GB2371800A - Amino acid transporter polypeptides - Google Patents

Abstract

An isolated amino acid transporter polypeptide comprising <SL> <LI>(i) the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or <LI>(ii) a variant of (i) which has amino acid transporter activity or <LI>(iii) a fragment of (i) or (ii) which has amino acid transporter activity. </SL> The polypetide referred to as HIPHUM0000141 is expressed primarily in kidney and skeletal muscle and also in parts of the brain. Vectors and host cells expressing the polynucleotide and antibodies specific for the polypeptide are also claimed. The polypeptide may be a screening target for the identification of pharmaceutical agents that may be used in the treatment of psychiatric disorders, anxiety, schizophrenia and drug dependence.

Description

NOVEL PROTEIN Field of the Invention The present invention relates to amino acid 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+/CI-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 NelCl-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+/Cl- -dependent carrier family. Hydropathic analysis of the primary sequences of vesicular monoamine and acetylcholine transporters predict 12 putative transmembrane segments, while vesicular inhibitory amino acid transporters predict 10 transmembrane domains.

The D. melanogaster gene Cl 13384 encodes a product with the function of transporter. This fly gene is located on arm 2L and has a transcription unit length of 3635 bp. This gene has three transcripts. The deduced amino acid sequence contains a transmembrane region which is found in many amino acid transporters, including UNC-47. Other members of this family include proline transporters and amino acid permeases.

Summary of the Invention A novel amino acid transporter polypeptide, referred to herein as HIPHUM 0000141, is now provided. HIPHUM 0000141 is shown to be primarily expressed in kidney and skeletal muscle. The polypeptide is also shown to be expressed in the following areas of the brain: substantia nigra, amygdala and thalamus. The novel amino acid transporter polypeptide is a screening target for the identification and development of novel pharmaceutical agents, including modulators of amino acid transporter polypeptide activity. These agents may be used in the treatment and/or prophylaxis of disorders such as psychiatric disorders, including bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.

Accordingly, the present invention provides an isolated amino acid transporter polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; (ii) a variant of (i) which has amino acid transporter activity; or (iii) a fragment of (i) or (ii) which has amino acid transporter 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 or SEQ ID NO: 3 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 amino acid 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 amino acid transporter activity and/or expression; a compound which or modulates amino acid 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 amino acid 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 amino acid transporter activity in the manufacture of a medicament for the treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of amino acid transporter activity.

Preferably the disorder is selected from psychiatric disorders, including 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 0000141.

SEQ ID NO: 2 is the amino acid sequence alone of HIPHUM 0000141.

SEQ ID NO: 3 shows the nucleotide and amino acid sequence of a splice variant of HIPHUM 0000141.

SEQ ID NO: 4 shows the amino acid sequence alone of a splice variant HIPHUM 0000141.

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 amino acid transporter polypeptide, referred to herein as HIPHUM 0000141, and variants thereof Sequence information for HIPHUM 0000141 and a splice variant thereof is provided in SEQ ID NO: 1 and SEQ ID NO: 3 (nucleotide and amino acid) and in SEQ ID NO: 2 and SEQ ID NO: 4. A polypeptide of the invention thus consists essentially of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 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'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 0000141. The essential character of HIPHUM 0000141 can be defined as follows: HIPHUM 0000141 is a amino acid transporter polypeptide. Preferably a variant polypeptide is one which binds to the same ligand or substrate as HIPHUM 0000141. Preferably the polypeptide has amino acid transporter activity. A polypeptide having a same essential character as HIPHUM 0000141 be identified by monitoring for a function of the amino acid transporter selected from neurotransmitter transporter activity, and in particular amino acid transporter activity. A full length variant preferably includes a transmembrane region associated with transport of amino acids. A variant transporter may be identified by looking for substrate binding or transport, in particular of amino acids. A variant may be a splice variant..

Typically, polypeptides with more than about 65% identity preferably at least 80% or at least 90% and particularly preferably at least 95% at least 97% or at least 99% identity, with the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 4, 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 0000141 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 amino acid 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 CSTM NQ Polar-charged DE 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 0000141. 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 ligandbinding 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 amino acid transporter polypeptide, more preferably with another member of the family of amino acid transporter polypeptides.

