GB2365432A

GB2365432A - Neurotransmitter transporter polypeptide

Info

Publication number: GB2365432A
Application number: GB0012199A
Authority: GB
Inventors: Cinzia Felicita Sala; Goerg Christian Terstappen
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2000-05-19
Filing date: 2000-05-19
Publication date: 2002-02-20
Also published as: GB0012199D0

Abstract

The present invention provides an isolated neurotransmitter transporter polypeptide comprising <SL> <LI>(i) the amino acid sequence of SEQ ID NO: 2 or <LI>(ii) a variant thereof which has sodium:neurotransmitter symporter activity or <LI>(iii) a fragment of (i) or (ii) which has sodium:neurotransmitter symporter activity. </SL>

Description

<Desc/Clms Page number 1> NEW POLYPEPTIDE Field of the Invention The present invention relates to neurotransmitter transporter polypeptides. Background of the Invention In most systems termination of chemical neurotransmission is achieved by rapid uptake of the released neurotransmitter by specific neurotransmitter transporters into the synaptic terminal or the surrounding glial cells. At least two distinct superfamilies of transporters can be distinguished: the plasma membrane transporters that operate on the plasma membrane of neuronal and glial cells, and the vesicular membrane transporters that function in the uptake of neurotransmitters into synaptic vesicles. The superfamily of plasma membrane transporters can be further divided into two families depending on their ionic dependence: Na+/Cl"-dependent transporters and Na@/K±dependent transporters.

The gene products of the Na`/Cl" transporter family are highly conserved. In mammals they can be grouped into subfamilies: monoamine transporters, amino acid transporters and "orphan transporters". The subfamilies with known substrates are distinguished not only by their sequence homology, but also by their substrate specificity and pharmacology, whereas for "orphan transporters" the transported substrates to date are unknown. The Na/Cl- transporters show a common structure of 12 transmembrane domains (12TM) with a single large loop in the external face of the membrane with potential glycosylation sites. The orphan transporter structure deviates from the norm by having two potential glycosylated loops outside the membrane.

The Na'/K'-dependent transporters function on the plasma membranes especially in excitatory amino acid (eg. glutamate) transport. The family members display 6 to 10 hydrophobic transmembrane domains and share no sequence homology with the Na+/CI" -dependent carrier family. Hydropathic analysis of the primary sequences of vesicular neurotransmitter transporters predict 12 putative transmembrane segments.

The rat NTT4 protein belongs to the sodium: neurotransmitter symporter

family. Its function is not yet known, it is an orphan transporter. Its subcellular localization is in the plasma membrane, and it is found exclusively in the central nervous system where it is more abundant in the cerebellum and frontal cortex. Summary of the Invention A novel neurotransmitter transporter polypeptide, referred to herein as HIPHUM 0000029, is now provided. The novel neurotransmitter transporter polypeptide is a screening target for the identification and development of novel pharmaceutical agents, including modulators of neurotransmitter transporter polypeptide activity. These agents may be used in the treatment and/or prophylaxis of disorders such as psychiatric disorders such as bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.

Accordingly, the present invention provides an isolated neurotransmitter transporter polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2; (ii) a variant thereof which has sodium:neurotransmitter symporter activity; or (iii) a fragment of (i) or (ii) which has sodium:neurotransmitter symporter activity.

According to another aspect of the invention there is provided a polynucleotide encoding a polypeptide of the invention which polynucleotide includes a sequence comprising: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c).

The invention also provides:

- an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention; - a host cell comprising an expression vector of the invention; - a method of producing a polypeptide of the invention which method comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide; - an antibody specific for a polypeptide of the invention; - a method for identification of a substance that modulates neurotransmitter transporter activity and/or expression, which method comprises contacting a polypeptide, polynucleotide, expression vector or host cell of the invention with a test substance and determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by the said polynucleotide, thereby to determine whether the test substance modulates neurotransmitter transporter activity and/or expression; - a compound which or modulates neurotransmitter transporter activity and which is identifiable by the method referred to above; - a method of treating a subject having a disorder that is responsive to neurotransmitter transporter stimulation or modulation, which method comprises administering to said subject an effective amount of substance of the invention; and - use of a substance that stimulates or modulates neurotransmitter transporter activity in the manufacture of a medicament for the treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of neurotransmitter transporter activity.

Preferably the disorder is selected from psychiatric disorders such as bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.

Brief Description of the Sequences SEQ ID NO: 1 shows the nucleotide and amino acid sequences of human protein HIPHUM 0000029.

SEQ ID NO: 2 is the amino acid sequence alone of HIPHUM 0000029.

Detailed Description of the Invention Throughout the present specification and the accompanying claims the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The present invention relates to a human neurotransmitter transporter polypeptide, referred to herein as HIPHUM 0000029, and variants thereof. Sequence information for HIPHUM 0000029 is provided in SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2. A polypeptide of the invention thus consists essentially of the amino acid sequence of SEQ ID NO: 2 or of a variant of that sequence, or of a fragment of either thereof.

