FR2660659A1 - Polypeptides having a dopaminergic receptor activity, nucleic acids encoding these polypeptides and use of these polypeptides for screening substances active on these polypeptides - Google Patents

Abstract

The subject of the invention is new polypeptides having a dopaminergic receptor activity containing: @ the sequence of 446 amino acids of Figure 1, @ or a fragment of this sequence, this fragment being such that * either it contains, nevertheless, the sites contained in this sequence and whose presence is necessary so that, when this fragment is exposed at the surface of a cell, it is capable of binding dopamine, its agonists or antagonists specifically and measurably, for example by means of a radioactive ligand, * or it is capable of being recognised by antibodies which also recognise the abovementioned sequence of 446 amino acids, but recognise neither the domaminergic receptor D-1, nor the dopaminergic receptor D-2, * or it is capable of generating antibodies recognising the abovementioned sequence of 446 amino acids but recognising neither the dopaminergic receptor D-1, nor the dopaminergic receptor D-2. s

Description

POLYPEPTIDES HAVING DOPANINERGIC RECEPTOR ACTIVITY, NUCLEIC ACIDS ENCODING CE8
POLYPEPTIDES AND USE OF CE8 POLYPEPTIDES FOR
THE SCREENING OF 8UB8TANCE8 ACTIVE ON CE8 POLYPEPTIDES.

 The subject of the invention is polypeptides having dopaminergic receptor activity and the genes coding for these polypeptides.

 The invention also relates to - vectors containing the genes coding for polypeptides having dopaminergic receptor activity, - cells transformed to express the abovementioned genes on which it is possible to evaluate the activity of agonist agents or antagonists of said receptors, - to nucleotide probes capable of hybridizing with the genes coding for the abovementioned polypeptides, which probes would be capable of being used for the in vitro diagnosis of conditions, in particular neurological, psychiatric, cardiovascular or neuroendocrine, - to polyclonal and monoclonal antibodies directed against the above-mentioned polypeptides and usable for an analytical purpose to purify said polypeptides, for in vitro diagnosis, or for a therapeutic purpose, in particular in the affections implying the dopaminergic systems, - with kits (or kits) to study the degree of affinity of certain substances for the above-mentioned polypeptides - to medicaments intended more particularly for the treatment of psychiatric, neurological, cardiovascular or neuroendocrine affections and containing substances active on the above-mentioned polypeptides having a dopaminergic receptor activity, or containing substances active on cells transfected by genes or fragments of genes coding for said polypeptides with dopaminergic receptor activity, - to drugs active on dopaminergic receptors already identified but for which one would like to reduce certain side effects attributable to their interaction with said polypeptides.

Hitherto, it was recognized that the various effects of dopamine resulted from its interaction with two types of receptors commonly known as Dl and D-2 receptors (Kebabian and Calne, (1979) (1)). Among these, only the sequence of the D-2 receptor was known (Bunzow et al., (1988) (2)) and two isoforms of the D-2 receptor (sometimes designated
D-2A and D-2B or even D-2 (415) and D-2 (444) resulting from the alternative splicing of the messenger RNA of the gene for said receptor had been described (Giros et al., (1989) ( 3); Dal Toso et al., (1989) (4)).

 The D-2 receptor belongs to the family of receptors comprising seven transmembrane domains, coupled to a G protein and having a certain degree of homology with the receptors of the rhodopsin family which are expressed by the photoreceptors of the retina.

 The existence of dopaminergic receptors distinct from the Dl and D-2 receptors had been suspected by various authors on indirect observations (see in particular Schwartz et al., (1984) (5)) but had never been proven because these n receptors 'had never been isolated, identified as to their structure, or entirely defined by their pharmacological properties. In particular, the existence of autoreceptors regulating the synthesis and / or release of dopamine and / or even the activity of dopaminergic neurons had been demonstrated, but it was commonly accepted that they were identical to the D-2 receptors in that concerns their pharmacology.

 The object of the invention is to provide access to new polypeptides having dopaminergic receptor activity, which is similar neither to that of dopaminergic D-1 receptors, nor to that of dopaminergic D-2 receptors.

 The invention also relates to methods for screening for new drugs acting on new polypeptides having a dopaminergic receptor action or the interaction of which with the new polypeptides, on the contrary, and intended, inter alia, for the treatment of disorders psychiatric, neurological, cardiovascular or neuroendocrine.

In particular, the new polypeptide of the invention having dopaminergic receptor activity
- contains the 446 amino acid sequence of Figure 1 or a fragment of this sequence, this fragment being such that
* or it nevertheless contains the sites contained in this sequence and the presence of which is necessary so that, when this fragment is present in a membrane, it is capable of binding dopamine to its agonists or antagonists in a specific and measurable manner, for example at using a radioactive ligand according to the technique described in Sokoloff et al.