Such fragments of HIPHUM 0000141 or a variant thereof can also be used to raise anti-HIPHUM 0000141 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM 0000141 polypeptide and may otherwise not demonstrate the substrate or ligand binding or other properties of HIPHUM 0000141.

Polypeptides of the invention may be chemically modified, e. g. posttranslationally 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 0000141 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 and SEQ ID NO: 3. 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: I or SEQ ID NO: 3.

A polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA 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 or SEQ ID NO: 3 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 or SEQ ID NO: 3. The intensity of interaction may be measured, for example, by radiolabelling the probe, e. g. with 32p. 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 oC. If lower stringency is required suitable conditions include 2 x SSC at 60 oc.

The coding sequence of SEQ ID NO : I or SEQ ID NO : 3 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 or SEQ ID NO: 3 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 0000141 polypeptide activity. Alternatively, a polynucleotide encodes a ligand-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM 0000141 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 or SEQ ID NO: 3 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 or SEQ ID NO: 3 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 or SEQ ID NO: 3.

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 (1984) 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 (1990) J. Mol. Biol. 215: 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 (1992) Proc. Natl. Acad.

Sci. USA 89: 10915-10919) 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. Natl. 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 0000141, 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: I or SEQ ID NO: 3.

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 -actin promoters, may be used. Tissuespecific 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 0000141 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 a mouse. A transgenic nonhuman 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: 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 0000141 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 (1988)"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

64 "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 amino acid transporter polypeptides and in particular which bind to HIPHUM 0000141 such as a ligand or substrate for the polypeptide. Screening methods may also be used to identify agonists or antagonists which may modulate amino acid transporter polypeptide activity, inhibitors or activators of HIPHUM 0000141 activity, and/or agents which up-regulate or downregulate HIPHUM 0000141 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 amino acid 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 0000141, and incubating such cells with the test substance optionally in the presence of HIPHUM 0000141 ligand. Alternatively an antibody may be used to complex HIPHUM 0000141 and thus mediate HIPHUM 0000141 activity. Test substances may then be added to assess the effect on such activity. Cells expressing HIPHUM 0000141 constitutively may be provided for use in assays for HIPHUM 0000141 function.

Additional test substances may be introduced in any assay to look for inhibitors of ligand or substrate binding or inhibitors of HIPHUM 0000141-mediated activity.

Assays may also be carried out to identify substances which modify HIPHUM 0000141 polypeptide expression, for example substances which up-or down-regulate expression. Such assays may be carried out for example by using

antibodies for HIPHUM 0000141 to monitor levels of HIPHUM 0000141 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM 0000141 expression include using a reporter gene construct driven by the HIPHUM 0000141 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 0000141 polypeptide either in lavis 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 amino acid 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 amino acid transporter agonist or amino acid 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 InM to 1000uM, preferably from luM to 1OOM, more preferably from I gM

to lOuM. 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 0000141 polypeptides of the invention to identify mutations in HIPHUM 0000141 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 0000141 expression. Polynucleotides such as SEQ ID NO: 1 or SEQ ID NO: 3 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 0000141 genes. This may comprise determining the level of an HIPHUM 0000141 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, "PCRIn 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 0000141, and qualitative aspects of HIPHUM 0000141 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM 0000141 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: 189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87: 18741878), 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 0000141 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM 0000141 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 amino acid 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, including 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, 17th 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 0000141 or a variant thereof which inhibits HIPHUM 0000141 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 Ipg to 1 mg, preferably to Ipg to I Oug nucleic acid for particle mediated gene delivery and long to Img for other routes.

The following Examples illustrate the invention.

Example 1 : Characterisation of the sequence A amino acid transporter polypeptide, designated as HIPHUM 0000141 has been identified. The nucleotide and amino acid sequences of the polypeptide have been determined. These are set out below in SEQ ID NOs: 1 and 2. A splice variant has also been identified having the nucleotide and amino acid sequences set out in SEQ ID NO's 3 and 4. Suitable primers and probes were designed and used to analyse tissue expression. HIPHUM 0000141 was found to be primarily expressed in kidney and skeletal muscle. The polypeptide is also shown to be expressed in the following areas of the brain: substantia nigra, amygdala and thalamus. This distribution pattern can be seen in figure 1, where the distribution has been compared with other brain specific proteins, and also housekeeping genes.