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention. Routine methods, can be employed to purify and/or synthesise the proteins according to the invention. Such methods are well understood by persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2"d Edition, CSH Laboratory Press, 1989, the disclosure of which is included herein in its entirety by way of reference.

The term "variant" refers to a polypeptide which has a same essential character or basic biological functionality as HIPHUM 0000029. The essential character of HIPHUM 0000029 can be defined as follows: HIPHUM 0000029 is a neurotransmitter transporter polypeptide. Preferably a variant polypeptide is one which binds to the same ligand or substrate as HIPHUM 0000029. Preferably the polypeptide has sodium: neurotransmitter symporter activity. A polypeptide having a same essential character as HIPHUM 0000029 may be identified by monitoring for a fiuiction of the neurotransmitter transporter selected from sodium:neurotransmitter

symporter activity, uptake of neurotransmitter transporter substrates. HIPHUM 0000029 is member of the orphan transporter subfamily of Na-/Cl' transporters. A variant of HIPHUM 0000029 may have orphan transporter-like activity. A full length variant polypeptide is preferably one which includes twelve transmembrane domains and may additionally include two potential glycosylated loops outside the membrane.

Typically, polypeptides with more than about 65% identity preferably at least 80%, at least 90% or at least 95% and particularly preferably at least 96%, at least 97%, at least 98% or at least 99% identity, with the amino acid sequences of SEQ ID NO: 2, are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains a basic biological functionality of the HIPHUM 0000029 polypeptide.

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. The modified polypeptide generally retains activity as a neurotransmitter transporter. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

ALIPHATIC Non-polar GAP ILV Polar-uncharged CST M NQ Polar-charged D E KR AROMATIC H F W Y

Shorter polypeptide sequences are within the scope of the invention. For example, a peptide of at least 20 amino acids or up to 50, 60, 70, 80, 100, 150 or 200 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality of HIPHUM 0000029. In particular, but not exclusively, this aspect of the invention encompasses the situation when the protein is a fragment of the complete protein sequence and may represent a ligand- binding region (N-terminal extracellular domain) or an effector binding region (Cterminal intracellular domain). Such fragments can be used to construct chimeric polypeptides, preferably with another twelve transmembrane polypeptide, more preferably with another member of the family of neurotransmitter transporter polypeptides. Such fragments of HIPHUM 0000029 or a variant thereof can also be used to raise anti-HIPHUM 0000029 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM 0000029 polypeptide and may otherwise not demonstrate the substrate or ligand binding or other properties of HIPHUM 0000029.

Polypeptides of the invention may be chemically modified, e.g. post- translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term "polypeptide" of the invention.

The invention also includes nucleotide sequences that encode for HIPHUM 0000029 or variant thereof as well as nucleotide sequences which are complementary thereto. The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Nucleotide sequence information is provided in SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al, 1989.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a eDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with "P. Selective hybridisation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989. For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at 60'C up to 65'C. If lower stringency is required suitable conditions include 2 x SSC at 60 C.

The coding sequence of SEQ ID NO: 1 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends. A polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included. The modified polynucleotide generally encodes a polypeptide which has a HIPHUM 0000029 polypeptide activity. Alternatively, a polynucleotide encodes a ligand-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM 0000029 activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more

preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1.

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (l984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al (l990) J. Mol. Biol. 2l5:403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (l992) Proc. Mad. Acad.

Sci. USA 89: 10915-109l9) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Nad. Acad Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence

if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

The nucleotides according to the invention have utility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo. The nucleotides may be involved in recombinant protein synthesis or indeed as . therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides complementary to those encoding HIPHUM 0000029, or antisense sequences, may also be used in gene therapy.

Polynucleotides of the invention may be used as a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.

Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO: 1.

The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to

allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as (3-actin promoters, may be used. Tissue- specific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modified to express the HIPHUM 0000029 polypeptide or a variant thereof. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T, CHO, HeLa and COS cells. Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosylation and cell surface expression of a polypeptide. Expression may be achieved in transformed oocytes. A polypeptide of the invention may be expressed in cells of a transgenic non-human animal, preferably amouse. A transgenic non-

human animal expressing a polypeptide of the invention is included within the scope of the invention. A polypeptide of the invention may also be expressed in Xenopus laevis oocytes, in particular for use in an assay of the invention.

According to another aspect, the present invention also relates to antibodies, specific for a polypeptide of the invention. Such antibodies are for example useful in purification, isolation or screening methods involving immunoprecipitation techniques or, indeed, as therapeutic agents in their own right.