(1980) (6) and in Martres et al. (1985) (7).

* or it is capable of being recognized by antibodies which also recognize the aforementioned sequence of 446 amino acids, but recognize neither the dopaminergic receptor D-1, nor the dopaminergic receptor D-2
* either it is capable of generating antibodies which recognize the aforementioned sequence of 446 amino acids but recognize neither the dopaminergic receptor Dl nor the dopaminergic receptor
D-2.

 Recognition of the above sequence of 446 amino acids by the above antibodies - or of the above fragment by the above antibodies - means that the above sequence forms a complex with one of the above antibodies.

 The formation of the antigen complex (that is to say 446 amino acid sequence or aforementioned fragment) antibody and the detection of the existence of a complex formed can be done by conventional techniques (such as those using a labeled tracer by radioactive isotopes or an enzyme).

 With regard to the definition of "fragments containing the sites" and meeting the above definition, reference is made below in the description.

 Among the fragments defined above, preference is given to the fragment or fragments produced by alternative splicing of the messenger RNA produced by the gene coding for the 446 amino acid sequence defined above.

 The invention also relates to chimeric proteins in which the polypeptide as defined above or parts thereof are joined to a chain of amino acids heterologous with respect to this polypeptide.

 The polypeptides and chimeric proteins of the invention may be glycosylated and may or may not contain disulfide bridges.

 In the following description, the polypeptides of the invention will, for simplicity, be designated by "D-3 receptors".

 An advantageous D-3 receptor of the invention consists of the chain of amino acids 1 to 446, shown in Figure 1.

 This D-3 receptor is considered to have seven hydrophobic transmembrane regions separated by hydrophilic intra and extracellular loops.

 The invention also relates to the variant polypeptides which correspond to the above-defined polypeptides comprising certain localized mutations, without the polypeptides losing the properties of the dopaminergic D-3 receptor. Among these variants, there may be mentioned those which are recognized by antibodies recognizing the transmembrane regions, as well as those which are recognized by antibodies recognizing the regions other than the transmembrane regions.

 The invention also relates to nucleic acids which comprise or consist of a sequence of nucleotides coding for any of the D-3 receptors defined above.

 More particularly, the invention relates to the nucleic acid which comprises the sequence of nucleotides shown in FIG. 2, extending from the end constituted by the nucleotide in position 1 to that constituted by the nucleotide in position 1863.

 The invention particularly relates to the nucleic acid represented in FIG. 2 comprising or consisting of the chain extending from the end constituted by the nucleotide in position 442 to that constituted by the nucleotide in position 1779.

 The above-defined nucleic acid corresponds to the coding part of the gene corresponding to the polypeptide shown in Figure 1.

 Also forming part of the invention are nucleic acids which vary from those defined above and which contain certain localized mutations insofar as these variant nucleic acids hybridize with the nucleic acids previously defined or with the nucleic probes defined below. under the hybridization conditions defined below in the description.

 The nucleic acids of the invention can be prepared either by a chemical process or by other processes.

 A suitable mode of preparation of the nucleic acids (comprising a maximum of 200 nucleotides - or bp, in the case of double-stranded nucleic acids) of the invention by chemical means comprises the following stages - the synthesis of DNA using the automated p-cyanethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325, 1986, - the cloning of the DNAs thus obtained in an appropriate plasmid vector and the recovery of the DNAs by hybridization with an appropriate probe.

A method of preparation, by chemical route, of nucleic acids of length greater than 200 nucleotides - or bp (in the case of double-stranded nucleic acids) comprises the following stages: - assembly of chemically synthesized oligonucleotides provided at their ends with different restriction sites, the sequences of which are compatible with the amino acid chain of the natural polypeptide according to the principle described in
Proc. Nat. Acad. Sci. USA 80; 7461-7465, 1983, - the cloning of the DNAs thus obtained in an appropriate plasmid vector and the recovery of the desired nucleic acid by hybridization with an appropriate probe.

Another method for preparing the nucleic acids of the invention from mRNA comprises the following steps - preparation of cellular RNA from any tissue expressing the dopaminergic D-3 receptor according to the techniques described by Maniatis et al. (1982) (8), and
Ausubel FM et al. (1989) (9), - recovery and purification of mRNAs by passage of total cellular RNAs by chromatography with an immobilized oligodT, - synthesis of a strand of cDNA from mRNAs purified according to the technique described in Gene 25: 263, 1983, - cloning of the nucleic acids thus obtained into an appropriate plasmid vector and recovery of the sought nucleotide sequence using an appropriate hybridization probe.