The chromosomal localization has been mapped to 5q32-q33.

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 ICso 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 lysats 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 ICo values for instance.

SEQUENCE LISTING < 110 > GLAXO GROUP LTD < 120 > NOVEL PROTEIN < 130 > P80199 < 140 > < 141 > < 160 > 4 < 170 > Patent In Ver. 2. 1 < 210 > 1 < 211 > 1619 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (80).. (1528) < 400 > 1 tacactagac accttcctgc ttccctcctt ccagagcaga cctctttgtc accccgagct 60 ccttgtttct taagcagtc atg tct gtg aca aaa agt act gag ggt ccc cag 112 Met Ser Val Thr Lys Ser Thr Glu Gly Pro Gln 1 5 10 gga gcc gtt gcc ate aaa ttg gac ctt atg teg cct cct gaa agt gcc 160 Gly Ala Val Ala Ile Lys Leu Asp Leu Met Ser Pro Pro Glu Ser Ala 15 20 25 aag aag ttg gag aac aag gac tct aca ttc ttg gat gaa agt cct tea 208 Lys Lys Leu Glu Asn Lys Asp Ser Thr Phe Leu Asp Glu Ser Pro Ser 30 35 40 gag tea gca ggc ttg aag aag ace aag ggc ata aca gtg ttc cag gcc 256 Glu Ser Ala Gly Leu Lys Lys Thr Lys Gly Ile Thr Val Phe Gln Ala 45 50 55 ttg att cac ctg gtg aaa ggc aac atg ggc aca ggg ate ctg gga cta 304 Leu Ile His Leu Val Lys Gly Asn Met Gly Thr Gly Ile Leu Gly Leu 60 65 70 75 ccc etc get gtg aag aac gcg ggc ate ctg atg ggc cca etc agt ctg 352 Pro Leu Ala Val Lys Asn Ala Gly Ile Leu Met Gly Pro Leu Ser Leu 80 85 90 ctg gtg atg ggc ttc att gee tgc cac tgt atg cac ate ctg gtc aag 400 Leu Val Met Gly Phe Ile Ala Cys His Cys Met His Ile Leu Val Lys 95 100 105 tgt gcc cag cgc ttc tgt aag agg ctt aac aag ccc ttt atg gac tat 448 Cys Ala Gln Arg Phe Cys Lys Arg Leu Asn Lys Pro Phe Met Asp Tyr