Antibodies may be raised against specific epitopes of the polypeptides according to the invention. Such antibodies may be used to block ligand binding to the polypeptide. An antibody, or other compound, "specifically binds" to a protein when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other proteins. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

Antibodies of the invention may be antibodies to human polypeptides or fragments thereof. For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample, which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

A sample may be for example a tissue extract, blood, serum and saliva. Antibodies of the invention may be bound to a solid support and/or packaged into

kits in a suitable container along with suitable reagents, controls, instructions, etc. Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo HIPHUM 0000029 imaging.

Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (l988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the "immunogen".

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

An important aspect of the,present invention is the use of polypeptides

according to the invention in screening methods. The screening methods may be used to identify substances that bind to neurotransmitter transporter polypeptides and in particular which bind to HIPHUM 0000029 such as a ligand or substrate for the polypeptide. Screening methods may also be used to identify agonists or antagonists which may modulate neurotransmitter transporter polypeptide activity, inhibitors or activators of HIPHUM 0000029 activity, and/or agents which up-regulate or down- regulate HIPHUM 0000029 expression.

Any suitable format may be used for the assay. In general terms such screening methods may involve contacting a polypeptide of the invention with a test substance and monitoring for binding of the test substance to the polypeptide or measuring polypeptide activity. A polypeptide of the invention may be incubated with a test substance. Modulation of neurotransmitter transporter activity may be determined. In a preferred aspect, the assay is a cell-based assay. Preferably the assay may be carried out in a single well of a microtitre plate. Assay formats which allow high throughput screening are preferred.

Modulator activity can be determined by contacting cells expressing a polypeptide of the invention with a substance under investigation and by monitoring an effect mediated by the polypeptide. The cells expressing the polypeptide may be in vitro or in vivo. The polypeptide of the invention may be naturally or recombinantly expressed. Preferably, the assay is carried out in vitro using cells expressing recombinant polypeptide. Preferably, control experiments are carried out on cells which do not express the polypeptide of the invention to establish whether the observed responses are the result of activation of the polypeptide.

The binding of a test substance to a polypeptide of the invention can be determined directly. For example, a radiolabelled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored. Typically, the radiolabelled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined by repeating the experiment in the presence of a saturating concentration

of a non-radioactive ligand.

Alternatively, the uptake of a substrate may be monitored using a radiolabelled substance. Typically, the assays are carried out in a cellular system, either in a recombinant system or a synaptosomal preparation. The radiolabelled substrate is incubated with the cellular system. Subsequently uptake of the substrate can be determined by lysing the cells and measuring the amount of intracellular labelled substrate.

Assays may be carried out using cells expressing HIPHUM 0000029, and incubating such cells with the test substance optionally in the presence of HIPHUM 0000029 ligand. Alternatively an antibody may be used to complex HIPHUM 0000029 and thus mediate HIPHUM 0000029 activity. Test substances may then be added to assess the effect on such activity. Cells expressing HIPHUM 0000029 constitutively may be provided for use in assays for HIPHUM 0000029 function. Additional test substances may be introduced in any assay to look for inhibitors of ligand or substrate binding or inhibitors of HIPHUM 0000029-mediated activity.

Assays may also be carried out to identify substances which modify HIPHUM 0000029 polypeptide expression, for example substances which up- or down- regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM 0000029 to monitor levels of HIPHUM 0000029 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM 0000029 expression include using a reporter gene construct driven by the HIPHUM 0000029 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide. Further possible assays could utilise membrane fractions from overexpression of HIPHUM 0000029 polypeptide either in X. laevis oocytes or cell lines such as HEK293, CHO, COS7 and HeLa cells and assessment of displacement of a radiolabelled ligand.

Additional control experiments may be carried out. Assays may also be carried out using known ligands of other neurotransmitter transporter polypeptides to identify ligands or substrates which are specific for polypeptides of the invention. An assay may be carried out to assess the effect of a test substance on the uptake of a labelled substrate. An assay of the invention may be carried out using a known neurotransmitter transporter agonist or neurotransmitter transporter antagonist to

provide a comparison with a compound under test.

Suitable test substances which can be tested in the above assays include combinatorial libraries, defined chemical entities and compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e.g. phage display libraries) and antibody products.

Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or activation tested individually. Test substances may be used at a concentration of from 1 nM to 1 OOO#LM, preferably from 1 p,M to 1 OO@.M, more preferably from 1 p,M to 10 1zM. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the polypeptide. Alternatively a test substance which acts as an activator may produce 50% of the maximal activity produced using a known activator.

Another aspect of the present invention is the use of polynucleotides encoding the HIPHUM 0000029 polypeptides of the invention to identify mutations in HIPHUM 0000029 genes which may be implicated in human disorders. Identification of such mutations may be used to assist in diagnosis or susceptibility to such disorders and in assessing the physiology of such disorders. Polynucleotides may also be used in hybridisation studies to monitor for up- or down-regulation of HIPHUM 0000029 expression. Polynucleotides such as SEQ ID NO: 1 or fragments thereof may be used to identify allelic variants, genomic DNA and species variants.