To prepare the nucleic acids of the invention, the chemically synthesized oligonucleotide hybridization probes are the following
that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 1, to that constituted by the nucleotide in position 441,
that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 442, to that constituted by the nucleotide in position 537,
that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 718, to that constituted by the nucleotide in position 753, that - that defined by the acid sequence nucleic acid shown in Figure 2, extending from the end formed by the nucleotide at position 820, to that formed by the nucleotide at position 888, that - that defined by the nucleic acid sequence shown in Figure 2, s extending from the end constituted by the nucleotide at position 958, & that constituted by the nucleotide at position 996, that - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide at position 1068, to that constituted by the nucleotide at position 1240, that - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide at position 1068, to that constituted by the nucleotide at position 1566, or their complementary nucleotide sequence; these are sequences which have been derived from those of FIG. 2 and they can be used under the hybridization conditions described by Maniatis et al. (1982) (8).

 The synthesis of the cDNA strand and its subsequent amplification in vitro can also be carried out using the PCR (Polymerase Chain Reaction) method, as described for example by Goblet et al. (1989) (10), using two chemically synthesized amplimers defined from the sequence of Figure 2. Suitable amplimers are for example: that defined in Figure 2 from nucleotide 410 to nucleotide 433 and that defined in Figure 2 from nucleotide 1800 to nucleotide 1825.

The amplified nucleic acid fragment can then be cloned according to the techniques described in
Ausubel FM et al. (1989) (11).

 The invention also relates to the recombinant vectors, in particular for cloning and / or expression, in particular of the plasmid, cosmid, phage, or virus type, containing a nucleic acid of the invention in one of its nonessential sites. for its replication.

 A suitable vector of the invention, contains at one of its sites which are not essential for its replication, the elements necessary to promote the expression of a polypeptide according to the invention, in a cellular host and possibly a promoter recognized by the polymerases. of the cell host, in particular an inducible promoter and optionally a signal sequence and an anchoring sequence.

 The invention also relates to a cellular host transformed by a recombinant vector defined above comprising the regulatory elements allowing the expression of the nucleotide sequence coding for one of the polypeptides according to the invention in this host.

 By cellular host is meant any organism capable of being maintained in culture.

One of the microorganisms used can consist of a bacterium, in particular Escherichia
Coli.

An organism of choice is constituted by a eukaryotic organism such as CHO cells (Chinese
Hamster Ovary) or COS-7 (African green monkey kidney fibroblast transformed by the SV-40 virus).

 However, other organisms can be used just as easily, naturally provided that each of them has vectors, in particular plasmids, capable of replicating therein and nucleotide sequences insertable into these vectors and capable, when followed in these vectors by an insert coding for a polypeptide of the invention, to ensure the expression of this insert in the chosen organisms and their transport in the membranes of these cellular hosts.

The invention also relates to antibodies specifically directed against one of the polypeptides of the invention, these antibodies being such that they neither recognize nor the dopaminergic receptor
Dl, nor the dopaminergic receptor D-2. In particular, these antibodies recognize the following amino acid sequences - that defined by the amino acid sequence represented in FIG. 1 extending from the end constituted by the amino acid in position 1 to that constituted by the amino acid at position 38, - that defined by the amino acid sequence shown in Figure 1 extending from the end formed by the amino acid at position 135 to that formed by the amino acid at position 147, - that defined by the amino acid sequence represented in FIG. 1 extending from the end constituted by the amino acid in position 175 to that constituted by the amino acid in position 187, - that defined by the sequence of amino acids shown in Figure 1 extending from the end formed by the amino acid in position 210 to that formed by the amino acid in position 375.

 To obtain the antibodies, one of the above polypeptides can be injected into animals.

 Monoclonal antibodies are prepared by cell fusion between myeloma cells and spleen cells of immunized mice, according to conventional methods.

 The antibodies of the invention can be used for the in vitro diagnosis of the tumor character or not of certain cells as well as the benign or malignant character of certain tumors, insofar as these elements are correlated with the pathological expression of the D- receptor. 3.

 Indeed, it has been observed that in certain tumors, in particular lung tumors, it is possible to detect the expression of dopaminergic receptors which should not normally be present (Sokoloff et al. (1989) (12)).

 This in vitro diagnostic method based on the biological sample capable of containing the dopaminergic receptor D-3 comprises - bringing an antibody of the invention into contact with the above-mentioned biological sample under conditions allowing the possible production of a complex immunological formed between the dopaminergic receptor D-3 or product which is derived therefrom and the antibody of the invention, - the detection of the above-mentioned immunological complex formed.

 The invention also relates to the nucleotide probes, synthetic or not, hybridizing with one of the nucleic acids defined above or their complementary sequences or their corresponding RNA, these probes being such that they hybridize neither with the gene nor with the RNA messengers of the D-1 and D-2 dopaminergic receptors.

 The probes of the invention comprise at least 10, advantageously 15 nucleic acids and can comprise at most the entire nucleotide sequence shown in FIG. 2.