110 115 120 ggg gac acg gtg atg cat gga cta gaa gcc aac ccc aac gcc tgg etc 496 Gly Asp Thr Val Met His Gly Leu Glu Ala Asn Pro Asn Ala Trp Leu 125 130 135 cag aat cac get cac tgg gga agg cat ate gtg age ttc ttc ctt att 544 Gln Asn His Ala His Trp Gly Arg His Ile Val Ser Phe Phe Leu Ile 140 145 150 155 ate ace caa ctt ggc ttc tgc tgt gtg tac att gtg ttt ttg get gat 592 Ile Thr Gln Leu Gly Phe Cys Cys Val Tyr Ile Val Phe Leu Ala Asp 160 165 170 aat tta aaa cag gta gtg gaa get gtt aat age aca ace aac aac tgc 640 Asn Leu Lys Gln Val Val Glu Ala Val Asn Ser Thr Thr Asn Asn Cys 175 180 185 tat tee aat gag acg gtg att ctg ace ccc ace atg gac teg cga etc 688 Tyr Ser Asn Glu Thr Val Ile Leu Thr Pro Thr Met Asp Ser Arg Leu 190 195 200 tac atg etc tee ttc ctg ccc ttc ctg gtg ctg ctg gtc etc ate egg 736 Tyr Met Leu Ser Phe Leu Pro Phe Leu Val Leu Leu Val Leu Ile Arg 205 210 215 aac etc agg ate ttg ace ate ttc tee atg ctg gcc aac ate age atg 784 Asn Leu Arg Ile Leu Thr Ile Phe Ser Met Leu Ala Asn He Ser Met 220 225 230 235 ctg gtc age ttg gtc ate ate ata cag tac att ace cag gaa ate cca 832 Leu Val Ser Leu Val Ile Ile Ile Gln Tyr Ile Thr Gln Glu Ile Pro 240 245 250 gac ccc age egg ttg cca ctg gta gca age tgg aag ace tac cct etc 880 Asp Pro Ser Arg Leu Pro Leu Val Ala Ser Trp Lys Thr Iyr Pro Leu 255 260 265 ttc ttc gga aca gcc att ttt tct ttt gaa age att ggt gtg gtt ctg 928 Phe Phe Gly Thr Ala Ile Phe Ser Phe Glu Ser Ile Gly Val Val Leu 270 275 280 cct ctg gaa aac aag atg aag aat gcc cgc cac ttc cca gcc ate ctg 976 Pro Leu Glu Asn Lys Met Lys Asn Ala Arg His Phe Pro Ala Ile Leu 285 290 295 tct ttg gga atg tee ate gtc act tee cta tac att ggc atg gcg get 1024 Ser Leu Gly Met Ser Ile Val Thr Ser Leu Tyr Ile Gly Met Ala Ala 300 305 310 315 ctg ggc tac ctg egg ttt gga gat gac ate aag gee age ata age ctt 1072 Leu Gly Tyr Leu Arg Phe Gly Asp Asp Ile Lys Ala Ser Ile Ser Leu 320 325 330 aac ctg cct aac tgc tgg ctg tac cag tct gtc aag ctt ctc tac att 1120 Asn Leu Pro Asn Cys Trp Leu Tyr Gln Ser Val Lys Leu Leu Tyr Ile 335 340 345

gcc ggc ate ctg tgc ace tat gcc ctg cag ttc tac gtc cct gca gaa 1168 Ala Gly Ile Leu Cys Thr Tyr Ala Leu Gln Phe Tyr Val Pro Ala Glu 350 355 360 ate ate ate ccc ttt gcc ate tee egg gtg tea aca cgc tgg gca ctg 1216 Ile Ile Ile Pro Phe Ala Ile Ser Arg Val Ser Thr Arg Trp Ala Leu 365 370 375 cct ctg gat ctg tee att cgc etc gtc atg gtc tgc ctg aca tgc etc 1264 Pro Leu Asp Leu Ser Ile Arg Leu Val Met Val Cys Leu Thr Cys Leu 380 385 390 395 ctg gcc ate etc ate ccc cgc ctg gac ctg gtc ate tee ctg gtg ggc 1312 Leu Ala Ile Leu Ile Pro Arg Leu Asp Leu Val Ile Ser Leu Val Gly 400 405 410 tee gtg agt ggc ace gcc ctg gcc etc ate ate cca ccg etc ctg gag 1360 Ser Val Ser Gly Thr Ala Leu Ala Leu Ile Ile Pro Pro Leu Leu Glu 415 420 425 gtc ace acg ttc tac tea gag ggc atg age ccc etc ace ate ttc aag 1408 Val Thr Thr Phe Tyr Ser Glu Gly Met Ser Pro Leu Thr Ile Phe Lys 430 435 440 gac gcc ctg ate age ate ctg ggc ttc gtg ggc ttt gtg gtg ggg ace 1456 Asp Ala Leu Ile Ser Ile Leu Gly Phe Val Gly Phe Val Val Gly Thr 445 450 455 tac cag gee ctg gac gag ctg etc aag tea gaa gac tct cac ccc ttt 1504 Tyr Gln Ala Leu Asp Glu Leu Leu Lys Ser Glu Asp Ser His Pro Phe 460 465 470 475 tee aac tee ace act ttt gtt egg tgagcctggc actgctcctt gcctaccagc 1558 Ser Asn Ser Thr Thr Phe Val Arg 480 acccgacttt taattatatg gatctctttt tttttttttt tttttttttg agacggagtt 1618 t 1619 < 210 > 2 < 211 > 483 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Ser Val Thr Lys Ser Thr Glu Gly Pro Gln Gly Ala Val Ala Ile 1 5 10 15 Lys Leu Asp Leu Met Ser Pro Pro Glu Ser Ala Lys Lys Leu Glu Asn 20 25 30 Lys Asp Ser Thr Phe Leu Asp Glu Ser Pro Ser Glu Ser Ala Gly Leu 35 40 45