The present invention provides a method for detecting variation in the

expressed products encoded by HIPHUM 0000029 genes. This may comprise determining the level of an HIPHUM 0000029 expressed in cells or determining specific alterations in the expressed product. Sequences of interest for diagnostic purposes include, but are not limited to, the conserved portions as identified by sequence similarity and conservation of intron/exon structure. The diagnosis may be performed in conjunction with kindred studies to determine whether a mutation of interest co-segregates with disease phenotype in a family.

Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate procedures are described in, for example, Nuovo, G.J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of a HIPHUM 0000029, and qualitative aspects of HIPHUM 0000029 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM 0000029 nucleic acid molecules may involve their amplification, e.g. by PCR (the experimental embodiment set forth in U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-l93), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874- 1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. 15 USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Particularly suitable diagnostic methods are chip-based DNA technologies such as those described by Hacia et al., 1996, Nature Genetics 14:441-447 and Shoemaker et al., 1996, Nature Genetics 14:450-456. Briefly, these techniques involve quantitative methods for analyzing large numbers of nucleic acid sequence

targets rapidly and accurately. By tagging with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization.

Following detection, the results seen in a given patient may be compared with a statistically significant reference group of normal patients and patients that have HIPHUM 0000029 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM 0000029 encoded product detected with various clinical states or predisposition to clinical states.

Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease states, which are responsive to regulation of neurotransmitter transporter polypeptide activity. The treatment may be therapeutic of prophylactic. The condition of a patient suffering from such a disease state can thus be improved.

In particular, such substances may be used in the treatment of psychiatric disorders such as bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.

Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation will depend upon several factors including the particular substance to be, administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 17"' Ed. 1985, the disclosure of which is included herein of its entirety by way of reference.

The substances may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition

of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM 0000029 or a variant thereof which inhibits HIPHUM 0000029 activity may be administered to the mammal. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid encoding the polypeptide may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of lpg to lmg, preferably to lpg to 10g nucleic acid for particle mediated gene delivery and 10.g to lrng for other routes.

The following Examples illustrate the invention. Example 1: Characterisation of the sequence A neurotransmitter transporter polypeptide, designated as HIPHUM 0000029 has been identified. The nucleotide and amino acid sequences of the polypeptide have been determined. These are set out below in SEQ ID NOs: l and 2. HIPHUM

0000029 has been mapped to a 6cM interval (D 1 S248-D 1 S2726) on chromosome 1p21. The closest known homologue to HIPHUM 0000029 is rat NTT4, which shares 96 % amino acid identity.

Example 2: Screening for substances which exhibit protein modulating activity For the identification of compounds which modulate transporters, two types of assays are typically being utilised: binding assays with the radiolabeled substrates as ligands and functional uptake assays using these radiolabeled substrates.

In a typical binding assay, the radiolabeled substrate (eg. tritiated substrate) is being incubated with a membrane preparation containing the transporter protein (either recombinant or native) in a suitable incubation buffer (typically in microtiter plates). After a period of incubation (typically between 30 min and 2hrs) total bound radiolabeled substrate is separated from unbound substrate by means of filtration or any other kind of suitable separation procedure. Nonspecific binding is usually being detected by adding a more than 100-fold excess of unlabeled substrate in parallel experiments. Radioactivity is measured using standard measurement equipment for radioactivity. Specific transporter binding is typically calculated as the difference between total binding minus nonspecific binding. When these kind of experiments are carried out in the presence of compounds to be tested (screening), transportermodulating properties of such compounds are typically revealed by their radioligand displacing activities. Ki and 'C50 values can be calculated in order to analyse their potency.

In a typical functional assay, the uptake of a radiolabeled substrate (eg. tritiated substrate) into an appropriate cellular system is being analysed. Such a cellular system may be a natural system in form of a synaptosomal preparation (for instance) or a recombinant system such as a recombinant cell line expressing the transporter under study. Typically, such a cellular system is incubated with the radiolabeled substrate for a short period (eg. 5 min) in a suitable incubation buffer (typically in microtiter plates). Subsequently, the incubation buffer is removed from the cells and cells are lysed by the addition of a lysing agent such as a detergent. Radioactivity of the cell lysates is then analysed using standard techniques. The amount of radioactivity detected in the cell lysate is a measure of the transport activity. If these kind of experiments are carried out in the presence of compounds to

be tested (screening), uptake inhibitors are identified by virtue of their uptake reducing capacity. On the other hand, uptake stimulators may be identified by their uptake increasing capacity. Respective potencies of such compounds can be calculated and expressed as 'C50 values for instance.