 For the shortest probes, that is to say from about 10 to about 100 nucleotides, suitable hybridization conditions are as follows: 900 mN NaCl, 90 mM tri-sodium citrate pH 7.0, 100 µg / ml salmon sperm DNA, 0.05% sodium pyrophosphate, 10-25% deionized formamide, 0.02% Ficoll (Pm 400,000), 0.02% serum albumin bovine, 0.02% polyvinylpyrrolidone, for 14 to 16 hours at 42 "C.

 For the longest probes, that is to say having more than about 100 nucleotides, suitable hybridization conditions are as follows: 600 mM NaCl, 60 mM tri-sodium citrate, 20 μg / ml of Salmon sperm DNA, 20 µg / ml yeast tRNA, 8 mM Tris-HCl pH 7.4, 40 to 60% deionized formamide, 0.02% Ficoll, 0.02% albumin bovine serum, 0.02% polyvinylpyrrolidone, 0.2% sodium dodecyl sulfate, 10% dextran sulfate.

 The invention relates in particular to the nucleotide probes defined above.

 The probes of the invention can be used as diagnostic tools, especially in vitro for neurological, psychiatric and cardiovascular disorders.

 The probes of the invention can also be used to detail the alternative splicing of the messenger RNA produced by the gene coding for the 446 amino acid sequence defined above, the existence or not of this splicing being able to be linked to neurological, psychiatric, cardiovascular or neuroendocrine pathologies.

 More particularly, the probes of the invention can be used to detect genetic anomalies, in particular polymorphisms or point mutations.

 As regards the detection of polymorphisms of the gene coding for the dopaminergic receptor D-3 in an individual, it can be carried out from a biological sample such as blood, taken from the individual according to a procedure comprising the following steps the treatment of the nucleic acid originating from the abovementioned biological sample carried out using a restriction enzyme under conditions allowing the obtaining of restriction fragments resulting from the cleavage of said nucleic acid at these sites of restriction recognized by said enzyme, - bringing a nucleotide probe of the invention into contact, capable of hybridizing with the abovementioned fragments under conditions allowing the possible production of hybridization complexes between said probe and the restriction fragments mentioned above, - detection of the above hybridization complexes, - measurement of the size of any polymorphic restriction fragments engaged in the above-mentioned hybridization complexes.

 As regards the in vitro diagnostic method for detecting point mutations of the nucleic acid or of a fragment of the nucleic acid coding for the dopaminergic receptor D-3 in an individual, it can be carried out according to the method comprising the following steps - bringing a nucleotide probe of the invention into contact with a biological sample, for example blood, under conditions allowing the possible production of a hybridization complex formed between the probe and the nucleic acid or a fragment of the above-mentioned nucleic acid, - the detection of the above-mentioned hybridization complex, - the sequencing of the nucleic acid or of the nucleic acid fragment involved in the above-mentioned hybridization complex, - the comparison of the sequence of the nucleic acid or of the nucleic acid fragment involved in the aforementioned hybridization complex with the sequence of the nucleic acid (not having a mutation) or of the fragment of the dopamine D-3 receptor nucleic acid (not showing a mutation).

 As for the detection of the alternative splicing of the messenger RNA produced by the gene coding for the D-3 receptor, it can be done by quantification of the messenger RNA contained in a tissue sample, using the one of the probes of the invention.

 The probes of the invention can also be used to detect the pathological expression of the dopaminergic receptor D-3, this expression being revealed by the presence of messenger RNA of the dopaminergic receptor D-3 in cells where it should not be present .

 This in vitro detection can be carried out from a biological sample taken from an individual, such as blood, according to a method which comprises the following stages - the possible prior amplification of the quantities of nucleotide sequence likely to be contained in a biological sample taken from a patient, using a DNA primer, - bringing the biological sample indicated above into contact with a nucleotide probe, under conditions allowing the production of a hybridization complex formed of said probe and said nucleotide sequence, - the detection of the above hybridization complex which may have formed.

 This pathological expression of the dopaminergic D-3 receptor can manifest itself in the case of certain tumors, and can also be correlated with the malignant or benign character of certain tumors.

 The polypeptides of the invention can be prepared by culture in an appropriate medium of a cellular host previously transformed by a recombinant vector containing one of the nucleic acids defined above and by recovery from the above-mentioned culture of the polypeptide produced by said host. transformed cell.

 Another process for the preparation of the polypeptides of the invention is characterized in that, starting preferably from the C-terminal amino acid, the successive aminoacyls are successively condensed two by two in the required order, or aminoacyls and fragments previously formed and already containing several aminoacyl residues in the appropriate order, or alternatively several fragments previously thus prepared, it being understood that care has been taken beforehand to protect all the reactive functions carried by these aminoacyles or fragments to the exception of the amine functions of one and carboxyl of the other or vice versa, which should normally intervene in the formation of peptide bonds, in particular after activation of the carboxyl function, according to the methods known in the synthesis of peptides and so on , step by step, up to the N-terminal amino acid.