Lys Lys Thr Lys Gly Ile Thr Val Phe Gln Ala Leu Ile His Leu Val 50 55 60 Lys Gly Asn Met Gly Thr Gly Ile Leu Gly Leu Pro Leu Ala Val Lys 65 70 75 80 Asn Ala Gly Ile Leu Met Gly Pro Leu Ser Leu Leu Val Met Gly Phe 85 90 95 Ile Ala Cys His Cys Met His Ile Leu Val Lys Cys Ala Gln Arg Phe 100 105 110 Cys Lys Arg Leu Asn Lys Pro Phe Met Asp Tyr Gly Asp Thr Val Met 115 120 125 His Gly Leu Glu Ala Asn Pro Asn Ala Trp Leu Gln Asn His Ala His 130 135 140

Trp Gly Arg His Ile Val Ser Phe Phe Leu Ile Ile Thr Gln Leu Gly 145 150 155 160 Phe Cys Cys Val Tyr Ile Val Phe Leu Ala Asp Asn Leu Lys Gln Val 165 170 175 Val Glu Ala Val Asn Ser Thr Thr Asn Asn Cys Tyr Ser Asn Glu Thr 180 185 190 Val Ile Leu Thr Pro Thr Met Asp Ser Arg Leu Tyr Met Leu Ser Phe 195 200 205 Leu Pro Phe Leu Val Leu Leu Val Leu Ile Arg Asn Leu Arg Ile Leu 210 215 220 Thr Ile Phe Ser Met Leu Ala Asn Ile Ser Met Leu Val Ser Leu Val 225 230 235 240

Ile Ile Ile Gln Tyr Ile Thr Gln Glu Ile Pro Asp Pro Ser Arg Leu 245 250 255 Pro Leu Val Ala Ser Trp Lys Thr Tyr Pro Leu Phe Phe Gly Thr Ala 260 265 270 Ile Phe Ser Phe Glu Ser Ile Gly Val Val Leu Pro Leu Glu Asn Lys 275 280 285 Met Lys Asn Ala Arg His Phe Pro Ala Ile Leu Ser Leu Gly Met Ser 290 295 300 Ile Val Thr Ser Leu Tyr Ile Gly Met Ala Ala Leu Gly Tyr Leu Arg 305 310 315 320 Phe Gly Asp Asp Ile Lys Ala Ser Ile Ser Leu Asn Leu Pro Asn Cys 325 330 335 Trp Leu Tyr Gln Ser Val Lys Leu Leu Tyr Ile Ala Gly Ile Leu Cys 340 345 350

Thr Tyr Ala Leu Gln Phe Tyr Val Pro Ala Glu Ile Ile Ile Pro Phe 355 360 365 Ala Ile Ser Arg Val Ser Thr Arg Trp Ala Leu Pro Leu Asp Leu Ser 370 375 380 Ile Arg Leu Val Met Val Cys Leu Thr Cys Leu Leu Ala Ile Leu Ile 385 390 395 400 Pro Arg Leu Asp Leu Val Ile Ser Leu Val Gly Ser Val Ser Gly Thr 405 410 415 Ala Leu Ala Leu Ile Ile Pro Pro Leu Leu Glu Val Thr Thr Phe Tyr 420 425 430 Ser Glu Gly Met Ser Pro Leu Thr Ile Phe Lys Asp Ala Leu Ile Ser 435 440 445