SEQUENCE LISTING < 110> GLAXO GROUP LIMITED < 120> NEW POLYPEPTIDE < 130> P79076 SER < 140> < 141> < 160> 2 < 170> PatentIn Ver. 2.1 < 210> 1 < 211> 2258 < 212> DNA < 213> Homo sapiens < 220> < 221> CDS < 222> (71)..(2254) < 400> 1 agctgacagc agctgaattc catcttctct gtgtgctggg gagcagggct acacggccca 60 ggtggcatca atg ccg aag aac agc aaa gtg acc cag cgt gaa cac agc 109 Met Pro Lys Asn Ser Lys Val Thr Gln Arg Glu His Ser 1 5 10 agt gag cat gtc act gag tcc gtg gcc gac ctg ctg gcc ctc gag gag 157 Ser Glu His Val Thr Glu Ser Val Ala Asp Leu Leu Ala Leu Glu Glu 15 20 25 cct gtg gac tat aag cag agt gta ctg aat gtg get ggt gag gca ggc 205 Pro Val Asp Tyr Lys Gln Ser Val Leu Asn Val Ala Gly Glu Ala Gly 30 35 40 45 ggc aag cag aag gcg ttg gag gag gag ctg gat gca gag gac cgg ccg 253 Gly Lys Gln Lys Ala Leu Glu Glu Glu Leu Asp Ala Glu Asp Arg Pro 50 55 60 gcc tgg aac agt aag ctg cag tac atc ctg gcc cag att ggc ttc tct 301 Ala Trp Asn Ser Lys Leu Gln Tyr Ile Leu Ala Gln Ile Gly Phe Ser 65 70 75 gtg ggc ctc ggc aac atc tgg agg ttc ccc tac ctg tgc cag aaa aat 349 Val Gly Leu Gly Asn Ile Trp Arg Phe Pro Tyr Leu Cys Gln Lys Asn 80 85 90 gga gga ggt get tac ctg gtg ccc tac ctg gtg ctg ctg atc atc atc 397 Gly Gly Gly Ala Tyr Leu Val Pro Tyr Leu Val Leu Leu Ile Ile Ile 95 100 105

G3 ggg atc ccc ctc ttc ttc ctg gag ctg get gtg ggt cag agg atc cgc 445 Gly Ile Pro Leu Phe Phe Leu Glu Leu Ala Val Gly Gln Arg Ile Arg 110 115 120 125 cgc ggc agc atc ggt gtg tgg cac tat ata tgt ccc cgc ctg ggg ggc 493 Arg Gly Ser Ile Gly Val Trp His Tyr Ile Cys Pro Arg Leu Gly Gly 130 135 140 atc ggc ttc tcc agc tgc ata gtc tgt ctc ttt gtg ggg ctg tat tat 541 Ile Gly Phe Ser Ser Cys Ile Val Cys Leu Phe Val Gly Leu Tyr Tyr 145 150 155 aat gtg atc atc ggg tgg agc atc ttc tat ttc ttc aag tcc ttc cag 589 Asn Val Ile Ile Gly Trp Ser Ile Phe Tyr Phe Phe Lys Ser Phe Gln 160 165 170 tac ccg ctg ccc tgg agt gaa tgt cct gtc gtc agg aat ggg agc gtg 637 Tyr Pro Leu Pro Trp Ser Glu Cys Pro Val Val Arg Asn Gly Ser Val 175 180 185 gca gtg gtg gag gca gag tgt gaa aag agc tca gcc act acc tac ttc 685 Ala Val Val Glu Ala Glu Cys Glu Lys Ser Ser Ala Thr Thr Tyr Phe 190 195 200 205 tgg tac cga gag gcc ttg gac atc tct gac tcc atc tcg gag agt ggg 733 Trp Tyr Arg Glu Ala Leu Asp Ile Ser Asp Ser Ile Ser Glu Ser Gly 210 215 220 ggc ctc aac tgg aag atg acc ctg tgc ctc ctc gtg gcc tgg agc atc 781 Gly Leu Asn Trp Lys Met Thr Leu Cys Leu Leu Val Ala Trp Ser Ile 225 230 235 gtg ggg atg get gtc gtt aag ggc atc cag tcc tcg ggg aag gtg atg 829 Val Gly Met Ala Val Val Lys Gly Ile Gln Ser Ser Gly Lys Val Met 240 245 250 tat ttc agc tcc ctc ttc ccc tac gtg gtg ctg gcc tgc ttc ctg gtc 877 Tyr Phe Ser Ser Leu Phe Pro Tyr Val Val Leu Ala Cys Phe Leu Val 255 260 265 cgg ggg ctg ttg ctg cga ggg gca gtt gat ggc atc cta cac atg ttc 925 Arg Gly Leu Leu Leu Arg Gly Ala Val Asp Gly Ile Leu His Met Phe 270 275 280 285 act ccc aag ctg gac aag atg ctg gac ccc cag gtg tgg cgg gag gca 973 Thr Pro Lys Leu Asp Lys Met Leu Asp Pro Gln Val Trp Arg Glu Ala 290 295 300 get acc cag gtc ttc ttt gcc ttg ggc ctg ggc ttt ggt ggt gtc att 1021 Ala Thr Gln Val Phe Phe Ala Leu Gly Leu Gly Phe Gly Gly Val Ile 305 310 315 gcc ttc tcc agc tac aat aag cag gac aac aac tgc cac ttc gat gcc 1069 Ala Phe Ser Ser Tyr Asn Lys Gln Asp Asn Asn Cys His Phe Asp Ala 320 325 330