 With regard to achieving expression of the dopaminergic D-3 receptors in a bacterium, such as E.

coli or in a eukaryotic cell such as a CHO cell, the following steps are carried out - the transformation of a competent cellular host with a vector, in particular a plasmid or phage, into which has previously been inserted a nucleotide sequence coding for the D-3 receptor (insert), under the control of regulatory elements, in particular a promoter recognized by the polymerases of the cell host and allowing the expression in the used cell host of said nucleotide sequence, - the culture of the cellular host transforms under conditions allowing the expression of said insert, and the transport of the D-3 receptor expressed towards the membrane so that the transmembrane sequences of the D-3 receptor are exposed on the surface of the cell host transforms.

 With regard to expression in eukaryotic cells, the regulatory elements may include the endogenous promoter of dopaminergic receptors or viral promoters such as those of SV40 viruses or of Rous sarcoma virus (RSV).

 As regards expression in E. coli, the regulatory elements may include the promoter of the lactose operon or of the tryptophan operon.

 The invention also relates to a method of detecting the capacity of a dopaminergic molecule to behave as a ligand with respect to a polypeptide of the invention, which method comprises - bringing the dopaminergic molecule into contact with a cellular host previously transformed by a vector itself modified by an insert coding for the above-mentioned polypeptide, this host carrying on its surface one or more specific sites of this polypeptide, if necessary after induction of the expression of this insert, this setting in contact being carried out under conditions allowing the formation of a bond between at least one of these specific sites and said molecule as soon as it turns out to actually have an affinity for this polypeptide, - the detection of the possible formation of a ligand-polypeptide complex.

 The invention also relates to a method for studying the affinity of a polypeptide of the invention for one or more determined ligands, which method comprises - the transformation of a competent cellular host with a vector, in particular a plasmid or a phage, into which a nucleotide sequence encoding the D-3 receptor (insert) had previously been inserted, under the control of regulatory elements, in particular a promoter recognized by the polymerases of the cellular host and allowing the expression in the used cellular host of said nucleotide sequence, - the culture of the cellular host transforms under conditions allowing the expression of said insert, and the transport of the D-3 receptor expressed towards the membrane so that transmembrane sequences of the D-3 receptor are exposed on the surface of the transformed cellular host, - the contacting of this cellular host with these determined ligands, - the d detection of a specific bond between said transformed cellular host and said determined ligands.

The method described above also makes it possible to identify the "fragments containing the sites" mentioned above and which contain only part of the 446 amino acid sequence of the
Figure 1.

 This identification consists in using inserts of a smaller size than the nucleic acid coding for the above sequence, in implementing the steps relating to the expression, the transport of the expression product and the exposure mentioned above. When the expression, the transport of the expression product and its exposure on the membrane, as well as the reaction with the ligands as defined above, are obtained, the fragments containing the essential sites can thus be determined. Consequently, the absence of reaction with the ligands, as previously defined, using fragments of size smaller than the complete sequence, would tend to show that certain essential sites have been eliminated.

 The invention also relates to a kit for the detection of the possible affinity of a ligand for a polypeptide of the invention, which kit comprises: - a culture of cellular hosts transformed by a modified vector as defined above or a culture of cellular hosts defined above, or a preparation of membranes of said hosts, - physical or chemical means for inducing the expression of the nucleotide sequence contained in the vector changes when the promoter placed upstream of this sequence is a promoter inducible by said physical or chemical means, and obtain a protein, - one or more control ligands having determined affinities for the above-mentioned polypeptide, - physical or chemical means for characterizing the biological activity of the expressed protein.

The invention also relates to a method for screening drugs for the treatment of neurological conditions (such as
Parkinson's), psychiatric (such as psychotic states), cardiovascular (such as high blood pressure) or neuroendocrine (such as dysfunctions of the hypothalamic-pituitary axis).

 The invention also relates to medicaments containing, as active substance, an active substance on at least one of the polypeptides according to the invention, having dopaminergic receptor activity or containing as active substance active substances on cells transfected by genes or the fragments of genes coding for at least one of the polypeptides with dopaminergic receptor activity according to the invention, in combination with a pharmaceutically acceptable vehicle.

 The invention also relates to medicaments containing, as active substance, a substance active on the dopaminergic receptors D-1 or D-2, but inactive on the polypeptides with dopaminergic receptor activity according to the invention.