Ile Leu Gly Phe Val Gly Phe Val Val Gly Thr Tyr Gln Ala Leu Asp 450 455 460 Glu Leu Leu Lys Ser Glu Asp Ser His Pro Phe Ser Asn Ser Thr Thr 465 470 475 480 Phe Val Arg < 210 > 3 < 211 > 1520 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (80).. (1429) < 400 > 3

tacactagac accttcctgc ttccctcctt ccagagcaga cctctttgtc accccgagct 60 ccttgtttct taagcagtc atg tct gtg aca aaa agt act gag ggt ccc cag 112 Met Ser Val Thr Lys Ser Thr Glu Gly Pro Gln 1 5 10 gga gcc gtt gcc ate aaa ttg gac ctt atg teg cct cct gaa agt gcc 160 Gly Ala Val Ala Ile Lys Leu Asp Leu Met Ser Pro Pro Glu Ser Ala 15 20 25 aag aag ttg gag aac aag gac tct aca ttc ttg gat gaa agt cct tea 208 Lys Lys Leu Glu Asn Lys Asp Ser Thr Phe Leu Asp Glu Ser Pro Ser 30 35 40 gag tea gca ggc ttg aag aag ace aag ggc ata aca gtg ttc cag gcc 256 Glu Ser Ala Gly Leu Lys Lys Thr Lys Gly Ile Thr Val Phe Gln Ala 45 50 55 ttg att cac ctg gtg aaa ggc aac atg ggc aca ggg ate ctg gga cta 304 Leu Ile His Leu Val Lys Gly Asn Met Gly Thr Gly Ile Leu Gly Leu

60 65 70 75 ccc etc get gtg aag aac gcg ggc ate ctg atg ggc cca etc agt ctg 352 Pro Leu Ala Val Lys Asn Ala Gly Ile Leu Met Gly Pro Leu Ser Leu 80 85 90 ctg gtg atg ggc ttc att gcc tgc cac tgt atg cac ate ctg gtc aag 400 Leu Val Met Gly Phe Ile Ala Cys His Cys Met His Ile Leu Val Lys 95 100 105 tgt gcc cag cgc ttc tgt aag agg ctt aac aag ccc ttt atg gac tat 448 Cys Ala Gln Arg Phe Cys Lys Arg Leu Asn Lys Pro Phe Met Asp Tyr 110 115 120 ggg gac acg gtg atg cat gga cta gaa gcc aac ccc aac gcc tgg etc 496 Gly Asp Thr Val Met His Gly Leu Glu Ala Asn Pro Asn Ala Trp Leu 125 130 135 cag aat cac get cac tgg gga agg cat ate gtg age ttc ttc ctt att 544 Gln Asn His Ala His Trp Gly Arg His Ile Val Ser Phe Phe Leu Ile 140 145 150 155 ate ace caa ctt ggc ttc tgc tgt gtg tac att gtg ttt ttg get gat 592 Ile Thr Gln Leu Gly Phe Cys Cys Val Tyr Ile Val Phe Leu Ala Asp 160 165 170 aat tta aaa cag gta gtg gaa get gtt aat age aca ace aac aac tgc 640 Asn Leu Lys Gln Val Val Glu Ala Val Asn Ser Thr Thr Asn Asn Cys 175 180 185 tat tee aat gag acg gtg att ctg ace ccc ace atg gac teg cga etc 688 Tyr Ser Asn Glu Thr Val Ile Leu Thr Pro Thr Met Asp Ser Arg Leu 190 195 200 tac atg etc tee ttc ctg ccc ttc ctg gtg ctg ctg gtc etc ate egg 736 Tyr Met Leu Ser Phe Leu Pro Phe Leu Val Leu Leu Val Leu Ile Arg 205 210 215 aac etc agg ate ttg ace ate ttc tee atg ctg gcc aac ate age atg 784 Asn Leu Arg Ile Leu Thr Ile Phe Ser Met Leu Ala Asn Ile Ser Met 220 225 230 235 ctg gtc age ttg gtc ate ate ata cag tac att ace cag gtt ctg cct 832 Leu Val Ser Leu Val Ile Ile Ile Gln Tyr Ile Thr Gln Val Leu Pro 240 245 250 ctg gaa aac aag atg aag aat gcc cgc cac ttc cca gcc ate ctg tct 880 Leu Glu Asn Lys Met Lys Asn Ala Arg His Phe Pro Ala Ile Leu Ser 255 260 265 ttg gga atg tee ate gtc act tee cta tac att ggc atg gcg get ctg 928 Leu Gly Met Ser Ile Val Thr Ser Leu Tyr Ile Gly Met Ala Ala Leu 270 275 280 ggc tac ctg egg ttt gga gat gac ate aag gcc age ata age ctt aac 976 Gly Tyr Leu Arg Phe Gly Asp Asp Ile Lys Ala Ser Ile Ser Leu Asn 285 290 295