gcc ctg gtg tcc ttc atc aac ttc ttc acg tca gtg ttg gcc acc ctc 1117 Ala Leu Val Ser Phe Ile Asn Phe Phe Thr Ser Val Leu Ala Thr Leu 335 340 345 gtg gtg ttt get gtg ctg ggc ttc aag gcc aac atc atg aat gag aag 1165 Val Val Phe Ala Val Leu Gly Phe Lys Ala Asn Ile Met Asn Glu Lys 350 355 360 365 tgt gtg gtc gag aat get gag aaa atc cta ggg tac ctt aac acc aac 1213 Cys Val Val Glu Asn Ala Glu Lys Ile Leu Gly Tyr Leu Asn Thr Asn 370 375 380 gtc ctg agc cgg gac ctc atc cca ccc cac gtc aac ttc tcc cac ctg 1261 Val Leu Ser Arg Asp Leu Ile Pro Pro His Val Asn Phe Ser His Leu 385 390 395 acc aca aag gac tac atg gag atg tac aat gtc atc atg acc gtg aag 1309 Thr Thr Lys Asp Tyr Met Glu Met Tyr Asn Val Ile Met Thr Val Lys 400 405 410 gag gac cag ttc tca gcc ctg ggc ctt gac ccc tgc ctt ctg gag gac 1357 Glu Asp Gln Phe Ser Ala Leu Gly Leu Asp Pro Cys Leu Leu Glu Asp 415 420 425 gag ctg gac aag tcc gtg cag ggc aca ggc ctg gcc ttc atc gcc ttc 1405 Glu Leu Asp Lys Ser Val Gln Gly Thr Gly Leu Ala Phe Ile Ala Phe 430 435 440 445 act gag gcc atg acg cac ttc ccc gcc tcc ccg ttc tgg tcc gtc atg 1453 Thr Glu Ala Met Thr His Phe Pro Ala Ser Pro Phe Trp Ser Val Met 450 455 460 ttc ttc ttg atg ctt atc aac ctg ggc ctg ggc agc atg atc ggg acc 1501 Phe Phe Leu Met Leu Ile Asn Leu Gly Leu Gly Ser Met Ile Gly Thr 465 470 475 atg gca ggc atc acc acg ccc atc atc gac acc ttc aag gtg ccc aag 1549 Met Ala Gly Ile Thr Thr Pro Ile Ile Asp Thr Phe Lys Val Pro Lys 480 485 490 gag atg ttc aca gtg ggc tgc tgt gtc ttt gca ttc ctc gtg ggg ctg 1597 Glu Met Phe Thr Val Gly Cys Cys Val Phe Ala Phe Leu Val Gly Leu 495 500 505 ttg ttc gtc cag cgc tcc gga aac tac ttt gtc acc atg ttc gat gac 1645 Leu Phe Val Gln Arg Ser Gly Asn Tyr Phe Val Thr Met Phe Asp Asp 510 515 520 525 tac tcg gcc acc ctg cca ctc act ctc atc gtc atc ctt gag aac atc 1693 Tyr Ser Ala Thr Leu Pro Leu Thr Leu Ile Val Ile Leu Glu Asn Ile 530 535 540 get gtg gcc tgg att tat gga acc aag agg ttc atg cag gag ctg acg 1741 Ala Val Ala Trp Ile Tyr Gly Thr Lys Arg Phe Met Gln Glu Leu Thr 545 550 555