Other characteristics and advantages of the invention appear in the following description and examples, in particular in relation to the drawings and tables in which - Figure 1 represents the amino acid sequences of the dopaminergic receptor D-2 (second line) of rat, aligned with that of the dopaminergic receptor
D-3 rat (first line). In the sequences of the dopaminergic receptor D-2, the amino acid residues identical and in position analogous to those of the dopaminergic receptor D-3 are marked by double lines (=).

To highlight the homologies, we carried out deletions in the two sequences, and these deletions are represented by the dotted spaces
In the N-terminal extracellular region of the D-3 receptor, the consensus sequences for the glycosylation sites linked to asparagine (NXS / T) are in positions 12 and 19.

 Figure 2 shows the nucleic acid sequence of the dopaminergic receptor D-3 in rats, and the corresponding amino acid sequence.

Figure 3 depicted from Northern analyzes
Blot of RNA from rat brain tissue. In Figure 3, references 1 to 10 correspond respectively to
1: hypothalamus,
2: medulla-pont,
3: olfactory bulb,
4: olfactory tubers,
5: striatum,
6: black substance,
7: cortex,
8: seahorse,
9: cerebellum,
10: pituitary gland.

EXAMPLE
Determination of the D-3 dopaminergic receptor sequence in rats -:
Oligonucleotides were synthesized from the dopaminergic receptor D-2 sequence (nucleotides 146-233, Bunzow et al. (1988) (2)), radiolabelled and used to screen a cDNA library of rat brain (Giros et al. (1989) (3)).

Among four million clones, one was isolated comprising an open partial reading phase which exhibited strong homologies with the dopaminergic receptor D-2, from the N-terminal part to the second transmembrane part.

 The DNA of this clone was radiolabeled and used to screen a rat genomic library. Among a million clones, three identical clones were isolated, different from the sequences of the D-2 receptor and of the aforementioned clone, but strongly homologous with these.

 To obtain the entire coding sequence, the "Polymerase Chain Reaction" (PCR) technique was used on rat brain mRNA with a degenerate primer contained in the seventh transmembrane part of the dopaminergic D-2 receptor (GAC (AG ) GCACTGTT (GC) AC (GA) TA (GC) AGCCA) and sequentially, three primers of the new sequence (nucleotides 442-467, 491-513 and 617-641 in Figure 2). A cDNA whose sequence covers nucleotides 641 to 1710 in Figure 2 was thus obtained.

 To obtain the end of the coding part, a rat genomic library was then screened with a terminal fragment of the cDNA obtained above by PCR.

Out of 450,000 clones, two partially overlapping positive clones were obtained, the sequence of which provided the end of the open reading phase, up to the stop codon TGA (nucleotides 1711 to 1782 of FIG. 2).

 To finally obtain the complete receptor sequence, PCR experiments using the mRNA of rat olfactory tubers were again carried out using as primers the nucleotides 410-432 in 5 'and 1823-1800 in 3'.

BIBLIOGRAPHY 1. Kekabian JW and Calne DB, "Multiple receptors
for dopamine ". Nature, 1979, 277: 93-96.

2. Bunzow JR, Van Tol HHN, Grandy DK, Albert
P., Salon J., Christie Mc D., Machida CA, Neve
KA and Civelli O. "Cloning and expression of a
D-2 dopamine receptor cDNA ". Nature, 1988, 336:
783-787.

3. Giros B., Sokoloff P., Martres MP, Riou JF,
Emorine LJ and Schwartz JC "Alternative
splicing direct the expression of two D-2
dopamine receptor isoforms ". Nature, 1989, 342:
923-926.

4. Dal Toso R., Sommer B., Ewert M., Herb A.,
Pritchett DB, Bach A., Shivers BD and Seeburg
PH "The dopamine D-2 receptor: two molecular
forms generated by alternative splicing ". The EMBO
Journal, 1989, 8: 4025-4034.

5. Schwartz JC, Delandre M., Martres MP, Sokoloff
P., Protais P., Vasse M., Costentin J., Laibe P.,
Wermuth CG, Gulat C. and Lafitte A. "Biochemical
and behavioral identification of discriminant
benzamide derivatives: new tools to differentiate
subclasses of dopamine receptors ". Catecholamines:
neuropharmacology and central nervous system.

Theoretical aspects. Alan R. Liss, Inc. NY 1984,
pp. 59-72.

6. Sokoloff P., Martres MP and Schwartz JC "Three
classes of dopamine receptor (D-2, D-3, D-4)
identified by binding studies with 3H-apomorphine
and 3H-domperidone ". Naunyn-Schmiedeberg's Arch.

 Pharmacol. 1980, 315: 89-102.

7. Martres MP, Bouthenet ML, Sales N., Sokoloff
P. and Schwartz JC "Widespread distribution of
brain dopamine receptors evidenced with 125I-
iodosulpride, a highly selective ligand ". Science,
1985, 228: 752-755.