ctg cct aac tgc tgg ctg tac cag tct gtc aag ctt etc tac att gcc 1024 Leu Pro Asn Cys Trp Leu Tyr Gln Ser Val Lys Leu Leu Tyr Ile Ala 300 305 310 315 ggc ate ctg tgc ace tat gcc ctg cag ttc tac gtc cct gca gaa ate 1072 Gly Ile Leu Cys Thr Tyr Ala Leu Gln Phe Tyr Val Pro Ala Glu Ile 320 325 330 ate ate ccc ttt gcc ate tee egg gtg tea aca cgc tgg gca ctg cct 1120 Ile Ile Pro Phe Ala Ile Ser Arg Val Ser Thr Arg Trp Ala Leu Pro 335 340 345 ctg gat ctg tee att cgc etc gtc atg gtc tgc ctg aca tgc etc ctg 1168 Leu Asp Leu Ser Ile Arg Leu Val Met Val Cys Leu Thr Cys Leu Leu 350 355 360 gcc ate etc ate ccc cgc ctg gac ctg gtc ate tee ctg gtg ggc tee 1216 Ala Ile Leu Ile Pro Arg Leu Asp Leu Val Ile Ser Leu Val Gly Ser 365 370 375 gtg agt ggc ace gcc ctg gee etc ate ate cca ccg etc ctg gag gtc 1264 Val Ser Gly Thr Ala Leu Ala Leu Ile Ile Pro Pro Leu Leu Glu Val 380 385 390 395 ace acg ttc tac tea gag ggc atg age ccc etc ace ate ttc aag gac 1312 Thr Thr Phe Tyr Ser Glu Gly Met Ser Pro Leu Thr Ile Phe Lys Asp 400 405 410 gcc ctg ate age ate ctg ggc ttc gtg ggc ttt gtg gtg ggg ace tac 1360 Ala Leu Ile Ser Ile Leu Gly Phe Val Gly Phe Val Val Gly Thr Tyr 415 420 425 cag gcc ctg gac gag ctg etc aag tea gaa gac tct cac ccc ttt tee 1408 Gln Ala Leu Asp Glu Leu Leu Lys Ser Glu Asp Ser His Pro Phe Ser 430 435 440 aac tee ace act ttt gtt egg tgagcctggc actgctcctt gcctaccagc 1459 Asn Ser Thr Thr Phe Val Arg 445 450 acccgacttt taattatatg gatctctttt tttttttttt tttttttttg agacggagtt 1519 t 1520 < 210 > 4 < 211 > 450 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Met Ser Val Thr Lys Ser Thr Glu Gly Pro Gln Gly Ala Val Ala Ile 1 5 10 15 Lys Leu Asp Leu Met Ser Pro Pro Glu Ser Ala Lys Lys Leu Glu Asn 20 25 30

Lys Asp Ser Thr Phe Leu Asp Glu Ser Pro Ser Glu Ser Ala Gly Leu 35 40 45 Lys Lys Thr Lys Gly Ile Thr Val Phe Gln Ala Leu Ile His Leu Val 50 55 60 Lys Gly Asn Met Gly Thr Gly Ile Leu Gly Leu Pro Leu Ala Val Lys 65 70 75 80 Asn Ala Gly Ile Leu Met Gly Pro Leu Ser Leu Leu Val Met Gly Phe 85 90 95 Ile Ala Cys His Cys Met His Ile Leu Val Lys Cys Ala Gln Arg Phe 100 105 110 Cys Lys Arg Leu Asn Lys Pro Phe Met Asp Tyr Gly Asp Thr Val Met 115 120 125