gag atg ctg ggc ttc cgc ccc tac cgc ttc tat ttc tac atg tgg aag 1789 Glu Met Leu Gly Phe Arg Pro Tyr Arg Phe Tyr Phe Tyr Met Trp Lys 560 565 570 ttc gtg tct cca cta tgc atg get gtg ctc acc aca gcc agc atc atc 1837 Phe Val Ser Pro Leu Cys Met Ala Val Leu Thr Thr Al a Ser Ile Ile 575 580 585 cag ctg ggg gtc acg ccc ccg ggc tac agc gcc tgg atc aag gag gag 1885 Gln Leu Gly Val Thr Pro Pro Gly Tyr Ser Ala Trp Ile Lys Glu Glu 590 595 600 605 get gcc gag cgc tac ctg tat ttc ccc aac tgg gcc atg gca ctc ctg 1933 Ala Ala Glu Arg Tyr Leu Tyr Phe Pro Asn Trp Ala Met Ala Leu Leu 610 615 620 atc acc ctc atc gtc gtg gcg acg ctg ccc atc cct gtg gtg ttc gtc 1981 Ile Thr Leu Ile Val Val Ala Thr Leu Pro Ile Pro Val Val Phe Val 625 630 635 ctg cgg cac ttc cac ctg ctc tct gat ggc tcc aac acc ctc tcc gtg 2029 Leu Arg His Phe His Leu Leu Ser Asp Gly Ser Asn Thr Leu Ser Val 640 645 650 tcc tac aag aag ggc cgc atg atg aag gac atc tcc aac ctg gag gag 2077 Ser Tyr Lys Lys Gly Arg Met Met Lys Asp Ile Ser Asn Leu Glu Glu 655 660 665 aac gat gag acc cgc ttc atc ctc agc aag gtg ccc agt gag gca cct 2125 Asn Asp Glu Thr Arg Phe Ile Leu Ser Lys Val Pro Ser Glu Ala Pro 670 675 680 685 tcc ccc atg ccc act cac cgt tcc tat ctg ggg ccc ggc agc aca tca 2173 Ser Pro Met Pro Thr His Arg Ser Tyr Leu Gly Pro Gly Ser Thr Ser 690 695 700 ccc ctg gag acc agc ggt aac ccc aat gga cgc tat ggg agc ggc tac 2221 Pro Leu Glu Thr Ser Gly Asn Pro Asn Gly Arg Tyr Gly Ser Gly Tyr 705 710 715 ctg ctg gcc agc acc cct gag tcg gag ctg tga ccac 2258 Leu Leu Ala Ser Thr Pro Glu Ser Glu Leu 720 725 < 210> 2 < 211> 727 < 212> PRT < 213> Homo sapiens < 400> 2 Met Pro Lys Asn Ser Lys Val Thr Gln Arg Glu His Ser Ser Glu His 1 5 10 15 Val Thr Glu Ser Val Ala Asp Leu Leu Ala Leu Glu Glu Pro Val Asp 20 25 30

Tyr Lys Gln Ser Val Leu Asn Val Ala Gly Glu Ala Gly Gly Lys Gln 35 40 45 Lys Ala Leu Glu Glu Glu Leu Asp Ala Glu Asp Arg Pro Ala Trp Asn 50 55 60 Ser Lys Leu Gln Tyr Ile Leu Ala Gln Ile Gly Phe Ser Val Gly Leu 65 70 75 80 Gly Asn Ile Trp Arg Phe Pro Tyr Leu Cys Gln Lys Asn Gly Gly Gly 85 90 95 Ala Tyr Leu Val Pro Tyr Leu Val Leu Leu Ile Ile Ile Gly Ile Pro 100 105 110 Leu Phe Phe Leu Glu Leu Ala Val Gly Gln Arg Ile Arg Arg Gly Ser 115 120 125 Ile Gly Val Trp His Tyr Ile Cys Pro Arg Leu Gly Gly Ile Gly Phe 130 135 140 Ser Ser Cys Ile Val Cys Leu Phe Val Gly Leu Tyr Tyr Asn Val Ile 145 150 155 160 Ile Gly Trp Ser Ile Phe Tyr Phe Phe Lys Ser Phe Gln Tyr Pro Leu 165 170 175 Pro Trp Ser Glu Cys Pro Val Val Arg Asn Gly Ser Val Ala Val Val 180 185 190 Glu Ala Glu Cys Glu Lys Ser Ser Ala Thr Thr Tyr Phe Trp Tyr Arg 195 200 205 Glu Ala Leu Asp Ile Ser Asp Ser Ile Ser Glu Ser Gly Gly Leu Asn 210 215 220 Trp Lys Met Thr Leu Cys Leu Leu Val Ala Trp Ser Ile Val Gly Met 225 230 235 240 Ala Val Val Lys Gly Ile Gln Ser Ser Gly Lys Val Met Tyr Phe Ser 245 250 255 Ser Leu Phe Pro Tyr Val Val Leu Ala Cys Phe Leu Val Arg Gly Leu 260 265 270 Leu Leu Arg Gly Ala Val Asp Gly Ile Leu His Met Phe Thr Pro Lys 275 280 285 Leu Asp Lys Met Leu Asp Pro Gln Val Trp Arg Glu Ala Ala Thr Gln 290 295 300 Val Phe Phe Ala Leu Gly Leu Gly Phe Gly Gly Val Ile Ala Phe Ser 305 310 315 320 Ser Tyr Asn Lys Gln Asp Asn Asn Cys His Phe Asp Ala Ala Leu Val 325 330 335