8. Maniatis et al., "Molecular cloning", Cold Spring
Harbor Laboratory, 1982.

9. Ausubel FM, Brent R., Kingston RE, Moore DD,
Smith JA, Seidman JG and Struhl K. (1989)
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1989 "One step amplification of transcripts in
total RNA using Polymerase Chain Reaction ".

11. Ausubel FM, Brent R., Kingston RE, Moore DD,
Smith JA, Seidman JG and Struhl K. (1989)
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JC "Presence of dopamine D-2 receptors in human
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Claims (19)

 1. Polypeptide having dopaminergic receptor activity containing  the sequence of 446 amino acids of Figure 1,  or a fragment of this sequence, this fragment being such that  * or it nevertheless contains the sites contained in this sequence and the presence of which is necessary so that, when this fragment is exposed on the surface of a cell, it is able to bind dopamine to its agonists or antagonists in a specific and measurable manner , for example by means of a radioactive ligand according to the technique described in Solokoff et al. D-1, nor the dopaminergic receptor D-2 or variant polypeptides which correspond to the above-defined polypeptides, comprising certain localized mutations, without the polypeptides losing the properties of dopaminergic receptor.  * either it is capable of generating antibodies recognizing the above-mentioned sequence of 446 amino acids but not recognizing either the dopaminergic receptor  * either it is capable of being recognized by antibodies which also recognize the above-mentioned sequence of 446 amino acids, but do not recognize either the dopaminergic receptor D-1, or the dopaminergic receptor D-2, (1980) (6) and Martres et al. (1985) (7),  2. Polypeptide according to claim 1, characterized in that it is constituted by the sequence, represented in FIG. 1, extending from the end constituted by the amino acid in position 1 to that constituted by the end in position 446.  3. Nucleic acid characterized in that it comprises or consists of a sequence of nucleotides coding for the polypeptides according to claims 1 or 2.  4. Nucleic acid according to claim 5, characterized in that it comprises or is constituted by the sequence of nucleotides represented in FIG. 2, extending from the end constituted by nucleotide 1 to that constituted by nucleotide 1863 , or consists of the sequence of nucleotides represented in FIG. 2, extending from the end constituted by nucleotide 442 to that constituted by nucleotide 1779.  5. Recombinant vector, in particular for cloning and / or expression, in particular of the plasmid, cosmid, phage or virus type, characterized in that it contains a nucleic acid according to one of claims 3 and 4 in one of its sites not essential for its replication.  6. Recombinant vector according to claim 5, characterized in that it contains, in one of its non-essential sites for its replication, elements necessary to promote the expression of an amino acid sequence according to claims 1 or 2 , in a cellular host and possibly a promoter recognized by the polymerases of the cellular host, in particular an inducible promoter and optionally a signal sequence and an anchoring sequence.  7. A cellular host transformed by a recombinant vector according to any one of claims 5 or 6 and comprising the regulatory elements allowing the expression of the nucleotide sequence coding for the polypeptide according to one of claims 1 or 2 in this host.  8. A transformed cell host according to claim 7, characterized in that it is chosen from bacteria, in particular E. coli.  9. transformed cellular host according to claim 7, characterized in that it is chosen from eukaryotic organisms such as cells CHO or COS-7.  10. Antibody characterized in that it (s) is (or are) directed (s) specifically against a polypeptide according to any one of claims 1 or 2, and in that it (s) does not recognize (or recognize) the dopaminergic receptor Dl, nor the dopaminergic receptor D-2 and in particular that or those recognizing the following amino acid sequences: - that defined by the amino acid sequence represented in FIG. 1 extending from the end constituted by the amino acid in position 1 to that constituted by the amino acid in position 38, - that defined by the amino acid sequence shown in Figure 1 extending from the end constituted by the amino acid in position 135 to that constituted by the amino acid in position 147, - that defined by the amino acid sequence shown in FIG. 1 extending from the end constituted by the amino acid in position 175 to that constituted by the amino acid in position 187, - that defined by the amino acid sequence shown in FIG. 1 extending from the end constituted by the amino acid in position 210 to that constituted by the amino acid in position 375.  11. Nucleotide probe characterized in that it hybridizes with one of the nucleic acids according to claims 3 and 4 or their complementary sequence under hybridization conditions such that it does not hybridize with genes or RNA messenger of the dopamine D1 or D-2 receptors, and in particular the probes chosen from the nucleotide probes following that - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 1, to that constituted by the nucleotide in position 441, that - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 442 to that constituted by the nucleotide in position 537, that - that defined by the nucleic acid sequence shown in Figure 2, extending from the end formed by the nucleotide in position 718, to that constituted by the nucleotide in position 753, that - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 820, to that constituted by the nucleotide in position 888,. - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 958, to that constituted by the nucleotide in position 996,. - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 1068, to that constituted by the nucleotide in position 1240,. - that defined by the nucleic acid sequence represented in FIG. 2, extending from the end constituted by the nucleotide in position 1068, to that constituted by the nucleotide in position 1566, or their complementary nucleotide sequence.  