His Gly Leu Glu Ala Asn Pro Asn Ala Trp Leu Gln Asn His Ala His 130 135 140 Trp Gly Arg His Ile Val Ser Phe Phe Leu Ile Ile Thr Gln Leu Gly 145 150 155 160 Phe Cys Cys Val Tyr Ile Val Phe Leu Ala Asp Asn Leu Lys Gln Val 165 170 175 Val Glu Ala Val Asn Ser Thr Thr Asn Asn Cys Tyr Ser Asn Glu Thr 180 185 190 Val Ile Leu Thr Pro Thr Met Asp Ser Arg Leu Tyr Met Leu Ser Phe 195 200 205 Leu Pro Phe Leu Val Leu Leu Val Leu Ile Arg Asn Leu Arg Ile Leu 210 215 220 Thr Ile Phe Ser Met Leu Ala Asn Ile Ser Met Leu Val Ser Leu Val 225 230 235 240

Ile Ile Ile Gln Tyr Ile Thr Gln Val Leu Pro Leu Glu Asn Lys Met 245 250 255 Lys Asn Ala Arg His Phe Pro Ala Ile Leu Ser Leu Gly Met Ser Ile 260 265 270 Val Thr Ser Leu Tyr Ile Gly Met Ala Ala Leu Gly Tyr Leu Arg Phe 275 280 285 Gly Asp Asp Ile Lys Ala Ser Ile Ser Leu Asn Leu Pro Asn Cys Trp 290 295 300 Leu Tyr Gln Ser Val Lys Leu Leu Tyr Ile Ala Gly Ile Leu Cys Thr

305 310 315 320 Tyr Ala Leu Gln Phe Tyr Val Pro Ala Glu Ile Ile Ile Pro Phe Ala 325 330 335

Ile Ser Arg Val Ser Thr Arg Trp Ala Leu Pro Leu Asp Leu Ser Ile 340 345 350 Arg Leu Val Met Val Cys Leu Thr Cys Leu Leu Ala Ile Leu Ile Pro 355 360 365

Arg Leu Asp Leu Val Ile Ser Leu Val Gly Ser Val Ser Gly Thr Ala 370 375 380 Leu Ala Leu Ile Ile Pro Pro Leu Leu Glu Val Thr Thr Phe Tyr Ser 385 390 395 400 Glu Gly Met Ser Pro Leu Thr Ile Phe Lys Asp Ala Leu Ile Ser Ile 405 410 415 Leu Gly Phe Val Gly Phe Val Val Gly Thr Tyr Gln Ala Leu Asp Glu 420 425 430 Leu Leu Lys Ser Glu Asp Ser His Pro Phe Ser Asn Ser Thr Thr Phe 435 440 445 Val Arg 450

Claims (16)

1. CLAIMS 1. An isolated amino acid transporter polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or (ii) a variant of (i) which has amino acid transporter activity or (iii) a fragment of (i) or (ii) which has amino acid transporter activity.
2. 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 or SEQ ID NO: 4.
3. 3. A polynucleotide encoding a polypeptide according to claim 1 or 2.
4. 4. A polynucleotide according to claim 3 which is a cDNA sequence.
5. 5. A polynucleotide encoding a amino acid transporter polypeptide which has amino acid transporter activity which polynucleotide comprises: (a) the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 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. 6. An expression vector comprising a polynucleotide according to any one of claims 3 to 5.
7. 7. A host cell comprising an expression vector according to claim 6.
8. 8. An antibody specific for a polypeptide according to claim 1 or 2.
9. 9. A method for the identification of a substance that modulates amino acid 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 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 amino acid transporter polypeptide activity and/or expression.
10. 10. A method according to claim 9 wherein the polypeptide is expressed in a cell.
11. 11. A substance which modulates amino acid transporter polypeptide activity and which is identifiable by a method according to claim 9 or 10.
12. 12. A method of treating a subject having a disorder that is responsive to amino acid transporter polypeptide modulation, which method comprises administering to said subject an effective amount of a substance according to claim

    11.
13. 13. A method according to claim 12 wherein the disorder is selected from psychiatric disorders, including bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.
14. 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 amino acid transporter polypeptide activity.
15. 15. A use according to claim 14 wherein the disorder is selected from psychiatric disorders, including bipolar disorders, unipolar depression, anxiety, schizophrenia, psychotic disorders and neurological/neurodegenerative disorders and drug dependence.
16. 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.