 2220

CAGTACATCA ATGGGCGTGG ATAGCGGTTT GACTCACGGG GATTTCCAAG TCTCCACCCC 2280

ATTGACGTCA ATGGGAGTTT GTTTTGGCAC CAAAATCAAC GGGACTTTCC AAAATGTCGT 2340

AACAACTCCG CCCCATTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA 2400

AGCAGAGCTC TCTGGCTAAC TAGAGAACCC ACTGCTTACT GGCTTATCGA AATTAATACG 2460

ACTCACTATA GGGAGACCCA AGCTGAATTC GTCGACCGCG GAGCTTCTAG AGATCCCTCG 2520

ACCTCGAGAT CCATTGTGCT GGCGCGGATT CTTTATCACT GATAAGTTGG TGGACATATT 2580

ATGTTTATCA GTGATAAAGT GTCAAGCATG ACAAAGTTGC AGCCGAATAC AGTGATCCGT 2640

GCCGGCCCTG GACTGTTGAA CGAGGTCGGC GTAGACGGTC TGACGACACG CAAACTGGCG 2700

GAACGGTTGG GGGTGCAGCA GCCGGCGCTT TACTGGCACT TCAGGAACAA GCGGGCGCTG 2760

CTCGACGCAC TGGCCGAAGC CATGCTGGCG GAGAATCATA CGCTTCGGTG CCGAGAGCCG 2820

ACGACGACTG GCGCTCATTT CTGATCGGGA ATCCCGCAGC TTCAGGCAGG CGCTGCTCGC 2880

CTACCGCCAG CACAATGGAT CTCGAGGGAT CTTCCATACC TACCAGTTCT GCGCCTGCAG 2940

GTCGCGGCCG CGACTCTAGA GGATCTTTGT GAAGGAACCT TACTTCTGTG GTGTGACATA 3000

ATTGGACAAA CTACCTACAG AGATTTAAAG CTCTAAGGTA AATATAAAAT TTTTAAGTGT 3060

ATAATGTGTT AAACTACTGA TTCTAATTGT TGTGGTATTT TAGATTCCAA CCTATGGAAC 3120

TTATGAATGG GAGCAGTGGT GGAATGCCTT TAATGAGGAA AACCTGTTTT GCTCAGAAGA 3180

AATGCCATCT AGTGATGATG AGGCTACTGC TGACTCTCAA CATTCTACTC CTCCAAAAAA 3240

GAAGAGAAAG GTAGAAGACC CCAAGGACTT TCCTTCAGAA TTGGTAAGTT TTTTGAGTCA 3300

TGCTGTGTTT AGTAATAGAA CTCTTGCTTG CTTTGCTATT TACACCACAA AGGAAAAAGC 3360

TGCACTGCTA TACAAGAAAA TTATGGAAAA ATATTTGATG TATAGTGCCT TGACTAGAGA 3420

TCATAATCAG CCATACCACA TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC 3480

TCCCCCTGAA CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG 3540

CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTAT 3600

CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGGATCC 3660

CGCCATGGTA TCAACGCCAT ATTTCTATTT ACAGTAGGGA CCTCTTCGTT GTGTAGGTAC 3720

CGCTGTATTC CTAGGGAAAT AGTAGAGGCA CCTTGAACTG TCTGCATCAG CCATATAGCC 3780

CCCGCTGTTC GACTTACAAA CACAGGCACA GTACTGACAA ACCCATACAC CTCCTCTGAA 3840

ATACCCATAG TTGCTAGGGC TGTCTCCGAA CTCATTACAC CCTACCAAGT GAGAGCTGTA 3900

ATTTCGCGAT CAAGGGCAGC GAGGGCTTCT CCAGATAAAA TAGCTTCTGC CGAGAGTCCC 3960

GTAAGGGTAG ACACTTCAGC TAATCCCTCG ATGAGGTCTA CTAGAATAGT CAGTGCGGCT 4020

CCCATTTTGA AAATTCACTT ACTTGATCAG CTTCAGAAGA TGGGCGAGGG CCTCCAACAC 4080 AGTAATTTTC CTCCCGACTC TTAAAATAGA AAATGTCAAG TCAGTTAAGG AGGAAGTGGA 4140

CTAACTGACG CAGCTGGCCG TGCGACATCC TCTTTTAATT AGTTGCTAGG CAACGCCCTC 4200

CAGAGGGCGT GTGGTTTTGC AAGAGGAAGC AAAAGCCTCT CCACCCAGGC CTAGAATGTT 4260

TCCACCCAAT CATTACTATG ACAACAGCTG TTTTTTTTAG TATTAAGCAG AGGCCGGGGA 4320

CCCCTGGGCC CGCTTACTCT GGAGAAAAAG AAGAGAGGCA TTGTAGAGGC TTCCAGAGGC 4380

AACTTGTCAA AACAGGACTG CTTCTATTTC TGTCACACTG TCTGGCCCTG TCACAAGGTC 4440

CAGCACCTCC ATACCCCCTT T ATAAGCAG TTTGGGAACG GGTGCGGGTC TTACTCCGCC 4500

CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT 4560

TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG 4620

AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AAAGCTAGCT TGGCGTAATC ATGGTCATAG 4680

CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCAC ACAACATACG AGCCGGAAGC 4740

ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC TCACATTAAT TGCGTTGCGC 4800

TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC TGCATTAATG AATCGGCCAA 4860

CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGC 4893 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TCACCCGGGG CGCGATGGCG GAGGAGTGTC T 31

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CCAGTCTAGA AATTCGTTGG ATATATCCCG 30

Claims

1. An isolated polypeptide comprising an insect diuretic hormone receptor or analog or fragment thereof which is substantially free from associated insect polypeptides and exhibits activity characteristic of a native insect diuretic hormone receptor.

2. A polypeptide according to Claim 1 wherein the insect diuretic hormone receptor is isolated from Manduca sexta.

3. A polypeptide according to Claim 1 which has the amino acid sequence shown in SEQ ID NO. 2 and analogs or fragments thereof.

4. A DNA sequence encoding an insect diuretic hormone receptor according to either of Claims 1 or 2.

5. A DNA sequence encoding an insect diuretic hormone receptor according to Claim 3 as shown in SEQ ID No- 1-

6. rDNA comprising a regulating element which is responsive to a ligand which binds to an insect diuretic hormone receptor or analog or fragment thereof according to Claim 1.

7. Cells transformed with an rDNA according to Claim 6.

8. An antibody and binding fragments thereof with binding specificity for an epitope characteristic for an insect diuretic hormone receptor or analog or fragment thereof according to Claim 1.

9. Use of an insect diuretic hormone receptor or analog or fragment thereof according to Claim 1 in a method for selecting ligands with binding specificity for a diuretic hormone binding domain of an insect diuretic hormone receptor or analog or fragment thereof.

10. The use of the insect diuretic hormone receptor according to Claim 9 in baculovirus.

11. Use of a hormone binding domain of an insect diuretic hormone receptor or analog or fragment thereof according to Claim 1 in selecting ligands specific for binding to a ligand binding domain of an insect diuretic hormone receptor or analog or fragment thereof.

12. Use of a hormone binding domain of an insect diuretic hormone receptor or analog or fragment thereof according to Claim 1 in selecting DNA sequences which are capable of being bound specifically by an insect diuretic hormone receptor or analog or fragment thereof.

13. A chimeric polypeptide comprising a diuretic hormone binding domain of an insect diuretic hormone receptor or analog or fragment thereof according to Claim 1 and at least one further polypeptide.

14. DNA encoding a chimeric polypeptide according to Claim 13.

15. A method of producing a polypeptide by introducing into a cell which is insensitive to ligands which bind to an insect diuretic hormone receptor or analog or fragment thereof according to Claim 1 a receptor for that ligand and gene encoding the polypeptide which is operably linked to a regulating element which is responsive to said ligand and exposing the cell thus transformed to said ligand.