Ser Phe Ile Asn Phe Phe Thr Ser Val Leu Ala Thr Leu Val Val Phe 340 345 350 Ala Val Leu Gly Phe Lys Ala Asn Ile Met Asn Glu Lys Cys Val Val 355 360 365 Glu Asn Ala Glu Lys Ile Leu Gly Tyr Leu Asn Thr Asn Val Leu Ser 370 375 380 Arg Asp Leu Ile Pro Pro His Val Asn Phe Ser His Leu Thr Thr Lys 385 390 395 400 Asp Tyr Met Glu Met Tyr Asn Val Ile Met Thr Val Lys Glu Asp Gln 405 410 415 Phe Ser Ala Leu Gly Leu Asp Pro Cys Leu Leu Glu Asp Glu Leu Asp 420 425 430 Lys Ser Val Gln Gly Thr Gly Leu Ala Phe Ile Ala Phe Thr Glu Ala 435 440 445 Met Thr His Phe Pro Ala Ser Pro Phe Trp Ser Val Met Phe Phe Leu 450 455 460 Met Leu Ile Asn Leu Gly Leu Gly Ser Met Ile Gly Thr Met Ala Gly 465 470 475 480 Ile Thr Thr Pro Ile Ile Asp Thr Phe Lys Val Pro Lys Glu Met Phe 485 490 495 Thr Val Gly Cys Cys Val Phe Ala Phe Leu Val Gly Leu Leu Phe Val 500 505 510 Gln Arg Ser Gly Asn Tyr Phe Val Thr Met Phe Asp Asp Tyr Ser Ala 515 520 525 Thr Leu Pro Leu Thr Leu Ile Val Ile Leu Glu Asn Ile Ala Val Ala 530 535 540 Trp Ile Tyr Gly Thr Lys Arg Phe Met Gln Glu Leu Thr Glu Met Leu 545 550 555 560 Gly Phe Arg Pro Tyr Arg Phe Tyr Phe Tyr Met Trp Lys Phe Val Ser 565 570 575 Pro Leu Cys Met Ala Val Leu Thr Thr Al a Ser Ile Ile Gln Leu Gly 580 585 590 Val Thr Pro Pro Gly Tyr Ser Ala Trp Ile Lys Glu Glu Ala Ala Glu 595 600 605 Arg Tyr Leu Tyr Phe Pro Asn Trp Ala Met Ala Leu Leu Ile Thr Leu 610 615 620 Ile Val Val Ala Thr Leu Pro Ile Pro Val Val Phe Val Leu Arg His 625 630 635 640

Phe His Leu Leu Ser Asp Gly Ser Asn Thr Leu Ser Val Ser Tyr Lys 645 650 655 Lys Gly Arg Met Met Lys Asp Ile Ser Asn Leu Glu Glu Asn Asp Glu 660 665 670 Thr Arg Phe Ile Leu Ser Lys Val Pro Ser Glu Ala Pro Ser Pro Met 675 680 685 Pro Thr His Arg Ser Tyr Leu Gly Pro Gly Ser Thr Ser Pro Leu Glu 690 695 700 Thr Ser Gly Asn Pro Asn Gly Arg Tyr Gly Ser Gly Tyr Leu Leu Ala 705 710 715 720 Ser Thr Pro Glu Ser Glu Leu 725

Claims

CLAIMS 1. An isolated neurotransmitter transporter polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (ii) a variant thereof which has sodium:neurotransmitter symporter activity or (iii) a fragment of (i) or (ii) which has sodium:neurotransmitter symporter activity.
2. A polypeptide according to claim 1 wherein the variant (ii) has at least 80% identity to the amino acid sequence of SEQ ID NO: 2.
3. A polynucleotide encoding a polypeptide according to claim 1 or 2.
4. A polynucleotide according to claim 3 which is a cDNA sequence.
5. A polynucleotide encoding a neurotransmitter transporter polypeptide which has sodium:neurotransmitter symporter activity which polynucleotide comprises: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto; . (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c).
6. An expression vector comprising a polynucleotide according to any one of claims 3 to 5.
7. A host cell comprising an expression vector according to claim 6.
8. An antibody specific for a polypeptide according to claim 1 or 2.
9. A method for the identification of a substance that modulates neurotransmitter transporter polypeptide activity and/or expression, which method comprises: (i) contacting a test substance and a polypeptide according to claim 1 or 2, a polynucleotide according to any one of claims 3 to 5, an expression vector according to claim 6 or a host cell according to

<Desc/Clms Page number 30>

claim 7, and (ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by said polynucleotide, thereby to determine whether the test substance modulates neurotransmitter transporter polypeptide activity and/or expression.
10. A method according to claim 9 wherein the polypeptide is expressed in a cell.
11. A substance which modulates neurotransmitter transporter polypeptide activity and which is identifiable by a method according to claim 9 or 10.
12. A method of treating a subject having a disorder that is responsive to neurotransmitter transporter polypeptide modulation, which method comprises administering to said subject an effective amount of a substance according to claim 11.
13. A method according to claim 12 wherein the disorder is selected from psychiatric disorders, bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.
14. Use of a substance as defined in claim 11 in the manufacture of a medicament for treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of neurotransmitter transporter polypeptide activity.
15. A use according to claim 14 wherein the disorder is selected from psychiatric disorders, bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.
16. A method of producing a polypeptide according to claim 1 or 2, which method comprises maintaining a host cell as defined in claim 7 under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide.