WO1993008214A1 - ADNc CODANT POUR LA PROTEINE BOVINE DE TRANSPORT DE LA DOPAMINE SENSIBLE A LA COCAINE - Google Patents

ADNc CODANT POUR LA PROTEINE BOVINE DE TRANSPORT DE LA DOPAMINE SENSIBLE A LA COCAINE Download PDF

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WO1993008214A1
WO1993008214A1 PCT/US1992/008961 US9208961W WO9308214A1 WO 1993008214 A1 WO1993008214 A1 WO 1993008214A1 US 9208961 W US9208961 W US 9208961W WO 9308214 A1 WO9308214 A1 WO 9308214A1
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cells
leu
dopamine
cdna
ala
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PCT/US1992/008961
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Ted B. Usdin
Beth J. Hoffman
Michael J. Brownstein
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The Government Of The United States Of America As Represented By The Department Of Health And Human Services
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the invention relates to a cDNA encoding a bovine dopamine (DA) transporter protein (DAT) , its sequence and applications thereof.
  • DA bovine dopamine
  • DAT transporter protein
  • Drugs which affect dopamine neurotransmission are used to treat psychotic syndromes including schizophrenia, as well as Parkinsons disease and other movement disorders. Reuptake into pre-synaptic terminals is thought to be the major means of terminating the synaptic action of monoamine neurotransmitters (Axelrod, J. , Science. 173, 598-606 (1971); Hertting, G. et al., Nature. 189, 66-68 (1961); Iversen, L. L. , Br. Med. Bull.
  • dopamine Horn, A. S., Prog. Neurobiol. 34, 387-400 (1990)
  • the dopamine reuptake site, or transporter has been characterized by measuring the uptake of [ 3 H]dopamine and the binding of radioligands (Horn, A. S., Prog. Neurobiol. 34, 387-400 (1990); Raiteri, M. et al. , in "Advances in Biochemical Psychomarmacology", pp. 35-36,
  • GABA ⁇ -aminobutyric acid
  • NE norepinephrine
  • HT serotonin
  • the invention constitutes a cDNA encoding a protein that is localized upon the surface of cells which confers upon such cells the property of transport of dopamine from the extracellular medium into the interior of the cell. It is one object of the invention to provide the cDNA for the dopamine transporter and diagnostic applications of the cDNA or portions thereof. It is a further object of the invention to create cells which express the protein encoded by the cDNA, either as a result of transient transfection with the cDNA or as a result of incorporation of the cDNA into the genome of a host cell line.
  • FIG. 1A-1D Nucleotide sequence of the bovine dopamine transporter (DAT) cDNA.
  • SUBST hydroxylase (lower panel) directed oligonucleotide probes to 12 mm sections of bovine substantia nigra. Arrows indicate the same groups of neurons in the two adjacent sections; midline is indicated by the asterisk.
  • An 8 day exposure on hyperfilm-BMax (Amersham) is printed as a negative (developed grains are white) .
  • FIG. 3 Hybridization of the DA transporter directed oligonucleotide probe to bovine RNA. Approximately 6 mg of once-selected polyA + RNA from the adrenal medulla (lane 1) , spleen (lane 2) , hypothalmus (lane 3) , cerebellum (lane 4) , caudate (lane 5) and 1.5 mg from the substantia nigra (lane 6) were loaded onto the gel.
  • FIG. 4A-4D The deduced amino acid sequence of the dopamine transporter (DAT) compared to the norepinephrine transporter and GABA transporter. Gaps have been added to obtain good alignment. Positions at which amino acids are identical are boxed. Predicted transmembrane domains are underlined. Potential N-linked glycosylation sites ( ⁇ ) , and sites for phosphorylation by cAMP-dependent protein kinase (*) and protein kinase C (#) on the DA transporter are indicated.
  • DAT dopamine transporter
  • Gaps have been added to obtain good alignment. Positions at which amino acids are identical are boxed. Predicted transmembrane domains are underlined. Potential N-linked glycosylation sites ( ⁇ ) , and sites for phosphorylation by cAMP-dependent protein kinase (*) and protein kinase C (#) on the DA transporter are indicated.
  • fusion proteins of, subfragments or variants of the DAT protein disclosed in the present application wherein the original amino acid sequence is modified or changed by insertion, addition, substitution, inversion or deletion of one or more amino acids are within the scope of the present invention, so far as they retain the essential binding or transport specificity as mentioned above.
  • Example 1 Isolation and Characterization of the cDNA Encoding the Bovine Dopamine Transporter.
  • a cDNA library of 4 X 10 6 recombinants was synthesized from bovine substantia nigra poly A + RNA using the vector pCDSP6T7 as described by Okayama (Okayama, H. et al., Methods in Enzymology 154, 3-28 (1987).
  • PCDSP6T7 was derived from pCD (Okayama, H. et al., ibid. ) by addition of recognition sites for bacteriophage SP6 and T7 RNA polymerases 5• and 3' to the cDNA insert site.
  • DNA prepared from 36 subdivisions of 5 X 10 5 clones from the amplified library was screened on Southern blots (Bonner, T. I.
  • GTAGGGGAACCGCCACACATT-3• (SEQ. ID. NO. 3) directed at a region highly conserved in noradrenaline (Pacholczyk, T. et al., Nature 350,
  • the bovine DAT cDNA clone contained an insert of 2340 nucleotides ( Figure 1) .
  • Figure 4 The deduced amino acid sequence ( Figure 4) predicts a protein of 693 residues, suggesting an unglycosylated molecular weight of approximately 70,000. This is consistent with size estimates from studies in which the dopamine transporter was labeled with photoaffinity probes and analyzed by SDS gel electrophoresis (Grigoriadis, D. E. et al. , J. Neurosci. 9, 2664-2670 (1989); Berger, S. P. et al. , Mol. Pharm. 39, 429-435 (1991)).
  • Example 2 Analysis of bovine DAT expression in vitro and in vivo.
  • the antisense oligonucleotide probe for in situ hybridization histochemistry was synthesized using the sequence from a region of the DA transporter cDNA with minimal similarity to the other cloned neurotransmitter transporters. There was strong hybridization of this probe to discrete cells in the bovine substantia nigra in areas corresponding to the A9 and A10 cell groups
  • SUBSTITUTESHEET the locus coeruleus (data not shown) which were also labeled by the probe for tyrosine hydroxylase mRNA.
  • the cDNA cloned does not encode either the bovine 5HT or NE transporters.
  • There is a significant GABA containing population in the substantia nigra pars reticulata (Mugnaini, E. & Oertel, W. H. in "Handbook of Chemical Neuroanatomy", pp. 436-622, Bjorklund, A. & Hokfelt, T. eds., c. 1985 by Elsevier, Amsterdam.) however the absence of hybridization in the caudate and elsewhere demonstrate that it is not a GABA transporter.
  • mRNA (Okayama, H. et al., 154, 2-28 (1987)) from bovine tissues was enriched by a single passage over oligo dT cellulose, separated on a 1.2% agarose/formaldehyde gel, and transferred by electrophoresis to Nytran (Schleicher and Schuell) membrane.
  • the oligonucleotide probe was labeled with [ 32 P]dATP using terminal deoxynucleotidyltransferase and incubated with the blot overnight at 37°C in 4 X SSPE (l X SSPE is 0.15M NaCl/O.OlM NaH 2 P0 4 /l.3mM EDTA, pH 7.4), 5 X Denhardts solution, 0.5mg/ml denatured salmon sperm DNA, 0.1% SDS, 250 mg/ml tRNA, 50% formamide. Final wash conditions were 1 X SSPE/0.1%SDS at 60°C.
  • the oligonucleotide used for in situ hybridization was labeled with 32 P and used to probe blots of RNA from several bovine tissues (Figure 3) .
  • a single major band of approximately 3.0 kb is present in RNA from the substantia nigra.
  • Much less intense bands of 4.0 and 5.8 kb are also detected in the substantia nigra sample.
  • a faint band of approximately the same size as the major band in the substantia nigra was seen in the sample from the hypothalmus. No additional bands were detected when a 1 kb fragment of the DA transporter was used as a probe.
  • Probing a blot of human substantia nigra RNA revealed a single band of approximately 4.5 kb.
  • uptake buffer 25mM Hepes, pH 7.4, 125 mM NaCl, 4.8mM KCl, 1.2m KH 2 P04, 1.3m CaCl 2 , 1.2mM MgS0 4 , 5.6mM glucose, l M Na ascorbate, 10 mM pargyline
  • uptake buffer 25mM Hepes, pH 7.4, 125 mM NaCl, 4.8mM KCl, 1.2m KH 2 P04, 1.3m CaCl 2 , 1.2mM MgS0 4 , 5.6mM glucose, l M Na ascorbate, 10 mM pargyline
  • uptake buffer 25mM Hepes, pH 7.4, 125 mM NaCl, 4.8mM KCl, 1.2m KH 2 P04, 1.3m CaCl 2 , 1.2mM MgS0 4 , 5.6mM glucose, l M Na ascorbate, 10 mM pargyline
  • Ki's were determined using lOOnM [H]DA, which typically gave 31-46,000 dpm total uptake, 1400-3600 dpm non-specific uptake, defined in the presence of 1 mM GBR12909, and 1200-3300 dpm in mock transfected cells. Inhibitors were added at concentrations ranging from 10 nM to 10 ⁇ M. Cells were solubilized in 0.5 N NaOH and radioactivity determined by liquid scintillation counting.
  • the DA transporter was expressed in tissue culture cells by simultaneously introducing a recombinant vaccinia virus encoding bacteriophage SP6 RNA polymerase and the DA transporter cDNA. These cells accumulated [ 3 H]DA in a dose dependent manner with an Km of 31.5 mM, which is significantly higher than that for the rat and human dopamine transporters (Horn, A. S., Prog. Neurobiol. 34, 387-400 (1990)). Inhibition constants for a series of compounds were determined from displacement curves as descried above (Table 1; (Cheng, Y. & Prusoff, W. H. , Biochem. Pharmacol. 22, 3099 (1973)).
  • GBR12909 a highly specific inhibitor of the DA transporter (Andersen, P. H., J. Neurochem. 48, 1887-1896 (1987); Andersen, P. H. , Eur. J. Pharm. 166, 493-504 (1989)) blocked [H]dopamine accumulation much more potently than did desipramine, which is more specific for the NE uptake system (Horn, A. S. in "The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines", pp. 195-214, Paton, D. M. ed. , c. 1976 by Raven Press, New York.) or fluoxetine (Lemberger, L. H. et al. , Clin. Pharmacol. Ther.
  • Example 4 Production of variant amino acid sequences within the bovine dopamine transporter polypeptide and testing of their activity.
  • the nucleotide and derived amino acid sequences, as well as the predicted structure, are very similar to those of the rat GABA (Guastella, J. et al.. Science. 249, 1303-1306 (1990)), human NE (Pacholczyk, T. et al.. Nature 350, 350-354 (1991)), and rat 5HT (Hoffman, B. J. , Mezey, E., and Brownstein, M. J. , Science, in press) transporters.
  • Three consensus sites for N-linked glycosylation are present in a region which may be a large extracellular loop; two or three such sites are present in the same region of the other neurotransmitter transporters.
  • Potential sites for phosphorylation by cyclic AMP-dependent protein kinase are present on predicted intracellular domains near the carboxyl terminus of the DA transporter.
  • SUBSTITUTE SHEET A possibility raised by the deduced peptide sequence of the dopamine transporter is regulation by cyclic AMP (or cyclic GMP)-dependent protein kinase since potential sites for phosphorylation by these enzymes are present on domains predicted to be intracellular. Stimulation of dopamine accumulation by dibutyryl cyclic AMP and forskolin in tissue culture cells has recently been reported (Kadowaki, K. et al., Neuroendocrinology 52, 256-261 (1990)). The cloned transporter presents an opportunity for detailed study of its interaction with other proteins and with second messenger systems and the possibility to identify regulatory influences on this important component of synaptic physiology.
  • cyclic AMP or cyclic GMP
  • the DAT cDNA is removed from the pCDSP6T7 vector by cleavage of the recombinant DNA with BamHI and purification of the DAT cDNA insert by elution from an agarose gel after electrophoretic separation from the vector DNA.