12. Method for detecting the capacity of a molecule to behave as a ligand with respect to a polypeptide according to any one of claims 1 or 2, characterized by - bringing the molecule into contact with a host cell previously transformed by a vector itself modified by an insert coding for the above-mentioned polypeptide, this host carrying on its surface one or more specific sites of this polypeptide, if necessary after induction of the expression of this insert, this setting in contact being made under conditions allowing the formation of a bond between at least one of these specific sites and said molecule as soon as it turns out to actually have an affinity for this polypeptide, - the detection of the possible formation of 'a ligand-polypeptide complex.  13. Method for studying the affinity of a polypeptide according to any one of claims 1 or 2, for one or more determined ligands, characterized by - the transformation of a competent cellular host with a vector, in particular a plasmid or a phage, into which had previously been inserted a nucleotide sequence or coding for the D-3 receptor (insert), under the control of regulatory elements, in particular a promoter recognized by the polymerases of the cellular host and allowing the expression in the used cellular host of said nucleotide sequence, - the culture of the transformed cellular host under conditions allowing the expression of said insert, and the transport of the D-3 receptor expressed towards the membrane in such a way that the D-3 receptor transmembrane sequences are exposed on the surface of the transformed cell host, - the contacting of this cell host with these determined ligands, - the d detection of an affine reaction between said transformed cellular host and said determined ligands.  14. A method of in vitro diagnosis of the pathological expression of the dopaminergic receptor D-3, in which one or more of any of the probes defined according to claim 11 is used, comprising the following steps - the possible prior amplification of the amounts of nucleotide sequence according to any one of claims 3 or 4 capable of being contained in a biological sample taken from a patient, using DNA primers, - bringing the biological sample indicated above into contact with a nucleotide probe according to claim 11, under conditions allowing the production of a hybridization complex formed from said probe and said nucleotide sequence, - the detection of the above hybridization complex which may have formed.  15. Method of in vitro diagnosis of genetic anomalies, in particular polymorphisms of the gene coding for the dopaminergic receptor D-3 in an individual, likely to be correlated to psychiatric, neurological, cardiovascular or neuroendocrine affections, from a sample biological, such as blood, taken from an individual, according to a process comprising the following steps - treatment of the nucleic acid from the above-mentioned biological sample carried out using a restriction enzyme under conditions making it possible to obtain restriction fragments derived from the cleavage of said nucleic acid at these restriction sites recognized by said enzyme, - bringing a nucleotide probe according to claim 11 into contact, capable of hybridizing with the above fragments mentioned in conditions allowing the possible production of hybridization complexes between said probe and the fragments of restrict ion mentioned above, - the detection of the above hybridization complexes, - the measurement of the size of any polymorphic restriction fragments involved in the above mentioned hybridization complexes.  16. A method of in vitro diagnosis of detection of point mutations of the nucleic acid or of a fragment of the nucleic acid coding for the dopaminergic receptor D-3 according to claims 3 or 4, in an individual, comprising the following steps - bringing a nucleotide probe according to claim 11 into contact with a biological sample, for example blood, under conditions allowing the possible production of a hybridization complex formed between the probe and the nucleic acid or the above-mentioned nucleic acid fragment, - the detection of the above hybridization complex, - the sequencing of the nucleic acid or of the nucleic acid fragment involved in the above hybridization complex, - the comparison of the sequence of the nucleic acid or the nucleic acid fragment involved in the aforementioned hybridization complex with the nucleic acid sequence (not showing a mutation) or of the nucleic acid fragment ( showing no mutation) of the dopaminergic D-3 receptor.  17. A method of in vitro diagnosis of the pathological expression of the dopaminergic receptor D-3, in which one or more antibodies according to claim 10 are used comprising the following steps - bringing an antibody into contact with claim 10 with the abovementioned biological sample under conditions allowing the possible production of an immunological complex formed between the dopaminergic receptor D-3 or product which is derived therefrom and the antibody of the invention, - the detection of the above-mentioned immunological complex formed.  18. Medicament containing as active substance an active substance on at least one of the polypeptides according to one of claims 1 or 2, having a dopaminergic receptor activity or containing as active substance active substances on cells transfected by genes or the fragments of genes coding for at least one of the polypeptides with dopaminergic receptor activity according to one of claims 1 or 2 in association with a pharmaceutically acceptable vehicle.  19. Medicament containing as active substance a substance active on the dopaminergic receptors D-1 or D-2, but inactive on the polypeptides with dopaminergic receptor activity according to one of claims 1 or 2.