  • the DAT insert is then ligated into the pSELECT vector (Promega, Madison, WI) and site-directed mutagenesis of the desired nucleotides is accomplished using the ALTERED SITES kit (Promega, Madison, WI) and a mutagenic oligonucleotide of the sequence: 5•-GATCGGGAGACCTGGGCCAAGAAGGCCGACTTC-3 • (SEQ. ID. NO. 6)
  • the mutant cDNA insert is then reisolated by separation of the BamHI fragments of the mutant pSELECT recombinant and recloned into a suitable mammalian cell expression vector, for instance, pCDNAl (InVitrogen, San Diego, CA) .
  • the expression plasmid, containing the mutant DAT cDNA is then transfected into a host cell, such as CHO cells, or CV-1 cells infected with a recombinant vaccinia virus expressing T7 polymerase (Hoffman, B. J., Mezey, E. and Brownstein, M. , submitted) .
  • a host cell such as CHO cells, or CV-1 cells infected with a recombinant vaccinia virus expressing T7 polymerase (Hoffman, B. J., Mezey, E. and Brownstein, M. , submitted) .
  • the thus transfected cells are then assayed for dopamine transporter activity using the uptake assay described above.
  • SUBSTITUTE SHEET Example 5 Creation of a permanent cell line expressing bovine DAT at the cell surface.
  • the DAT cDNA cloned in the plasmid vector pCDSP6T7 is useful as a means of constitutive expression of bovine DAT protein in mammalian cells.
  • CV-1 cells (other cell lines may be used as well) are transfected by CaP0 4 precipitation in a molar ration of 1 to 10 with pCDNEO as described in detail by Chen and Okayama (Chen, C. and Okayama, H., Mol . Cell Biol . 7, 2745 (1982)).
  • Cotransfectants and cells stably integrating neomycin resistant clones are selected by growth for three weeks in the antibiotic G418 (200-600 ⁇ g/ml) . At the end of four weeks, individual colonies are isolated and assayed for DAT expression by the uptake assay described above.
  • a cell line, CVSVDAT4 has been produced by the described procedure.
  • DNA isolated from white blood cells by standard protocols is analyzed by Southern blots using a number of restriction enzymes for Restriction Fragment Length Polymorphisms (RFLPs) showing relatively high frequency of population variants, using the stringencies defined by conditions required to obtain specific hybridization of the DAT probe to human genomic DNA.
  • RFLPs in this locus can then be examined for their distribution in several kindreds of normal and also in several kindreds of subjects diagnosed with cocaine abuse or Parkinson's disease. Any restriction fragment length polymorphic forms of the gene found in higher abundance in populations diagnosed as above, compared to a normal, control population are checked by ascertainment in other populations.
  • SUBSTITUTESHEET Example 7 Diagnosis of deficiency, mutant or overexpression of dopamine transporter by PCR mRNA obtained from tissue biopsy from a patient is subjected to quantitative reverse- transcript PCR (for example, see A. M. Wang, et al. PNAS USA s9717 (1989)) utilizing as primers oligonucleotides derived from the cDNA sequence of DAT.
  • Reassay for example, see A. M. Wang, et al. PNAS USA s9717 (1989) utilizing as primers oligonucleotides derived from the cDNA sequence of DAT.
  • Use of the 5' 17-mer, 5'- AAAGCTGGGTACCGGGC-3 ' , bases 77 through 93 of SEQ. I.D. NO. 1 (and Figure 1) as the upstream primer and 5'-GCTCTCGGCAGAGAACC-3', the reverse complement of bases 2547 to 2563 of SEQ. I.D.
  • NO. 1 (and Figure 1) , as the downstream primer, allows examination of the character of the majority of the DAT mRNA. Variance in the expression level can be ascertained by comparison of product yield with a normal control. Abnormal mRNA structures can be diagnosed by observation of a product band of a length different from the normal control. Point mutants can be observed by use of primers and conditions appropriate for detection of the mismatch between the mutant and normal alleles. For example, the "reverse dot blot" procedure for screening the expression of several mutant alleles in a single experiment, has been described for the CFTR gene, mutants of which cause cystic fibrosis (Erlich, H.A. , et al Science 252:1643 (1991).
  • the plasmid vector pFLAG system International Biotechnologies, Inc., New Haven, CT
  • pFLAG system produces the polypeptide of interest attached to a short protein sequence that allows purification of the fusion protein by use of a monoclonal antibody directed against a hydrophilic, and thus probably surface localized, octapeptide.
  • the dopamine transporter encoding insert from the DAT cDNA is obtained by BamHI digestion and purification of the 2.9 kilobasepair fragment by electrophoresis and elution from an agarose gel by standard techniques and cloned into the Multiple Cloning Site of the pFLAG vector (International Biotechnologies, Inc.) in such a manner as to place the DAT cDNA in the correct orientation and reading frame as to produce a DAT fusion peptide.
  • the appropriate E. coli host is transformed and colonies containing the DAT cDNA may be screened by colony hybridization using the DAT cDNA as probe. Positive clones are grown as large-scale cultures and the fusion protein is obtained in pure form by use of the monoclonal antibody affinity column as described by the manufacturer of the system.
  • SUBSTITUTE SHEET puri ication may be modified as needed to obtain sufficient yield and purity by means apparent to one skilled in the art.
  • Authentic DAT protein lacking the FLAG octapeptide is obtained by enterokinase cleavage of the fusion protein as described by the supplier of the FLAG system.
  • Example 9 Purification of DAT from tissues or from transformed mammalian cells.
  • protein isolated from transformed bacterial cells lacks post-translational modifications, such as sugar additions, that occur in mammalian cells
  • the purification of the protein from transformed CHO cells is discussed.
  • CHO cells transformed as described in Example 5 are subjected to a purification protocol as described for the purification of the GABA transporter (Radian, et al., J. Biol. Chem. 261, 15437-15441 (1987) with the modification that binding of labelled GBR12935 (New England Nuclear) is used to assay for the presence of DAT in the sample rather than labelled gamma-amino butyric acid.
  • the protocol is modified as required to allow the isolation of DAT as a distinct protein by techniques known to a practitioner of the art.
  • SUBSTITUTESHEET Example 10 A biosensor for the measurement of dopamine. cocaine. or analogs thereof in physiologic samples. Biosensors consisting of DAT protein or the ligand binding portions thereof and a piezoelectric crystal can be created and utilized for the measurement of DAT ligands in samples.
  • the literature describing the use of ligand binding proteins as components in biosensors is large and expanding. Review of the art is given by Luong, et al. (Luong, J.H.T, et al. Trends Biotehnol. 6, 310-3316 (1988)) and Wingard (Wingrad, L.B. jr. Ann. N.Y. Acad. Sci.

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Abstract

On décrit un ADNc codant pour la protéine bovine de transport de la dopamine. On présente des applications de cette séquence d'ADNc concernant le diagnostic de la maladie de Parkinson ou d'autres syndromes provenant d'un fonctionnement anormal de cette protéine de transport de la dopamine, et aussi des applications de cette protéine telle qu'exprimée dans des cellules hôtes à partir de l'ADNc.
PCT/US1992/008961 1991-10-24 1992-10-26 ADNc CODANT POUR LA PROTEINE BOVINE DE TRANSPORT DE LA DOPAMINE SENSIBLE A LA COCAINE WO1993008214A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990006047A2 (fr) * 1988-11-21 1990-06-14 Baylor College Of Medicine Systeme de fixation de neurotransmetteurs humains a affinite elevee

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990006047A2 (fr) * 1988-11-21 1990-06-14 Baylor College Of Medicine Systeme de fixation de neurotransmetteurs humains a affinite elevee

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Dialog Information Services, file 154, Medline, Dialog accession no. 07225704, Medline accession no.90132704, Bannon MJ et al: "Expression of a human cocaine-sensitive dopamine transporter in Xenopus laevis cocytes", J Neurochem Feb 1990, 54 (2) p706-8 *
Dialog Information stystem, file 154, Medline, Dialog accession no. 07606196, Medline accession no.91125196, Cao CJ: "Putative cocaine receptor in striatum is a glycoprotein with avtive thiol func- tion", Membr Biochem 1989, 8 (4) p207-20 *

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