LIMBIC SYSTEM-ASSOCIATED MEMBRANE PROTEIN AND DNA The present invention is directed to nucleic acids encoding a limbic-system associated membrane protein ("LAMP") and to purified proteins with LAMP activity. LAMP is a self- binding, antibody-like cell surface adhesion protein, the presence of which on one neuron of the limbic system stimulates the formation of connections with adjacent neurons.

Part of the work performed during the development of this invention utilized United States Government Funds under National Institutes of Health Grant 5 R37 MH45507. The government has certain rights in the invention.

A monoclonal antibody, 2G9, that binds to the cell surface of ceπain neurons, mostly limbic system or limbic system-connected neurons is known. Levitt, Science 223:299-301, 1984. This antibody has been confirmed to be specific for LAMP. Explanted neuroris of the septum express the 2G9 antigen and, in culture, normally will invade explanted tissue from the hippocampus. In 1989, it was reported that 2G9 blocked that invasive process. Keller et al.. Neuron 3:551-561, 1989. Efforts were made to clone the antigen using 2G9. In 1989, those efforts led to the submission to the Society of Neuroscience of an erroneous abstract reporting that the antigen had been cloned. Efforts to clone the antigen using the antibody have proved unsuccessful.

Immunochemical studies indicate that the antigen is a 64-68 kDa glycoprotein that is expressed by cortical and subcortical neurons of the limbic system. These brain areas form functional circuits involved in memory, learning, mood, affect, cognitive behavior and central autonomic regulation. The antigen is also expressed by neurons having interconnections with limbic brain areas. Early in development, the antigen is expressed on limbic-related neurons. During pathway formation and differentiation, the antigen is expressed transiently on neural growth cones and axons. Immunochemical data indicate that the antigen, now designated LAMP, is a cell-surface adhesion molecule involved in directing the growth and differentiation of certain neurons. LAMP, when purified using the monoclonal antibody and coated onto tissue culture plates, induces adhesion and the growth of neuritic processes of certain limbic neurons, but generally not of non-limbic neurons. Summary of the Invention

Despite sustained effoπs to isolate the gene for the 2G9 antigen, it is only now that the nucleic acid encoding LAMP has been identified. It is herein designated SEQ ID NO: l for the human nucleic acid corresponding to amino acids 8-332 and SEQ ID NO:2 for the entire protein-encoding rat nucleic acid. The portions of SEQ ID NO: 2 encompassing only the open

reading frame are designated SEQ ID NO:4. These nucleic acids encode all or part of polypeptides of 338 amino acids and indicate that the protein is highly conserved, since the human and rat protein sequences differ in only four amino acid residues (approximately 99% homologous). As indicated in Figure 2, the sequence data indicate that LAMP has a three domain immunoglobin structure. Studies with phosphatidyl inositol ("PI") specific phospholipase C indicate that LAMP is bound to the cell membrane via a PI linkage. Thus, LAMP is much like other adhesion molecules of the immunoglobin superfamily that are attached to the cell surface by a PI linkage, such as TAG-1, Thy-1 and a form of NCAM. LAMP differs from these adhesion molecules in the specificity of its target tissues. The immunoglobulin domains of LAMP make up the binding or self-adhesion domain of LAMP.

In a first embodiment, the invention relates to a substantially pure nucleic acid ^' comprising a nucleic acid encoding a polypeptide having at least about 90% homology to a LAMP self-binding domain; the same substantially pure nucleic acid, wherein the LAMP self- binding domain is from a mammalian LAMP protein; the same substantially pure nucleic acid, wherein the LAMP self-binding domain is from a human LAMP protein; the same substantially pure nucleic acid, wherein position 55 of the polypeptide is Leu or Val, position 91 is Ser or Ala, position 171 is Thr or Leu, and position 332 is Leu or Phe, or wherein the polypeptide is selected from the group consisting of SEQ ID NOs:l, 4 through 18; the same substantially pure nucleic acid, wherein position 55 of the polypeptide is Leu or Val, position 91 is Ser or Ala. position 171 is Thr or Leu, and position 332 is Leu or Phe. or wherein the polypeptide is selected from the group consisting of SEQ ID NOs:9 through 18; the same substantially pure nucleic acid, wherein position 55 is Leu or Val, position 91 is Ser or Ala, position 171 is Thr or Leu, and position 332 is Leu or Phe, or wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 11 through 18; the same substantially pure nucleic acid of claim 1. comprising SEQ ID NOs: l, 2 or 3.

In a second embodiment, the invention relates to a substantially pure nucleic acid comprising a nucleic acid encoding a functional LAMP self-binding domain differing from an animal LAMP self-binding domain by no more than about 10 nucleotide substitutions; the same substantially pure nucleic acid, wherein each said mutation causes a conservative substitution in the sequence of the encoded LAMP self-binding domain. The invention also relates to a substantially pure nucleic acid that hybridizes under stringent conditions with a nucleic acid selected from the group consisting of SEQ ID NOs: l, 2, and 4-20.

In a third embodiment, the invention relates to a PCR primer capable of directing the amplification of the substantially pure nucleic acid set forth as the first embodiment. In a fourth embodiment, the invention relates to a transformed eukaryotic or prokaryotic cell comprising the substantially pure nucleic acid set forth as the first embodiment. In a fifth embodiment, the invention relates to a vector capable of reproducing in a eukaryotic or prokaryotic cell comprising the substantially pure nucleic acid set forth as the first embodiment.

In a sixth embodiment, the invention relates to a substantially pure protein comprising a polypeptide comprising an amino acid sequence of at least about 70 amino acids and at least about 90% homology with a corresponding segment of an animal LAMP, with the proviso that the substantially pure protein is not rat or bovine LAMP; the same substantially pure protein, wherein the polypeptide has at least about 2 amino acid substitutions relative to rat or bovine LAMP; the same substantially pure protein, wherein the polypeptide has at least about 3 amino acid substitutions relative to rat or bovine LAMP; the same substantially pure protein, wherein the corresponding segment is at least about 95% homologous to that of an animal LAMP; the same substantially pure protein, wherein the corresponding segment is at least about 98% homologous to that of an animal LAMP; the same substantially pure protein, wherein position 55 of the polypeptide is Leu or Val, position 91 is Ser or Ala, position 171 is Thr or Leu, and position 332 is Leu or Phe, or wherein the polypeptide is selected from the group consisting of SEQ ID NOs: l 1 through 18. The invention also relates to a substantially pure protein having LAMP self-binding activity and at least about 90% homology to a corresponding sequence of at least about 70 amino acids of an animal LAMP and a purity with respect to other proteins of at least about 95 % .

In a seventh embodiment, the invention relates to an antigen comprising the substantially pure protein set forth as the sixth embodiment.

In an eighth embodiment, the invention relates to a method of preparing a LAMP- derived protein comprising,

(ai cloning a nucleic acid encoding a polypeptide having at least about 90% homology to a LAMP self-binding domain in an expression vector capable of intracellularly or extracellularly expressing an appropriate inserted nucleic acid encoding a protein in an appropriate eukaryotic or prokaryotic cell,

(b) transforming an appropriate eukaryotic or prokaryotic cell with the resulting expression vector, and

(c) isolating the protein from the cell or the cell culture medium.

In a ninth embodiment, the invention relates to a method of identifying a familial genetic polymorphism at or near the LAMP gene site comprising,

(a) probing DNA from an animal tissue from an animal for a marker for the genetic sequence in the DNA in the region of the LAMP gene, and (b) comparing the results with those obtained from comparable tissues from individuals that are or are not genetically predisposed to suffer from a disease involving the limbic system; the same method of identifying a familial genetic polymorphism, wherein step (a) comprises,

(1) digesting isolated DNA from the animal with a restriction endonuclease, thereby generating restriction fragments,

(2) separating the restriction fragments by size, and

(3) identifying the restriction fragments that hybridize with a substantially pure nucleic acid comprising a nucleic acid encoding a polypeptide having at least about 90% homology to a LAMP self-binding domain; the same method of identifying a familial genetic polymorphism, wherein step (a) comprises,

(1) providing a pair of PCR probes effective to amplify the LAMP region from the DNA;

(2) PCR amplifying the LAMP region from the DNA; and

(3) determining (i) all or a part of the sequence of the amplified LAMP region, or (ii) the length of the amplified LAMP region; the same method of identifying a familial genetic polymorphism, wherein step (a) comprises,

(1) fragmenting the DNA,

(2) ligating a defined duplex DNA to the fragmented DNA,

(3) providing a PCR primer effective to direct the amplification of the LAMP region from the DNA and a PCR primer effective to hybridize with the defined DNA and direct the amplification of the fragments ligated thereto,

(a) PCR amplifying the ligated DNA from the animal, and

(b) determining (i) all or a part of the sequence of the amplified DNA, or (ii) the length of the amplified LAMP region. In a tenth embodiment, the invention relates to a pharmaceutical composition for treating an animal exhibiting excessive neural growth in the limbic region comprising an effective amount of a LAMP antisense nucleic acid or an effective amount of a soluble analog of a LAMP polypeptide or nucleic acid and a pharmaceutically acceptable carrier; the same

pharmaceutical composition, wherein the animal has epilepsy, Alzheimer's disease or schizophrenia.

In an eleventh embodiment, the invention relates to a method of determining the amount of LAMP expression in a tissue sample comprising (a) contacting the tissue sample with a nucleic acid that binds to mRNA encoding LAMP under stringent conditions,

(b) washing the tissue sample to remove non-specific bindings of the nucleic acid, and

(c) determining the amount of bound nucleic acid. In a twelfth embodiment, the invention relates to a pharmaceutical composition for treating neuropathologies involving the limbic system comprising a pharmaceutically acceptable carrier and transformed neural stem cells, wherein the transformed neural stem cells are transformed with the substantially pure nucleic acid of claim 1 in an expression vector suitable for the neural stem cells. In a thirteenth embodiment, the invention relates to a substantially pure nucleic acid with cell-type specific promoter activity comprising a nucleic acid with at least about 60% homology to the -647 to -1 sequence of a LAMP 5' promoter, wherein the LAMP promoter confers cell-type specific expression; the same substantially pure nucleic acid, comprising a nucleic acid having at least about 60% homology with -647 to -1 of SEQ ID ΝO:I9. In a fourteenth embodiment, the invention relates to a method of drug discovery comprising,

(a) contacting a molecule with LAMP-expressing cells,

(b) incubating said contacted cells,

(c) measuring free internal calcium or adherence of said incubated cells, and (d) comparing said measurement inter se or to that taken on non-LAMP-expressing control cells that were contacted, incubated, and measured as in steps (a), (b), and (c).

Brief Description of the Drawines

Figure 1 shows a schematic representation of LAMP mRNA, including the open reading frame, its signal peptide, its hydrophobic C-terminus (for processing of the PI anchor), and eight consensus glycosylation sites.

Figure 2 shows a schematic representation of the immunoglobulin-like material repeats of LAMP and other adhesion molecules.

Figure 3 shows the cDNA and protein sequence for human and rat LAMP, plus the protein sequence of a fragment of mouse LAMP.

Figure 4 displays the neurite stimulatory effect of recombinant LAMP expressed at the surface of CHO cells. Figure 5 displays a portion of the mouse genomic sequence for the LAMP gene, including 647 residues of the 5 ' untranscribed portion of the gene.

Figure 6 shows an alternate comparison of the rat and human LAMP sequences.

Figures 7A, 7B, 7C, and 7D are graphs relating to the effects of alteration of Ca ²⁺ channels by certain antagonists (Figs. 7 A and 7B) or potassium depolarization (Figs. 7C and 7D).

Detailed Description of the Invention

As noted above, LAMP is a cell adhesion molecule that is associated with cells of the nervous system. As further elucidated herein, LAMP is known to be highly conserved over evolution, such that there are only differences in the amino acid sequence between humans and rats. Accordingly, the LAMP nucleic acids and proteins of the present invention have utility in embodiments relating at least to any mammal, preferably to humans. The broad use of the LAMP nucleic acids of the present invention is also indicated by the inclusion of LAMP in the immunoglobulin superfamily (IgSF), and in particular to the homologous relationship between LAMP and several adhesion molecules in the IgSF. The relatedness of LAMP to other IgSF molecules was uncovered by the FASTA computer program described by Pearson and Lipman, Proc. Nad. Acad. Sci. USA 85: 2444-2448, 1988, or determined by manual sequence comparisons. The highest percentages of structural identity, 55% and 54% , respectively, are

with the bovine and rat opioid-binding cell adhesion molecule ("OBCAM") and neurotrimin. The data on these and other sequence homologies are recited below:

Adhesion Homolog. Homolog. Percent Opimiz. Z value molecule residues of residues of identity score Adh. molec. LAMP

OBCAM ¹ 5-338 3-332 55.2 983 94.7

Neurotrimin

2 54.

Amalgam ³ 77-324 71 -307 31 .3 332 9.3

MAG ⁴ 234-407 126-305 30.4 221 9.5

Lachesin ⁵ 14-319 17-306 29.1 342 11.0

N-CAM ⁶ 260-499 86-310 27.0 282 20.0

SMP ⁷ 229-404 122-303 25.7 193 12.3

TAG-I ⁸ 274-505 77-305 25.5 234 9.4

FASC-II ⁹ 1 -317 2-305 23.9 247 9.3

LI ¹⁰ 247-500 37-294 21.3 180 11.6

Ng-CAM ^{1 1} 405-670 16-289 21.3 140 11.9

- The sequences of the rat and bovine proteins provide essentially the same results.

The data from the rat sequence are in the table. The sequences are reported in Schofield et al., EMBO J. 8: 489-495. 1989; Lipp an et al.. Gene 117: 249-254, 1992. The sequence is reported in Struyk, et al.. J. Neurosci., 15. 2141-2156. 1995. This comparison was done manually.

³ The sequence is reported in Seeger et al.. Cell 55: 589-600, 1988.

The sequence is reported in Arquint et al., Proc. Natl. Acad. Sci. U.S.A. 84: 600-604, 1987; Salzer et al.. J. Cell Biol 104: 957-965. 1987; and Ui, et al., Proc. Natl. Acad. Sci. 84: 4237-4241. 1987.

The sequence is reported in Karlstron et al.. Development 118: 509-522. 1993.

The sequence is reported in Cunningham et al., Science 236: 799-806, 1987.

The sequence is reported in Dulac et al.. Neuron 8: 323-334, 1992.

The sequence is reported in Furley et al.. Cell 61: 157-170, 1990.

The sequence is reported in Grenningloh et al., Cell 67: 45-57, 1991.

¹⁰ The sequence is reported in Moos et al., Nature 334: 701-773, 1988.

¹¹ The sequence is reported in Burgoon et al., J. Cell Biol. 112: 1017-1029, 1991. In the above table, the Z value is indicative of the statistical significance of the homology, with a value of 6 or more indicating probable significance, and a score of 10 or more strongly indicating significance.

The N-terminal of the open reading frame of SEQ ID NOs:l and 2 (see Figs. 1 and 3) has the characteristics of a signal peptide that is cleared away during protein synthesis. Nucleic Acids Res. 14:4683-4690, 1986. This portion of LAMP is represented by the hatched region in Fig. 1. Indeed, microsequencing shows that the N-terminal on processed LAMP is Val _:9 . LAMP has a hydrophobic C-terminal sequence consistent with that found on other Pi-linked proteins; this kind of sequence is cleaved away when a protein is inserted into a membrane via a PI linkage. See, Cross, Ann. Rev. Cell Biol. 6: 1-39, 1990; Ferguson and Williams, Ann. Rev. Biochem. 57:285-320, 1988; Gerber et al., J. Biol. Chem. 267:18168-12173, 1992. This C-terminal portion is represented by the dark, filled region of Fig. 1, with the PI linkage indicated by the "GPI" notation. The C-terminal of processed LAMP is at or near Asn ₃ | ₅ . LAMP is found in two forms, which is probably a consequence of differential splicing of the RNA transcript upon processing to form the messenger RNA. Both forms are found in both rats and humans. Details of the second form are provided below in Example 11.

SEQ ID NO:5 represents the sequences of human cDNA/protein for the 29-332 amino acid sequence of LAMP, while SEQ ID NO:6 is for the 29-338 amino acid sequence of rat LAMP. SEQ ID NO:7 represents the sequences of cDNA/protein for human of the 8-315 ammo acid sequence of human LAMP, while SEQ ID NO:8 represents the 1-315 ammo acid sequence of rat LAMP. SEQ ID NOs:9 and 10 represent the sequences of cDNA/protein for human and rat, respectively, of the 29-315 amino acid sequence of LAMP. The minimal LAMP self-binding domain is made up of one or more of the immunoglobulin internal repeats made up of amino acid sequences 46-118, 156-204 and 232-297 of LAMP, which are designated SEQ ID NOs: l l , 13 and 15, respectively, for human, and SEQ ID NOs.12. 14 and 16. respectively, for rat. The sequence encompassing all of these repeats or "loops". specifically amino acid residues 46-294, is designated SEQ ID NOs: 17 and 18, respectively, for human and rat LAMP.

The predicted molecular weight of unglycosylated, proteolytically processed LAMP is about 32 kDa. Indeed, in E. coli the expression of LAMP cDNA results in protein of apparent molecular weight of 32 kDa (by SDS-PAGE). In Chinese hamster ovary ("CHO") cells, the cDNA produces a protein of apparent molecular weight of 55 kDa, a value intermediate

between the unglycosylated molecular weight and molecular weight of the native protein. The extra molecular weight of the CHO-expressed and native proteins is due to glycosylations. Eight sites for N-linked glycosylations (having the sequence Asn-X-Ser/Thr) are found at Asn40, Asn66, Asnl36, Asnl48, Asn279, Asn287, Asn300, and Asn315. The glycosylation sites are indicated by the balloon symbols in Fig. 1.

As mentioned above, the human and rat amino acid sequences of LAMP are highly homologous, sharing about 99% sequence identity. The four differences are:

RAT HUMAN

1 Val 55 Leu 55

2 Ala 91 Ser 91

3 Leu 171 Thr 171

4 Phe 332 Leu 332

Accordingly, the invention relates to nucleic acids encoding proteins having at least about 90% homology to human LAMP, preferably at least about 95% homology, more preferably at least about 98% homology, still more preferably at least about 99% homology. In measuring homology, any suitable computer program may be used, including but not limited to the FASTA program and version 6.0 of the GAP program. The GAP program is available from the University of Wisconsin Genetics Computer Group and utilizes the alignment method of Needleman and Wunsch, J. Mol. Biol. 48, 443, 1970, as revised by Smith and Waterman Adv. Appl. Math. 2, 482, 1981.

In one embodiment, the nucleic acids of the invention when employed with suitable marker agents, are histochemical staining reagents. Such staining allows one skilled in the an to characterize developmental or structural anomalies in biopsy tissue pathology samples. Alternatively, such staining is used to mark a select set of neurons in tissue slides or other preserved specimens for use as anatomical or medical teaching tools.

In yet another embodiment, the nucleic acids of the invention can also be used to transform neural stem cells to program their development as limbic system neurons. These replacement limbic system neurons can be implanted to treat neuropathologies by reconnecting limbic circuits involved in cognition, mood, memory and learning, and cardiovascular regulation, providing therapies for such diseases as dementia (including without limitation Alzheimer's disease, multi-infarct dementia, dementia associated with Parkinson's disease), all forms of epilepsy, major depression, anxiety (including without limitation manic-depressive illness, generalized anxiety, obsessive-compulsive disorders, panic disorder and others),

schizophrenia and schizophrenaform disorders (including without limitation schizoaffecto disorder), cerebral palsy and hypertension.

The nucleic acids of the invention can be used to create LAMP-derived polypeptides that interact with LAMP located at a neuron cell surface to either stimulate the growth and differentiation activities of LAMP or to inhibit those activities. Particularly preferred are such polypeptides that are soluble LAMP analogs having binding domains effective to bind LAMP. The LAMP-inhibitory polypeptides that are encoded by these nucleic acids can be used to treat diseases characterized by abnormal growth and functioning of limbic neurons, such as epilepsy, Alzheimer's disease and schizophrenia. Antisense strategies to inhibit the expression of LAMP can also be used to treat these diseases.

Another use for the nucleic acids of the invention is to create targeting polypeptides for directing the delivery of biological agents to the limbic system. The polypeptides are useful targeting agents because they bind to LAMP found at the cell surfaces in the limbic system. Such targeting agents are bound covalently or noncovalently to a biological agent or a vehicle for delivering biological agents. Biological agents are those that can act on a cell, organ or organism, including, but not limited to, pharmaceutical agents and gene therapy agents. Numerous targetable delivery vehicles are known, including liposomes, ghost cells and polypeptide matrices. See, for example, Huang et al., Proc. Natl. Acad. Sci. USA, 84, 7851- 7855, 1987, Kreuter, Infection 19 Supp. 4, 224-228, 1991 or Michel et al., Research in Virology, 144, 263-267, 1993.

The first embodiment of the invention provides a substantially pure nucleic acid encoding all or a fragment of LAMP effective to bind to a native LAMP at the cell surface of a neuron. The nucleic acid is preferably derived from the nucleic acid for a LAMP from a mammalian animal, more preferably from a human. Preferably, the nucleic acid comprises a contiguous nucleic acid of at least about 220 nucleotides from SEQ ID NO: 1 or 4, more preferably, at least about 480 nucleotides, still more preferably, at least about 780 nucleotides. Preferably, the sequence comprises all of one of SEQ ID NOs:5 - 18.

The nucleic acid embodiments of the invention are preferably deoxyribonucleic acids DNAs, both single- and double-stranded, preferably double-stranded deoxyribonucleic acids. However, they can also be ribonucleic acids (RNAs), as well as hybrid R_NA:DNA double- stranded molecules.

Numerous methods are known to delete sequence from or mutate nucleic acid sequences that encode a protein and to confirm the function of the proteins encoded by these deleted or mutated sequences. Accordingly, the invention also relates to a mutated or deleted

version of a LAMP nucleic acid that encodes a protein that retains the ability to bind specifically to a native LAMP bound to a cell surface. The invention also relates to nucleic acids encoding a protein that has the differentiation regulatory activity of LAMP.

As used herein, a "LAMP nucleic acid" means all or part of the LAMP-encoding segment of nucleic acid found in a LAMP-expressing organism, or the complementary strand thereof. "LAMP-encoding nucleic acid" or "nucleic acid sequences for a LAMP" refers to any nucleic acid, whether native or synthetic, RNA or DNA, that encodes all or part of a LAMP. A "LAMP protein" is a LAMP homologous protein with the ability to bind a native LAMP and possessing the differentiation regulatory activity of a native LAMP. An "animal LAMP" is a LAMP expressed by a member of the animal kingdom. A "LAMP 5 ' promoter sequence" is a 5 ^' untranscribed region of a gene for a LAMP encoding gene from a LAMP expressing organism that is sufficient to direct transcription in a LAMP-expressing cell.

To construct non-naturally occurring LAMP-encoding nucleic acids, the native sequences can be used as a starting point and modified to suit particular needs. For instance, the sequences can be mutated to incorporate useful restriction sites. See Maniatis et al.

Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989). Such restriction sites can be used to create "cassettes", or regions of nucleic acid sequence that are facilely substituted using restriction enzymes and ligation reactions. The cassettes can be used to substitute synthetic sequences encoding mutated LAMP amino acid sequences. Alternately, the LAMP-encoding sequence can be substantially or fully synthetic. See, for example, Goeddel et al., Proc. Na . Acad. Sci. USA, 76, 106-110, 1979. For recombinant expression purposes, codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic LAMP-encoding nucleic acid.

Many deletional or mutational analogs of nucleic acid sequences for a LAMP are effective hybridization probes for LAMP-encoding nucleic acid. Accordingly, the present invention relates to nucleic acids that hybridize with such LAMP-encoding nucleic acids under stringent conditions. Preferably, the nucleic acid of the present invention hybridizes with the nucleic acid described as SEQ ID NO: 11 or 18 under stringent conditions.

"Stringent conditions" refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, for a nucleic acid of 100 nucleotides. such conditions will generally allow hybridization of nucleic acids having at least about 85% homology, preferably having at least about 90% homology. Such hybridization conditions are described by Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Press. 1989.

Nucleic acid molecules that hybridize to a LAMP-encoding nucleic acid under stringent conditions can be identified functionally, using methods outlined above, or by using the hybridization rules reviewed in Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989. Without limitation, examples of the uses for hybridization probes include: the histochemical uses described above; measuring mRNA levels, for instance, to identify a sample's tissue type or to identify cells that express abnormal levels of LAMP; and detecting polymorphisms in the LAMP gene. RNA hybridization procedures are described in Maniatis et al. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989). Rules for designing polymerase chain reaction ("PCR") primers are well known in the art, as reviewed in PCR Protocols, Cold Spring Harbor Press, 1991. Degenerate primers, i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acids that are highly related to, but not identical to, a LAMP nucleic acid. For instance, such degenerate primers can be designed from the human LAMP cDNA and used to amplify nucleic acids for LAMPs from non-human species, as illustrated in the examples. As discussed above, deletional or mutational methods of producing recombinant proteins that retain a given activity are well known. Thus, the embodiments of the present invention that relate to proteins also encompass analogs of LAMP that retain the ability to bind to native LAMP expressed at a cell surface. These analogs preferably lack no more than about 45 amino acid residues at the N-terminal end, more preferably no more than about 37 amino acid residues. At the C-terminal, the analogs preferably lack no more than about 44 amino acid residues, more preferably no more man about 34 amino acid residues. The remaining LAMP protein sequence preferably has no more than about 10 exchanges of amino acids (probably the result of point mutations), preferably no more than about 5 exchanged amino acids, more preferably no more than about 3 exchanged amino acids. Preferably, the analogs will have at least about 90% homology, preferably at least about 95%. more preferably at least about 98% , still more preferably at least about 99% , to any of the proteins of one of SEQ ID NO: 1-18, preferably of SEQ ID NO: 11-18.

The nucleic acid that encodes the LAMP proteins having deletions at the N- or C- terminus, or exchanged amino acids in the interior segment of the protein, also has. accordingly and respectively, deletions at its 5 ' or 3 ' end, or point mutations in the interior segment of the nucleic acid. The point mutations are preferably conservative point mutations, which of course would reflect limited or no alteration in the conformation or functioning of the encoded protein.

Mutational and deletional approaches can be applied to all of the nucleic acid sequences of the invention that express LAMP proteins. As discussed above, conservative mutations are preferred. Such conservative mutations include mutations that switch one amino acid for another within one of the following groups: 1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin;

3. Polar, positively charged residues: His, Arg and Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

A preferred listing of conservative substitutions is the following:

Original Residue

Substitution

Ala Gly, Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Ala, Pro

His Asn, Gin

He Leu, Val

Leu He, Val

Lys Arg, Gin, Glu

Met Leu, Tyr, He

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp, Phe

Val He, Leu

The types of substitutions selected is preferably, but not necessarily, based on the analysis of the frequencies of amino acid substitutions between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag. 1978, on the analyses of structure-forming potentials developed by Chou and Fasman, Biochemistry 13, 211 , 1974 and Adv. Enzymol, 47, 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Nad. Acad. Sci. USA 81 , 140-144, 1984; Kyte & Doolittle J. Molec. Biol. 157, 105-132, 1981, and Goldman et al. , Ann. Rev. Biophys. Chem. 15, 321-353, 1986.

Because the four identified substitutions between rat and human LAMP are clearly functionally acceptable, the invention provides LAMP proteins having any combination of these substitutions.

As discussed above, at least one of the immunoglobin-like internal repeats of LAMP is the minimal LAMP self-binding domain, i.e., the minimal segment of amino acids needed to specifically interact with LAMP. Additional LAMP-related polypeptides may serve to enhance this self-binding activity. The exact boundaries of the minimal self-binding domain and the adjacent segment that improves the efficiency of the minimal segment can be determined by gene expression methods well known to the art. For instance, the nucleic acid for the minimal segment can be inserted at a downstream portion of the gene for a cell-surface protein and the recombinant gene expressed as a cell-surface fusion protein. The ability of the fusion protein to interact with LAMP can be tested, for instance, by one of the methods outlined in the Examples.

The invention also provides for the LAMP or LAMP-related proteins having LAMP self-binding activity encoded by any of the nucleic acids of the invention in a purity of at least about 95% with respect to other proteins, preferably about 98%, more preferably about 99%. The purities are achieved, for example, by applying protein purification methods, such as those described below, to the culture medium or lysate of a recombinant cell according to the invention. The invention further provides substantially pure LAMP or analogs of LAMP, with the proviso that they are not rat or bovine LAMP. The substantially pure LAMP or analog of LAMP preferably comprises a polypeptide that is homologous to a 73 amino acid stretch of an animal LAMP, more preferably a 159 amino acid stretch, still more preferably a 252 amino acid stretch. The LAMP or analog of LAMP preferably comprises one, two or three of the immunoglobin-like repeats. The LAMP or LAMP analog protein is preferably at least about 90% , preferably about 95%, more preferably 98% , still more preferably about 99% homologous to the corresponding amino acid sequence of an animal LAMP. The protein has at least about 2, preferably about 3, more preferably about 4, amino acid substitutions relative to the 29 to 315 amino acid sequence of rat LAMP, where "substitutions" includes deletions. In a preferred embodiment, the protein has at least about 10. preferably about 20, more preferably about 40. yet more preferably about 80 substitutions relative to rat or bovine LAMP and comprises at least one of the immunoglobin-like repeats of LAMP, preferably the third repeat (amino acids 232 to 297 of rat LAMP).

The invention further provides antigens comprising the proteins of the above paragraph. These antigens are used to produce antibodies with specificities that, for instance, differ from the specificity of antibodies against rat LAMP.

The methods by which the LAMP analogs of the above paragraphs, which include deletional and mutational analogs are constructed are discussed above with respect to LAMP or LAMP analog nucleic acids. These nucleic acids can be used to create organisms or cells that produce LAMP or LAMP analogs. Purification methods, including facilitative molecular biology methods, are described below.

One simplified method of isolating polypeptides synthesized by an organism under the direction of one of the nucleic acids of the invention is to recombinantly express a fusion protein wherein the fusion partner is facilely affinity purified. Any suitable fusion partner is used in the context of the present invention. For instance, a preferred fusion partner is glutathione s-transferase, which is available on commercial expression vectors (e.g., vector pGEX4T3, available from Pharmacia, Uppsala, Sweden). The fusion protein is preferably purified on a glutathione affinity column (for instance, that available from Pharmacia).

Alternatively, the recombinant polypeptides are affinity purified without such a fusion partner by using the 2G9 antibody, as described in Zacco et al., J. Neuroscience 10, 73-90, 1990 or another LAMP-specific antibody or previously purified LAMP, which has self-adhesion activity. If fusion proteins are used, the fusion partner is preferably removed by partial proteolytic digestion approaches that preferentially attack unstructured regions such as the linkers between the fusion partner and LAMP. The linkers are preferably designed to lack structure, for instance using the rules for secondary structure-forming potential developed, for instance, by Chou and Fasman, Biochemistry 13, 211, 1974 and Chou and Fasman. Adv. in Enzymol. 47, 45-147, 1978. In another embodiment, the linker is designed to incorporate protease target amino acids, such as, for trypsin, arginine and lysine residues. To create the linkers, standard synthetic approaches for making oligonucleotides are preferably employed together with standard subcloning methodologies. Procedures that utilize eukaryotic cells, particularly mammalian cells, are preferred because such cells will post-translationally modify the protein to create molecules highly similar to or functionally identical to native proteins. Additional purification techniques are applied, as appropriate, including without limitation, preparative electrophoresis, FPLC (Pharmacia, Uppsala. Sweden), HPLC (e.g., using gel filtration, reverse-phase or mildly hydrophobic columns), gel filtration, differential precipitation (for instance, "salting out" precipitations), ion-exchange chromatography and affinity chromatography.

A protein or nucleic acid is "isolated" in accordance with the invention in that the molecular closing of the nucleic acid of interest, for example, involves taking LAMP nucleic acid from a cell, and isolating it from other nucleic acids derived from the same cell. This isolated nucleic acid may then be inserted into a host cell, which may be yeast or bacteria, for example, or another of the same cell. A protein or nucleic acid is "substantially pure" in accordance with the invention if it is predominantly free of other proteins or nucleic acids, respectively. A macromolecule, such as a nucleic acid or a protein, is predominantly free if it constitutes at least about 50% by weight of the given macromolecule in a composition. Preferably, the protein or nucleic acid of the present invention constitutes at least about 60% by weight of the total proteins or nucleic acids, respectively, that are present in a given composition thereof, more preferably about 80%, still more preferably about 90%, yet more preferably about 95%, and most preferably about 100% . Such compositions are referred to herein as being proteins or nucleic acids that are 60% pure, 80% pure, 90% pure, 95% pure, or 100% pure, any of which are substantially pure. One aspect of the present invention is directed to the use of "antisense" nucleic acid to treat neurological diseases, including epilepsy. The approach involves the use of an antisense molecule designed to bind nascent mRNA (or "sense" strand) for a LAMP, thereby stopping or inhibiting the translation of the mRΝA, or to bind to the LAMP gene to interfere with its transcription. For discussion of the design of nucleotide sequences that bind genomic DΝA to interfere with transcription, see Helene, Anti-Cancer Drug Design 6, 569, 1991. Once the sequence of the mRΝA sought to be bound is known, an antisense molecule can be designed that binds the sense strand by the Watson-Crick base-pairing rules, forming a duplex structure analogous to the DΝA double helix. Gene Regulation: Biology of Antisense RNA and DNA, Erikson and Ixzant, eds., Raven Press, New York, 1991; Helene, Anti-Cancer Drug Design, 6:569 (1991); Crooke, Anti-Cancer Drug Design 6, 609, 1991.

A serious barrier to fully exploiting this antisense technology is the problem of efficiently introducing into cells a sufficient number of antisense molecules to effectively interfere with the translation of the targeted mRNA or the function of DNA. One method that has been employed to overcome this problem is to covalently modify the 5' or the 3' end of the antisense polynucleic acid molecule with hydrophobic substituents. These modified nucleic acids generally gain access to the cells interior with greater efficiency. See, for example, Boutorin et al., FEBS Lett. 23, 1382-1390, 1989; Shea et al. Nucleic Acids Res. 18, 3777-3783, 1990. Additionally, the phosphate backbone of the antisense molecules has been modified to remove the negative charge (see, for example. Agris et al., Biochemistry 25, 6268, 1986;

Cazenave and Helene in Antisense Nucleic Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol, eds., p. 47 et seq. , Marcel Dekker, New York, 1991) or the purine or pyrimidine bases have been modified (see, for example, Antisense Nucleic Acids and Proteins: Fundamentals and Applications , Mol and Van der Krol, eds., p. 47 et seq.. Marcel Dekker, New York, 1991; Milligan et al. in Gene Therapy For Neoplastic Diseases, Huber and Laso, eds., p. 228 et seq.. New York Academy of Sciences, New York, 1994). Other methods to overcome the cell penetration barrier include incorporating the antisense polynucleic acid sequence into an expression vector that is inserted into the cell in low copy number, but which, when in the cell, can direct the cellular machinery to synthesize more substantial amounts of antisense polynucleic molecules. See, for example, Farhood et al., Ann. N. Y. Acad. Sci. 716, 23, 1994. This strategy includes the use of recombinant viruses that have an expression site into which the antisense sequence has been incorporated. See, e.g., Boris-Lawrie and Temin, Ann. N.Y. Acad. Sci., 716:59 (1994). Others have tried to increase membrane permeability by neutralizing the negative charges on antisense molecules or other nucleic acid molecules with polycations. See, e.g. Wu and Wu, Biochemistry, 27:887-892, 1988; Behr et al., Proc. Nad. Acad Sci U.S.A. 86:6982-6986, 1989.

The invention also encompasses the use of gene therapy approaches to insert a gene (1) expressing a LAMP protein into de-differentiated limbic system-derived tumor cells or into stem cells, (2) expressing a LAMP-directed anti-sense molecule, or (3) a limbic system-targeted gene functionally linked to a LAMP promoter.

For gene therapy, medical workers incorporate, into one or more cell types of an organism, a DNA vector capable of directing the synthesis of a protein missing from the cell or useful to the cell or organism in greater amounts. The methods for introducing DNA to cause a cell to produce a new protein or a greater amount of a protein are called "transfection" methods. See, generally, Neoplastic Diseases, Huber and Lazo. eds.. New York Academy of Science, New York. 1994; Feigner, Adv. Drug Deliv. Rev., 5: 163 (1990): McLachlin, et al.. Progr. Nucl. Acids Res. Mol. Biol. , 38:91 (1990); Karlsson, 5. Blood. 78:2481 (1991); Einerhand and Valerio, Curr. Top. Microbiol. Immunol. , 177:217-235 (1992): Makdisi et al.. Prog. Liver Dis. , 10: 1 (1992); Litzinger and Huang, Biochim. Biophys. Acta. 1113:201 (1992); Morsy et al., J.A.M.A., 270:2338 (1993); Dorudi et al., British J. Surgery. 80:566 (1993). A number of the above-discussed methods of enhancing cell penetration by antisense nucleic acid are generally applicable methods of incorporating a variety of nucleic acids into cells. Other general methods include calcium phosphate precipitation of nucleic acid and incubation with the target cells (Graham and Van der Eb. Virology, 52:456. 1983), co-

incubation of nucleic acid, DEAE-dextran and cells (Sompayrac and Danna, Proc. Natl. Acad. Sci., 12:7575, 1981), electroporation of cells in the presence of nucleic acid (Potter et al., Proc. Natl. Acad. Sci. , 81:7161-7165, 1984), incorporating nucleic acid into virus coats to create transfection vehicles (Gitman et al., Proc. Natl. Acad. Sci. U.S.A., 82:7309-7313, 1985) and incubating cells with nucleic acid incorporated into liposomes (Wang and Huang, Proc. Natl. Acad. Sci., 84:7851-7855, 1987). One approach to gene therapy is to incorporate the gene sought to be introduced into the cell into a virus, such as a herpes virus, adenovirus, parvovirus or a retrovirus. See, for instance, Akli et al., Nature Genetics 3, 224, 1993.

The nucleic acid compositions of the invention, including antisense and gene therapy compositions, can be administered orally, topically, rectally, nasally, vaginally, by inhalation, for example by use of an aerosol, or parenterally, i.e. intramuscularly, subcutaneously, intraperitoneallly, intraventricularly, or intravenously. The polynucleotide compositions are administered alone, or they are combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceutical practice. For the oral mode of administration, the polynucleotide compositions are used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. In the case of tablets, carriers that are used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants, such as starch, and lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols When aqueous suspensions are required for oral use, the polynucleotide compositions are combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents are added. For parenteral administration, sterile solutions of the conjugate are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes are controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids are delivered by ocular delivery systems known to the art, such as applicators or eye droppers. Such compositions include mucomimetics, such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives, such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. For pulmonary administration, diluents and/or carriers are selected as appropriate for formation of an aerosol.

Generally, the polynucleotide compositions are administered in an effective amount. An effective amount is an amount effective to either (1) reduce the symptoms of the disease

sought to be treated or (2) induce a pharmacological change relevant to treating or preventing the disease sought to be treated or prevented.

For viral gene therapy vectors, dosages are generally from about 1 μg to about 1 mg of nucleic acid per kg of body mass. For non- infective gene therapy vectors, dosages are generally from about 1 μg to about 100 mg of nucleic acid per kg of body mass. Antisense oligonucleotide dosages are generally from about 1 μg to about 100 mg of nucleic acid per kg of body mass.

The stem cells that are useful in neural stem cell replacement therapy include human cortical and subcortical fetal brain cells, porcine fetal brain cells, human subventricular zone cells and glial progenitor cells, including O2A cells (which are progenitors for all glial cell types, including astrocytes and oligodendrocytes).

The biological agents that can be usefully targeted to the limbic system include, without limitation neurotransmitter biosynthetic enzymes (such as tyrosine hydroxylase), neurotransmitter transporters (such as the GAB A transporter), neurotransmitter receptors (such as type la, lb, II or III dopamine receptors, a and β adrenergic receptors and 5-HT receptors), neurotrophic and growth factors (such as NGF, BDNF, NT-3, NT-4, NT-5, TGF3, basic FGF and GDNF), neurotrophic factor receptors, protein kinases (such as MAP kinases and protein kinase C) and protein phosphatases. Further agents include, without limitation, antidepressants, neuroleptics, anti-epileptics and antagonists of neurotransmitter receptors (such as type la, lb, II or III dopamine receptors, a and β adrenergic receptors and 5-HT receptors). Such agents include antisense therapeutic agents, including antisense gene therapy agents.

LAMP promoter, meaning promoters linked to LAMP nucleic acid in the genome of an animal that is sufficient to confer limbic system-specific expression, in addition to the promoter defined in SEQ ID NO:20, is isolated by probing a genomic library with one of the LAMP nucleic acids of the invention. See, Maniatis et al.. Molecular Cloning, Cold Spring Harbor Press, 1989. The ability of the promoter to confer cell-specific expression is confirmed by transfection experiments and experiments that create transgenic animals, ]d. The invention relates to such LAMP promoters and mutated analogs that retain cell-type specific function.

5' LAMP promoter sequences are isolated using a genomic library containing relatively short inserts appropriate for PCR amplification. This library is made by first digesting genomic DNA with a relatively non-specific restriction endonuclease, such as one that recognizes a 4-6 base pair ("bp") sequence, or by shearing genomic DNA until relatively short (e.g. 1000-3000 bp) fragments are obtained. The genomic fragments are subcloned into a vector. The vector DNA is PCR amplified with a first vector-based primer that primes DNA synthesis towards the

insert site and a second primer, based on the 5' sequence of SEQ ID NO: 1 or SEQ ID NO:2, that primes DNA synthesis in the 5' direction. In this way, a LAMP promoter sequence is rapidly synthesized.

The promoter sequences of the invention include all sequences within 200 base pairs of the transcription start site that affect the efficiency with which LAMP is transcribed.

Preferably, the promoter will contain sufficient sequence to confer neural-specific transcription, still more preferably, limbic system-specific expression. Included in such a promoter sequence is the first intron of the LAMP gene, the sequence of which, for mouse LAMP, is made up of nucleotides 1033 through 1851 in SEQ ID NO:20. This intron, shown in Fig. 5, has a consensus binding sites for the Hox-1.3 and AP-1 transcription factors (beginning at nucleotides 1,312 and 1,282, respectively) and a TATA box consensus sequence (centered at about nucleotide 1,821). The first 350 nucleotides of the 5 ' portion of the promoter has CREB, AP- 1, SP-1 and TATA consensus sequences beginning at nucleotides 313, 359, 524 and 617, respectively (or, relative to the start of transcription, at -340, -294, -129 and -36, respectively). The preferred embodiment comprises a sequence that is homologous to the -647 to -1 portion of a LAMP promoter, more preferably the

-350 to -1 portion, yet more preferably the -130 to -1 portion. The -647 to -1, -350 to -1 or - 130 to -1 promoter sequence preferably has at least about 60% homology sequence of a LAMP promoter, more preferably at least about 70%, yet more preferably 80%, still more preferably at least about 90%. An example of such a sequence, derived from mouse genomic DNA, is represented in Fig. 5, where the arrow represents the dominant transcription start site. (+ 1).

The gene therapy agents that are usefully targeted to the limbic system include, without limitation, genes or antisense therapeutics for protein kinases (such as. without limitation, protein kinase C), enzymes including neurotransmitter biosynthesizing enzymes (such as, without limitation, acetylcholine synthesizing enzymes), neurotransmitter transporters, neurotrophins (i.e., factors that provide neural cells with nutritive support) and growth factors (such as, without limitation, NGF, BDNF, NT-3, NT-4, NT-5, TGF/3, basic FGF and GDNF), ion channels (such as. without limitation, calcium and sodium channels), neurotransmitter receptors (such as, without limitation, dopamine receptors), neurotrophic factor and receptors (such as NGF, 0-DNF, NT-3, NT-4, NT-5, TGF/3, basic FGF and GDNF) neurotrophic factor receptors, protein kinases (such as MAP kinases and protein kinase C) and protein phosphatases. In Alzheimer's, genes for acetyl choline synthesis aid to replace diminished acetylcholine levels in the limbic system. Genes for growth factors and neurotrophic factors help keep neurons from succumbing to neurodegenerative diseases that affect the limbic system.

The invention also provides a useful marker protein to examine the genome of families with genetic disorders to determine whether the affected members share a polymorphism at or adjacent to the LAMP gene. To identify polymorphisms, several techniques are used, including without limitation examining for variable number tandem repeats in the DNA at or adjacent to the LAMP gene, probing for restriction length polymorphisms, probing for variations in the length of PCR-amplified fragments, and examining the nucleotide sequence at or near the LAMP. These and other methods are described in the text by Victor McKusak. Mandelian Inheritance in Man: A Catalog of Human Genes and Genetic Disorders, 11th edition, Johns Hopkins University Press, 1994, and in a series entitled Genes edited by B. Lewin and published by Wiley & Sons. PCR methods are usefully employed for examining sequence information. In connection with this aspect of the invention, "probing" means examining genomic DNA for any useful indicator of a polymorphism.

The invention also relates to methods of measuring a LAMP mRNA from a tissue or staining a tissue for a LAMP mRΝA. Useful methods of measuring mRNA include Southern blot analysis, dot blot analysis, nuclear transcription analysis, histochemical staining for mRΝA and polymerase chain reaction amplification methods. See generally, Ausubel et al.. Current Protocols in Molecular Biology, Wiley Press, 1993; PCR Protocols, Cold Spring Harbor Press, 1991; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.. Cold Spring Harbor Press, 1989. For in-situ nucleic acid hybridization techniques, see Baldino et al., Methods in Enzymology 168, 761-777, 1989; Meson et al.. Methods in Enzymology 168. 753- 761. 1989; Harper et al.. Methods in Enzymology 151, 539-551. 1987; Angerer et al.. Methods in Enzymology 152. 649-661, 1987; Wilcox et al.. Methods in Enzymology 124, 510-533. 1986. Methods of assessing mRNA amounts are useful in diagnosing abnormalities associated with epilepsy and schizophrenia. For these uses, biopsy tissue and CSF fluid are usefully assayed. PCR methods of amplifying nucleic acid utilize at least two primers. One of these primers is capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction. The other is capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence initially is hypothetical, but is synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer. Conditions for conducting such amplifications, particularly under preferred stringent conditions, are well known in the an. See, for example. PCR Protocols, Cold Spring Harbor Press, 1991. Appropriate nucleic acid primers are ligated to the nucleic acid sought to be amplified to provide the hybridization

partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.

The samples that are assayed or stained for nucleic acid encoding LAMP include, without limitation, cells or tissues (including nerve tissues), protein extracts, nucleic acid extracts and biological fluids such as cerebra fluid, serum and plasma. Preferred samples are nervous system-derived samples.

All treatment methods of the invention, including pharmaceutical compositions, are applicable to animals in general, although mammals - particularly humans - are preferred treatment subjects. The present invention also relates to a method of drug discovery comprising:

(a) contacting a small molecule with LAMP-expressing cells;

(b) incubating the contacted cells;

(c) measuring LAMP activity of the incubated cells; and

(d) comparing the measurement inter se or to that taken on non-LAMP-expressing control cells that were contacted, incubated, and measured as in steps (a), (b), and (c). The method allows for the identification of a new class of antipsychotics, i.e., those drugs that have an effect on LAMP activity. Such drugs may be any suitable molecule, including without limitation peptides, proteins, small organic compounds, small organic-inorganic compounds, simple or complex sugars, steroids, and others. The drug can be a naturally-occurring compound, or it can be synthetic. The drug can be identified from a combinatorial library, or derived from a rational drug discovery approach. The LAMP-expressing cells of the invention are any suitable cells, including primary brain cells, LAMP-transfected CHO cells, and the like. The measurement of LAMP activity effected in the context of the present invention is any suitable measurement, including without limitation measurement of free internal calcium, adhesion, self-adhesion, and others. For example, a reporter gene, such as beta-galactosidase, linked to the LAMP gene of the cells set forth in step (a) allows measurement of beta- galactosidase activity to be an indicator of LAMP activity. Use of other reporter genes as known in the art are included in other embodiments of the present invention. Preferred measurements are of free internal calcium, as described in Example 12, and adhesion, as disclosed in Pimenta et al., Neuron. 15. 287-297 (1995). The measurement of step (c) is interpreted by comparison to the same measurement of a control culture or cell, wherein the control culture or cell preferably contains or is the same sort of cell except for the lacking of LAMP activity. In the case of transfected cells used in this method, the preferred control cell

is the same host transfected with the same tranfection vehicle as was used to transfect LAMP, but without the LAMP insert.

The drug discovery method of the present invention preferably has an initial screening for looking for agents or compounds having LAMP activity-altering characteristics. Preferably such a first screen uses LAMP-transfected CHO cells, using an apparatus that facilitates the testing of thousands of different compounds. A second screen preferably uses primary brain cells, wherein the measurements are scored by inter se comparisons. Alternatively, the first screen is repeated for the positive compounds identified in the first screen. Further development of a drug requires animal studies, toxicity studies, and phased clinical trials. The invention is described in more detail, but without limitation, by reference to the examples set forth below.

Example 1 - Isolation of Rat LAMP cDNA

LAMP was purified from adult rat hippocampal membranes as described by Zacco et al., J. Neurosci. 10: 73-90, 1990, using the 269 antibody as an affinity reagent. The purified protein was electrophoresed by SDS-PAGE and electroblotted onto a PVDF membrane in preparation for microsequencing as described by Matsudaira, J. Biol. Chem. 262: 10035-10038,

1987. The electroblotted LAMP was sequenced on an Applied Biosystems 470A gas phase sequencer equipped with a 120A on-line PTH analyzer as described by Henzel et al., J. Chromatogr. 404: 41-52, 1987. The N-terminal sequence was determined to be

VRSVDFNRGTDNITVRQGDTA [SEQ ID NO:21], using the single letter amino acid abbreviations.

The sequences DFNRGTD and ITVRQGD. both found in the N-terminal sequence. were used to design two separate batches of redundant oligonucleotide hybridization probes. The batches were as follows:

Group 1 : GAYTTYAAYCGIGGIACIGAY [SEQ ID NO:22] GAYTTYAAYAGRGGIACIGAY [SEQ ID NO:23] Group 2: ATHACIGTICGICARGGIGAY [SEQ ID NO:24] ATHACIGTIAGRCARGGIGAY [SEQ ID NO:25] where R = A/G, Y = C/T, I = Inosine and H = A/C/T. These probes were end-labeled with ³² P and used to screen an adult rat hippocampus cDNA library cloned in λgtl l (Clontech, Palo

Alto, CA). The methodology used was as described by Maniatis, Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor Press, 1989 and Hockfield et al., Molecular Probes of the Nervous System, Cold Spring Harbor Press, 1994, as was all the methodology of the

Examples, unless otherwise specified. The insert DNA from lambda plaques that were positive

for both probes was amplified by PCR using the λgtl l forward and reverse primers available from Promega (Madison, WI). Amplified inserts were subcloned into pCR II (Invitrogen, San Diego, CA) and sequenced by the dideoxy chain termination method. Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977. Four identical cDNA inserts had the sequence of SEQ ID NO:2.

Example 2 - Human LAMP cDNA

The following PCR primer pairs were designed based on the sequence of the rat LAMP cDNA of Figure 3 and were used to amplify human cerebral cDNAs of the sizes indicated in the parentheses below:

Pair Primer 1: GAYTTYAAYCGIGGIACIGAY nt 152-172 H-l [SEQ ID NO:26]

Primer 2: TGCCAGCAGCCACAGTGGTA nt 1020-1039 [SEQ ID NO:27]

(Size of the sequence amplified: 888 bp.)

Pair Primer 1 : CGAATCCAGAAGGTGGATGT nt 344-363 H-2 [SEQ ID NO:28]

Primer 2: TGCCAGCAGCCACAGTGGTA nt 1020-1039 [SEQ ID NO:29]

(Size of the sequence amplified: 696 bp.)

Pair Primer 1 : CGAATCCAGAAGGTGGATGT nt 344-363 H-3 [SEQ ID NO:30]

Primer 2: GTAGTGTTCCTCAGTGACGT nt 891-910 [SEQ ID NO:31]

(Size of sequence amplified 566 bp.)

Pair Primer 1 : nt 56-75 H-9 CGGAATTCATGGTCGGGAGAGTTCAACC [SEQ ID NO:32]

Primer 2: GTAGTGTTCCTCAGTGACGT nt 891-910 [SEQ ID NO:33]

(Size of the sequence amplified: 854 bp.)

Pair Primer 1 : nt 56-75 H-8 CGGAATTCATGGTCGGGAGAGTTCAACC [SEQ ID NO:34]

Primer 2: TCAACCAGGCCACTTTCGAG nt 232-251 [SEQ ID NO:35]

(Size of the sequence amplified: 195 bp.)

Pair Primer 1 : TCTAAGAGCAATGAAGCCAC nt 725-744 H-5 [SEQ ID NO:36]

Primer 2: TTAACATTTGCTGAGAAGGC nt 1053-1072 [SEQ ID NO:37]

(Size of the sequence amplified: 347 bp.)

The sequences amplified from human cerebral cortex cDNA by these primers were cloned into pCR II (Invitrogen. San Diego, CA) or pGEM T (Promega, Madison, WI). The inserts were sequenced by the chain termination method and the assembled sequence designated SEQ ID NO: l (see Fig. 3). The amplified regions are indicated in Fig. 1.

Example 3 - Northern Blot Analysis of LAMP Expression

Total cellular RNA was isolated from various tissues of adult, Sprague-Dawley rats and the poly-adenylated fraction was isolated therefrom using the PolyATract™ isolation system (Promega, Madison, WI). For each tissue-type, 3 μg of poly(A)+ RNA was separated on an agarose-formaldehyde gel, transferred to a nylon membrane (Nytran™, Schleicher & Schuel, Keene, NH), UV cross-linked and hybridized overnight under stringent conditions with one of the ³² P probes described below. The first probe was an RNA transcript, produced with T7 RNA polymerase, of a pCR II vector containing the rat cDΝA insert that had been linearized with Bal I. This process produced a probe of the 519-1238 sequence of Figure 3 (rat). A sense control probe was prepared with SP6 RNA polymerase acting on a Νar I-digested rat LAMP PCRII template (Νt. 1-527 of Figure 3). An 53 base oligonucleotide probe corresponding to nucleotides 973-1025 of SEQ ID ΝO:2 was labelled by adding a ³² P poly(A) ^~ tail.

The first LAMP probe identified a 1.6 and a 8.0 kb transcript in Hippocampus, perirhinal cortex and cerebellum, but identified no transcripts in kidney, lung or liver. The oligonucleotide probe, derived from a region with little homology with OBCAM, also hybridized with the 1.6 and 8.0 kb transcripts.

Example 4 - Histochemistrv For these studies, the 53 base oligonucleotide probe was labelled with an ³⁵ S poly(A) tail. Tissue slices, fixed with 4% formaldehyde, were hybridized with the probe at 58 °C, at a probe concentration of 10 ⁶ cpm/ml in hybridization buffer (50% formamide, 10% dextran sulfate, 0.2 M NaCI, 1 X Denhardfs solution, 10 mM Tris, 1 mM EDTA, pH 8.0). Stringent condition post-hybridization washes included 1 hour in 1 X SSC at 60°C. A control 53 base oligonucleotide probe, comprising the sequence complementary to the primary probe, was used on corresponding control tissue slices, but produced no signal. Signal was detected by autoradiography.

In a coronal section through the for brain dissected from a day E16 rat embryo, intense hybridization in the limbic perirhinal cortical region (pr) and hypothalamus (hy), and a small signal over background in the non-limbic dorsal sensorimotor cortex, were noted. A section from day E20 showed LAMP expression high in the perirhinal region of the cortex (pr) and sparse in the dorsal, non-limbic cortex. LAMP expression in the embryo was high in the hippocampus (h) and midthalamic region, including the mediodorsal nucleus of the thalamus (md). In sections from adult rat brain, hybridization was high in the perirhinal cortex (pr).

amygdala (a), hypothalamus and medial thalamic region (md), but sparse in the sensorimotor cortex. When a riboprobe spanning regions of the LAMP cDNA with high homology with OBCAM and neurotrimin was used, additional staining was observed.

These LAMP staining patterns are closely analogous to, though not identical with, the patterns observed with the 2G9 antibody. Chesselet et al., Neurosci. 40: 725-733, 1991; Levitt, Science 223: 299-301, 1984.

Example 5 - Recombinant Expression of LAMP in Chinese Hamster Ovarv Cells

The rat cDNA was subcloned into the EcoRI site of the eukaryotic expression vector pcDNA3 (Invitrogen, San Diego, CA). CHO cells were transfected with 10-15 μg of this subcloned vector or, to create control cells, with the pcDNA3 vector lacking an insert. Transfection was accomplished using the calcium phosphate co-precipitation method. Ishiura et al., Mol. Cell Biol. 2, 607-616, 1982: 5463-5467, 1977. Stably transformed cells were selected by growth in the presence of G418 (Life Technologies, Grand Island, NJ) and subcloned by limiting dilution (to select a genetically homogeneous colony). The LAMP cDNA transfected cells were designated CHO _L , while the control transformants were designated CHO _Veclor .

To confirm the cell-surface expression of LAMP, the CHO _L cells were incubated with mouse anti-LAMP, washed four times with DMEM/ 10% FCS, incubated with FITC conjugated donkey anti-mouse antibody (Jackson, Immunoresearch, West Grove, PA), fixed with 4% formaldehyde, and mounted in glycerol/PBS with 5% propyl gallate.

To test whether CHO _L cells can bind external LAMP, fluorescent synthetic beads of 2 μm diameter, which have reactive sites for covalently linking protein (Covasphere™ beads, Duke Scientific, Palo Alto, CA), were coated with native LAMP that had been released from hippocampal membranes with Pi-specific phospholipase C. The beads were incubated with the recombinant cells and the extent of binding to the cell surface determined.

The CHO _L cells were found to have cell-surface LAMP immunoreactivity, while the CHO _Veclor cells did not. When the CHO _L cells were treated with Pi-specific phospholipase C and the released proteins were analyzed by Western blot, a LAMP immunoreactive band of 55 kDa was identified. The released protein from hippocampal membranes has an apparent molecular weight of 64-68 kDa, probably reflecting a greater degree of glycosylation. The LAMP-coated Covasphere™ beads were found to bind the CHO _L cells.

Example 6 - Differentiation Promotion bv the CHO _L Cells

The growth of various embryonic cell populations on substrata of CHOL or CHO _Vect „ was tested. The first two cell populations were LAMP-expressing cells from (A) the hippocampus and (B) the perirhinal cortex. Non-LAMP-expressing cells from (C) the olfactory 5 bulb and (D) the visual cortex were also tested. Primary neurons from E16 embryos were prepared as outlined by Ferri and Levitt, Cerebral Cortex 3; 187-198, 1993. In some experiments, the cells were marked by adding lipophilic dye PKH26 (Sigma Chemical Co., St. Louis, MO); if they were not so marked, an antibody stain was used later in the experiment to identify neural cells. The cells were plated in DMEM/ 10% FCS at a density of 5 X 10 ³

10 cells/ml, 1ml per cm ² , onto coverslips on which there were monolavers of transformed CHO cells. After 48 hours in culture, the cells attached to the coverslips were fixed with ^- % formaldehyde and, if the neural cells were not dye-marked, stained for neural cells with anti- MAP2, as described in Ferri and Levitt, Cerebral Cortex 3: 187-198, 1993. For each category in the experiment, six coverslips were examined and the longest neuron in a randomly selected

15 field of 10-15 process-bearing cells was measured.

When grown on the CHO _L cells, the LAMP-expressing cells exhibited extensive neurite growth within 24 hours, with well-differentiated morphologies, often including long neurites. These cells grew poorly on CHO _v - _ta - cells (see Figure 5). When the neural cells were pre- treated with LAMP antibody, the length of the neurites extended by the CHO _L cells was

20 significantly reduced (Figure 5). The olfactory and visual cells bound the CHO _L substratum, but differentiated poorly, extending shorter neurites. Additionally, these olfactory and visual cells grew equivalently on CHO _vec -. and CHO _L cells.

Example 7 - Interference in Post Natal Development of an 25 Intrahippocampal Circuit bv Antibodies to LAMP

Newborn Sprague-Dawley rats were injected intraventricularly with Fab fragments of anti-LAMP (n= 15). control anti-paramyosin IgG (n= 14), and anti-Ll (n=5). Anti-Ll , which binds to developing axons, was as described by Sweadner, J. Neurosci. 3: 2504-2517, 1983.

30 All antisera was purified on a protein A column using a protein A affinity enhancement buffer (the MAPSII buffer system used as recommended by the supplier. Biorad Labs, Hercules, CA). Fab fragments were prepared from the antisera by digestion with immobilized papain (Pierce, Rockford, IL) and purified by protein-A affinity chromatography. The Fab fragments (10 μg in 10 μl of saline) were injected on postnatal day 0, 2, 4 and 6 into the cisterna magna using a

35 32-gauge needle. On day 9, the animals were sacrificed by transcardial perfusion with 4.9%

sodium sulfide in 0.1 M phosphate buffer (pH 7.4). Brains were fixed in Carnoy's solution together with 1.2% sodium sulfide. Paraffin sections of the brains were prepared for mossy fiber staining using the Timm method. Haug, Adv. Anat. Embryol. Cell Biol. 47: 1-71, 1973. Subfields were analyzed for density of innervation using the Bioquant OS/2 image analysis system (R & M Biometrics, Nashville, TN).

The excitatory glutaminergic mossy fiber projection of granule cells to pyramidal neurons of the hippocampus express LAMP during development. Zacco et al., J. Neurosci. 10: 73-90, 1990; Keller and Levitt, Neuroscience 28: 455-474, 1989. The anti-LAMP treatment, but not the other antibody treatments, resulted in an uncharacteristically diffuse pattern for this mossy fiber projection. Detailed examination found many misdirected fibers. Quantitatively, the treatment resulted in a six fold increase in the area occupied by mossy fiber projections.

Example 8 - Isolation of the 5 ^* Untranscribed Region of the LAMP Gene

Plaques from a genomic library derived from the 129/Rej mouse strain and cloned into Lambda Fix II (Stratagene, LaJolla, CA) were screened with a ³² P-labelled probe generated by randomly priming a plasmid containing nucleotides 1-453 of SEQ ID NO:2. Two positive clones were isolated and sequenced to generate the sequence information of Fig. 5 (SEQ ID NOs: 19 and 20).

Example 9 - Cell-tvpe Specific Expression of CAT Vectors

The nucleotide sequence (nt 1-864) of the mouse genomic SEQ ID NO: 19 was amplified by PCR using the following primers:

Primer 1 : CCGAAGCTTCTGCAGTATGCCTTCCTATCCATGTGTATG [SEQ ID N0.38] Primer 2: ATATCTAGATAGTGGTACCGAGTTGTTCCGCGGTGGACTGCGTGTGCGC [SEQ ID NO:39] and subcloned into the promoterless pCTA-Basic Vector (Promega, Madison, WI) which contains the CAT (Chloramphenicol acetyltransferase) reporter gene. Cell lines were transiently transfected with the pCAT-864 construct, and as a control, with a vector containing an active promoter and the cat gene (Promega, Madison, WI) using the calcium phosphate or the DEAE-Dextran method (Ausubel et al.. Current Protocols in Molecular Biology, Wiley Press, 1993).

Promoter activity on induction of the cat gene was measured by the CAT activity assay using 14C-chloramphenicol and acetyl CoA as substrates for CAT, and separating the acetylated 14C-chloramphenicol products by thin layer chromatography. The product was

detected by autoradiography (Ausubel et al.. Current Protocols in Molecular Biology, Wiley Press, 1993) and quantitated by liquid scintillation counting.

This fragment of the genomic DNA induces expression of the cat gene in SN56 cells (a limbic neuronal cell line described in H.J. Lee et al., Dev. Brain Res. 52, 219-228, 1990), but not in N2A cells (a neuroblastoma cell line) or CHO cells (a non-neuronal cell line).

Example 10 - Construction of Deletion Mutants

To examine the structure-function relationships of the various domains of LAMP, a number of deletion mutants were constructed. Nucleotides 17 through 1000 of SEQ ID NO:2 (which encodes amino acids 1 - 315) was generated by PCR amplification. The primers had overhang regions that were designed to create terminal restriction sites which were used to insert the amplified sequence into the pcDNA3 expression vector. The 3 ' primer overhang also included sequence for encoding six repeats of histidine followed by a stop codon. CHO cells were transformed with the recombinant expression vector and cultured. The recombinant protein produced by these cells was exported into the culture medium. To purify the protein, the medium was passed over an ionic nickel column (Ni-NTA, available from Qiagene, Chatsworth, CA), which bound the poly-His tail of the recombinant protein.

An analogous strategy was used to create recombinant proteins for nucleotides 374 through 1001 (amino acids 107-315, including the second and third immunoglobulin-like region) and nucleotides 713 through 907 (amino acids 220 - 284, including substantially all of the third immunoglobulin-like domain). These peptide sequences were expressed as fusion proteins together with a maltose binding domain. To do this, the sequences were subcloned into the pMaltose vector available from New England Biolabs (Beverly, MA) and the recombinant vectors were used to transform bacteria. The fusion proteins were isolated from bacterial lysates by column chromatography on an amylose affinity resin (available from New England Biolabs).

These proteins are tested by, for instance, the methods outlined above to determine whether they had LAMP binding activity and/or the ability to promote the formation of neurites.

Example 11 - Identification and Analysis of a Variant Rat LAMP cDNA

Clone 6c is a rat LAMP cDNA that was identified in identical fashion to the rat LAMP cDNA of SEQ ID NO.2, as set forth in Example 1. Using standard cloning and sequencing methods as set forth and referred to above, clone 6c was shown to have an insert of 69

nucleotides, which was inserted in the rat LAMP cDNA between nucleotides 974 and 975, as recited in SEQ ID NO:2. The inserted nucleotides, recited from 5' to 3 ', were shown to be: agcgtgttttacccacagtcccccaccctattcaagaaattggt accaccgtgcacttcaagcaaaaag [SEQ ID NO: 40] Insertion of SEQ ID NO:40 just after position 974 of SEQ ID NO: 2 changed the codon that included position 974 from AGA (which encodes arginine) to AAG (which encodes lysine). The corresponding amino acid sequence encoded by the insert in clone 6c, starting from nucleotide position 974 as recited in SEQ ID NO:2, is

K R V L P T V P H P I Q E I G T T V H F K Q K G [SEQ ID NO.41] The remainder of the protein encoded by clone 6c is the same as m SEQ ID NO.2, starting with amino acid 308 to the carboxy terminus; the sequence of the LAMP variance disclosed here is SEQ ID NO.3.

A probe that included SEQ ID No.40 was used in a reverse transcπptase-polymerase chain reaction (RT-PCR) experiment to determine whether the same LAMP variant was present and expressed in human cortical tissue. Using the RT-PCR protocol as recited in Current Protocols in Molecular Biology, vol. 3 (New York' John Wiley & Sons, 1995), it was shown that humans and rats both express LAMP genes that have the same insertion.

Example 12 - Inhibition Bv Ca ^"1*2 Channel Antagonists or Potassium Depolarization of Neurite Outgrowth on CHO Cells Expressing LAMP

The effect of long-term application of nifedφine, ω-conotoxin and the combination of both on neurite growth on CHO cells transfected with LAMP or vector alone was observed To set up this study, the rat lamp cDNA [SEQ ID NO.2] was subcloned from pCR II (as set forth in Example 1) into the EcoRI site of the eukariotic expression vector pcDNA (Invitrogen) CHO cells were transfected with pcDN A-lamp as described by Pimenta et al., Neuron. .15, 287-297 (1995) Primary neurons from E17 embryos were prepared as described by Fern and Levitt, Cerebral Cortex, 3, 197-198 (1993); also see Zhukareva and Levitt, Development, Hi, 1161-1172 (1995) and Pimenta et al., supra Cells were plated in DMEM/10% FCS at a density of 5 x 10 ³ cells/ml on confluent monolayers of CHO cells transfected with pcONA-lamp or pcDNA only (control) After 4 hours in culture, ω-conotoxin (Sigma Chemical Co , 1 μg/ml), nifedipme (Sigma Chemical Co.; 10 μg/ml) or a combination of both were added to the culture In some experiments, KC1 at a final concentration 30 mM was used during all times of cultuπng. After 48 hours in culture, coverslips were fixed with 4% formaldehyde, stained with the neuronal marker antι-MAP2 (Fern and Levitt, supra, Zhukareva and Levitt, supra)

mounted, and examined under a fluorescent microscope. Quantitative analysis was performed as described by Pimenta et al., supra. The length of the longest neurite was measured on each of fiver coverslips prepared for each category in three different experiments. A Bioquant image analysis system was used to digitize and measure the images. The results are summarized in Fig. 7 wherein each of Figs. 7A. 7B, 7C and 7D have a y-axis labeled and representing " % cells with neurite length longer than X" and an x-axis labeled and representing "neurite length, μm". Each point on the graphs represents the percentage of total cells with neurites longer than X. In Fig. 7A, the curve representing results based on control cells is labeled with open squares (D); cells treated with conotoxin, closed diamonds ( ♦ ); cells treated with nifedipine, open circles (O); and cells treated with a combination of nifedipine and conotoxin, closed triangles ( A ). In Fig. 7B, the curve representing results based on control cells is labeled with open squares (D); cells treated with conotoxin, closed diamonds ( ♦ ); cells treated with nifedipine, open circles (O); and cells treated with a combination of nifedipine and conotoxin, closed triangles ( A ). In Fig. 7C, the curve representing results based on control cells is labeled with open squares (□); and the cells treated with 25 mM KCl, closed diamonds ( ♦ ). And in Fig. 7D, the curve representing results based on first control cells (i.e., CHO-pC DNA 3) is labeled with closed squares (■); cells treated with 25 mM KCl, closed diamonds ( ♦ ); and cells treated with second control cells (i.e., CHO-LAMP), open squares (D). Fig. 7 A demonstrates that blocking of L-type calcium channels with 10 μ of nifedipine was sufficient to completely abolish the LAMP effect. As to the treatment with 0.5 μg/ml ω-conotoxin, the blocking effect was not so prominent, suggesting the minor role of N-type of calcium channel in LAMP-stimulated neurite outgrowth. Combination of both drugs did not have additional blocking effect on the neurite outgrowth. The control experiments are in agreement with the results from Williams et al., Molecular and Cellular Neuroscience. 6, 69-79 (1992). demonstrating that these agents do not modulate neurite outgrowth over parental cells transfected with vector only (See Fig. 7B).

The results of the studies directed at the question of whether K ⁺ -depolarization can mimic LAMP-dependent neurite outgrowth are shown in Figs. 7C and 7D. The effect of culturing embryonic hippocampal neurons in the presence of a depolarizing concentration of potassium was the object of this aspect of the study. Potassium depolarization is known to cause an influx of calcium mainly through L-type calcium channels (Harper et al., Cell Adhesion and Commun.. 2. 441-453 (1994). Neurons cultured on the monolayers of control CHO cells in the presence of 35 mM KCl demonstrated a substantial increase in neurite

outgrowth that was comparable in magnitude to the outgrowth on the LAMP-transfected cells in the presence of a depolarizing concentration of KCl was not significantly different from the one under the normal conditions of culturing (i.e., 5 mM of KCl; Fig 7C). The result of this experiment shows that potassium depolarization fully mimics the LAMP-induced outgrowth from hippocampal neurons in culture.

Example 13 - LAMP-induced Changes in Internal Calcium Concentration

To examine whether LAMP protein can cause Ca ² * changes in hippocampal neurons, soluble recombinant LAMP was purified using standard means and applied to the neurons growing on laminin-coated coverslips, using the following protocol:

Freshly dissociated E17 neurons were plated on 25-mm glass coverslips coated with poly-L-lysine in DMEM/10% FCS. After cells were allowed to attach, the media was changed for N2 defined media (Bottenstein and Sato, Proc. Natl. Acad. Sci. USA. 76, 514-517 (1974)) with B27 supplement (Gibco). After 3, 5 and 7 days in culture, coverslips were rinsed several times with buffer A: 125 mM NaCI; 5 mM KCl; 1 mM MgSO ₄ ; 2 mM CaCl ₂ ; 10 mM glucose; 25 mM HEPES; at pH 7.3. The rinsed coverslips were then loaded witJi a sonicated combination of cell permeable AM form of 2.5 μM fluo-3 and 5 μM Pluronic (Molecular Probes, Inc., Eugene, OR) in buffer A at 37° for 20 minutes. After several washes with the same buffer, the coverslips were incubated in buffer A for another 30 minutes to allow the dye to be esterified, and mounted in a specially-designed chamber capable of holding up to 35 μl of solution on the stage of a Zeiss Axovert microscope (BioRad) with a 20 objective. Soluble form of recombinant LAMP was purified using a Ni-affinity column. Purified LAMP (100 μg/ml) or buffer alone was added into the chamber. A time series of images was obtained every minute during 40 minutes of incubation at room temperature using BioRad MRC-600 laser scanning confocal imaging system. The fluo-3 was excited with 488 nm line of argon ion laser. The filter set appropriate for single labeled FITC samples with excitor filter 488 DF 10, dichromic reflector 510 LP and emission filter 515 LP were used. Five frames were averaged with Kalman filter. Images were processed using OptimasS software (Optimas Corp.). Mean fluorescence of pixels that reach the threshold value were measured within the boundaries of neurons and plotted using Origin 3.73 software (Microcal Software Inc.).

The base line of internal calcium concentration (represented as [Ca ²⁺ ],) in the neurons incubated with buffer alone was stable during the experiment, indicating that cells remained intact and did not suffer from phototoxicity during fluorescence monitoring. Also, after application of 80 mM of KCl at the end of each experiment, the fluorescence substantially

increased (two-three-fold), indicating the viability of neurons. In the initial experiments, neurons cultured for three days were used. Application of LAMP (100 μg/ml) to hippocampal neurons caused the sustained elevation in [Ca ²⁺ ] in 25 % of the cells examined. When LAMP was applied to the hippocampal neurons growing for 5 or 7 days in culture, the number of cells responding increased to 33.4% , with typical delayed response (See Table 1). The average latency after LAMP application was 9.5 ± 4.5 minutes in all experiments regardless of the age of cultured cells. Further attempts to investigate the origin of [Ca ^:+ ]j increase and to do measurements with cells maintained in Ca ²⁺ -free medium failed because cells changed their morphology and lifted off the coverslips after 20 minutes of incubation. Table 1 . Summary of calcium responses to LAMP application in embryonic hippocampal neurons.

% cells % cells responding with different normalized peak F / F. values responding w ⁱ th Ca-. 1 .25 < F/F. < 1 .5 1.5 < F/F. < 1.75 1.75 < F/F. < 2.0 F/F. > 2.0 Respond ^ιncrθase latency.

Min. * *

Control > 1 % (144)

LAMP 35.7% 15.1 % 6.3% 6.5% 5.7% 9.5 ± 4.5 (158)

Number of cells tested are given in the parentheses.

^* Normalized peak represents the maximum calcium increase during the experiment divided by the baseline value.

* ^* The respond latency was defined as the time between the application of LAMP and the beginning of respond after approximation of data.

Example 14 - LAMP involvement in Schizophrenia

Schizophrenia has both positive and negative symptoms, namely hallucinations, delusions and thought disorder on the one hand, and social withdrawal and flattening of affect on the other. A major difficulty in the management of patients suffering from schizophrenia is that no molecular model exists by which one could rationally identify drugs useful in treatment. The psychotomimetic drug phencyclidine (PCP) has been noted to induce the same positive and negative symptoms of schizophrenia in non-schizophrenic human individuals, in a manner that is indistinguishable from the expression of these symptoms in schizophrenic patients. Accordingly, the pharmacological effect of PCP is related to the underlying pathophysiology of schizophrenia. See Olney et al.. Arch. Gen. Psychiatry. 52. 998-1007 (1995). Rats were chemically administered PCP once a day at dosages of 10 mg per kg per day. and compared to control rats that received no PCP. Using standard laboratory techniques, treated and control rats were sacrificed at day 8, their brains dissected and frozen, and sections thereof prepared for in situ hybridization using LAMP specific probes disclosed herein.

PCP-treated rats were thus shown to have up-regulated the concentration of LAMP messenger RNA located in the cingulate cortex relative to control animals. Accordingly, LAMP activity is altered under conditions known to create psychotic behavioral states in humans.

Example 15 - Drug Discovery for Schizophrenia

Because PCP treatment was shown to result in altered regulation of LAMP mRNA concentration in rats, modification of LAMP activity is hypothesized to provide a therapeutic effect for schizophrenics. Small molecules, such as peptides, oligonucleotides, simple or complex sugars, organic compounds, and other agents, as generated in combinatorial libraries and the like, for example, are tested for impact on LAMP activity in the following test procedure:

CHO cells transfected by LAMP, as set forth in Example 5, are distributed into the wells of a 96-well plate, 1 x 10 transfected cells per 90 μl pere well. Sets of three wells receive the same agent, delivered from a dilution series of 1 mg/ml, 100 μg/ml, and 10 μg/ml, wherefrom 10 μl of each is added to each of the three wells of the set, respectively. The cells are then incubated for 2 hours, and then observed for fluorescence, using the same method as set forth in Example 13. As noted above, the fluorescence is directly correlated with free internal calcium concentration, which in turn correlates with LAMP activity. Agents shown to cause a lowering of free internal calcium concentration are then selected for a second screen, as follows:

Instead of LAMP-transferred CHO cells, the second-stage screen uses primary animal brain cells dissected from the cingulate cortex. These cells already express LAMP. All other aspects of the second-stage screen are identical to the first-stage screen. In another approach, because LAMP-LAMP homotypic interactions have been shown to promote cell adhesion (Pimenta et al., supra), agents that modify LAMP activity at this level are also hypothesized to provide a therapeutic effect in schizophrenia. Such agents are identified in a screening assay in which LAMP-transfected CHO cells adhere, as described in Pimenta et al., supra, based upon which the facilitation or inhibition of LAMP-mediated adhesion is tested.

Subsequent development involves subjecting positive compounds as to efficacy in model animal systems for schizophrenia, followed by toxicity studies geared toward human use of the LAMP-class of antipsychotic, including schizophrenia-indicated, drugs identified by the present invention.

(1) GENERAL INFORMATION

(i) APPLICANT: Levitt, Pat Ressler, Pimenta, Aurea, Fischer,

Itzhak, Zhukareva, Victoria

(ii) TITLE OF INVENTION: Limbic System-Associated Membrane Protein and DNA

(iii) NUMBER OF SEQUENCES:

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE : Dechert Price & Rhoads

(B) STREET: P.O. Box 5218

(C) CITY: Princeton

(D) STATE: New Jersey

(E) COUNTRY: USA

(F) ZIP: 08543-5218

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch, 1.44 Mb storage

(B) COMPUTER: IBM-compatible

(C) OPERATING SYSTEM: DOS 5.0

(D) SOFTWARE: WordPerfect (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

<B) FILING DATE: March 29, 1996

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Allen Bloom

(B) REGISTRATION NUMBER: 29,135

(C) REFERENCE/DOCKET NUMBER: 317743-102 WO (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (609) S20-3214

(B) TELEFAX: (609) 520-3259

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 977 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LIBRARY: CDNA

(K) RELEVANT RESIDUES IN SEQ ID NO: 1 :FROM 1 TO 977 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

G GAT CGG AAA CAG TTG CCA CTG GTC CTA CTG AGA TTG CTC TGC CTT 46 Asp Arg Lys Gin Leu Pro Leu Val Leu Leu Arg Leu Leu Cys Leu 10 15 20

CTT CCC ACA GGA CTG CCT GTT CGC AGC GTG GAT TTT AAC CGA GGC 91 Leu Pro Thr Gly Leu Pro Val Arg Ser Val Asp Phe Asn Arg Gly 25 30 35

ACG GAC AAC ATC ACC GTG AGG CAG GGG GAC ACA GCC ATC CTC AGG 136 Thr Asp Asn lie Thr Val Arg Gin Gly Asp Thr Ala lie Leu Arg 40 45 50

TGC GTT CTA GAA GAC AAG AAC TCA AAG GTG GCC TGG TTG AAC CGT 181 Cys Val Leu Glu Asp Lys Asn Ser Lys Val Ala Trp Leu Asn Arg 55 60 65

TCT GGC ATC ATT TTT GCT GGA CAT GAC AAG TGG TCT CTG GAC CCA 226 Ser Gly lie lie Phe Ala Gly His Asp Lys Trp Ser Leu Asp Pro 70 75 80

CGG GTT GAG CTG GAG AAA CGC CAT TCT CTG GAA TAC AGC CTC CGA 271 Arg Val Glu Leu Glu Lys Arg His Ser Leu Glu Tyr Ser Leu Arg 85 90 95

ATC CAG AAG GTG GAT GTC TAT GAT GAG GGT TCC TAC ACT TGC TCA 316 lie Gin Lys Val Asp Val Tyr Asp Glu Gly Ser Tyr Thr Cys Ser 100 105 110

GTT CAG ACA CAG CAT GAG CCC AAG ACC TCC CAA GTT TAC TTG ATC 361 Val Gin Thr Gin His Glu Pro Lys Thr Ser Gin Val Tyr Leu lie 115 120 125

GTA CAA GTC CCA CCA AAG ATC TCC AAT ATC TCC TCG GAT GTC ACT 406 Val Gin Val Pro Pro Lys lie Ser Asn He Ser Ser Asp Val Thr 130 135 140

GTG AAT GAG GGC AGC AAC GTG ACT CTG GTC TGC ATG GCC AAT GGC 451 Val Asn Glu Gly Ser Asn Val Thr Leu Val Cys Met Ala Asn Gly 145 150 155

CGT CCT GAA CCT GTT ATC ACC TGG AGA CAC CTT ACA CCA ACT GGA 496 Arg Pro Glu Pro Val He Thr Trp Arg His Leu Thr Pro Thr Gly 160 165 170

AGG GAA TTT GAA GGA GAA GAA GAA TAT CTG GAG ATC CTT GGC ATC 541

Arg Glu Phe Glu Gly Glu Glu Glu Tyr Leu Glu He Leu Gly He 175 180 185

ACC AGG GAG CAG TCA GGC AAA TAT GAG TGC AAA GCT GCC AAC GAG 586 Thr Arg Glu Gin Ser Gly Lys Tyr Glu Cys Lys Ala Ala Asn Glu 190 195 200

GTC TCC TCG GCG GAT GTC AAA CAA GTC AAG GTC ACT GTG AAC TAT 631

Val Ser Ser Ala Asp Val Lys Gin Val Lys Val Thr Val Asn Tyr 205 210 215

CCT CCC ACT ATC ACA GAA TCC AAG AGC AAT GAA GCC ACC ACA GGA 676

Pro Pro Thr He Thr Glu Ser Lys Ser Asn Glu Ala Thr Thr Gly 220 225 230

CGA CAA GCT TCA CTC AAA TGT GAG GCC TCG GCA GTG CCT GCA CCT 721

Arg Gin Ala Ser Leu Lys Cys Glu Ala Ser Ala Val Pro Ala Pro 235 240 245

GAC TTT GAG TGG TAC CGG GAT GAC ACT AGG ATA AAT AGT GCC AAT 766

Asp Phe Glu Trp Tyr Arg Asp Asp Thr Arg He Asn Ser Ala Asn 250 255 260

GGC CTT GAG ATT AAG AGC ACG GAG GGC CAG TCT TCC CTG ACG GTG 811

Gly Leu Glu He Lys Ser Thr Glu Gly Gin Ser Ser Leu Thr Val 265 270 275

ACC AAC GTC ACT GAG GAG CAC TAC GGC AAC TAC ACC TGT GTG GCT 856

Thr Asn Val Thr Glu Glu His Tyr Gly Asn Tyr Thr Cys Val Ala 280 285 290

GCC AAC AAG CTG GGG GTC ACC AAT GCC AGC CTA GTC CTT TTC AGA 901

Ala Asn Lys Leu Gly Val Thr Asn Ala Ser Leu Val Leu Phe Arg 295 300 305

CCT GGG TCG GTG AGA GGA ATA AAT GGA TCC ATC AGT CTG GCC GTA 946

Pro Gly Ser Val Arg Gly He Asn Gly Ser He Ser Leu Ala Val 310 315 320

CCA CTG TGG CTG CTG GCA GCA TCT CTG CTC T 977

Pro Leu Trp Leu Leu Ala Ala Ser Leu Leu 325 330

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1238 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:2 :FROM 1 TO 123B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GTGGCCAGCA GCGCGCACAC GCGAGTCCAC CGCTGACCAA CTCGCCGAGG 50

CCACC ATG GTC GGG AGA GTT CAA CCT GAT CGG AAA CAG TTG CCA CTG 97 Met Val Gly Arg Val Gin Pro Asp Arg Lys Gin Leu Pro Leu 1 5 10

GTC CTA CTG AGA CTG CTC TGC CTT CTT CCC ACA GGA CTG CCC GTT 142 Val Leu Leu Arg Leu Leu Cys Leu Leu Pro Thr Gly Leu Pro Val 15 20 25

CGC AGC GTG GAT TTT AAC CGA GGC ACG GAC AAC ATC ACC GTG AGG 187 Arg Ser Val Asp Phe Asn Arg Gly Thr Asp Asn He Thr Val Arg 30 35 40

CAG GGG GAC ACG GCC ATC CTC AGG TGT GTG GTA GAA GAC AAG AAC 232 Gin Gly Asp Thr Ala He Leu Arg Cys Val Val Glu Asp Lys Asn 45 50 55

TCG AAA GTG GCC TGG TTG AAC CGC TCT GGC ATC ATC TTC GCT GGA 277 Ser Lys Val Ala Trp Leu Asn Arg Ser Gly He He Phe Ala Gly 60 65 70

CAC GAC AAG TGG TCT CTG GAC CCT CGG GTT GAG CTG GAG AAA CGC 322 His Asp Lys Trp Ser Leu Asp Pro Arg Val Glu Leu Glu Lys Arg 75 80 85

CAT GCT CTG GAA TAC AGC CTC CGA ATC CAG AAG GTG GAT GTC TAT 367 His Ala Leu Glu Tyr Ser Leu Arg He Gin Lys Val Asp Val Tyr 90 95 100

GAT GAA GGA TCC TAC ACA TGC TCA GTT CAG ACA CAG CAT GAG CCC 412 Asp Glu Gly Ser Tyr Thr Cys Ser Val Gin Thr Gin His Glu Pro 105 110 115

AAG ACC TCT CAA GTT TAC TTG ATT GTA CAA GTT CCA CCA AAG ATC 457 Lys Thr Ser Gin Val Tyr Leu He Val Gin Val Pro Pro Lys He 120 125 130

TCC AAC ATC TCC TCG GAT GTC ACT GTG AAT GAG GGC AGC AAT GTA 502 Ser Asn He Ser Ser Asp Val Thr Val Asn Glu Gly Ser Asn Val 135 140 145

ACC CTG GTC TGC ATG GCC AAT GGG CGC CCT GAA CCT GTT ATC ACC 547 Thr Leu Val Cys Met Ala Asn Gly Arg Pro Glu Pro Val He Thr 150 155 160

TGG AGA CAC CTT ACA CCA CTT GGA AGA GAA TTT GAA GGA GAA GAA 592 Trp Arg His Leu Thr Pro Leu Gly Arg Glu Phe Glu Gly Glu Glu 165 170 175

GAA TAT CTG GAG ATC CTA GGC ATC ACC AGG GAA CAG TCA GGC AAA 637 Glu Tyr Leu Glu He Leu Gly He Thr Arg Glu Gin Ser Gly Lys 180 185 190

TAT GAG TGC AAG GCT GCC AAC GAG GTC TCC TCC GCG GAT GTC AAA 682 Tyr Glu Cys Lys Ala Ala Asn Glu Val Ser Ser Ala Asp Val Lys 195 200 205

CAA GTC AAG GTC ACT GTG AAC TAT CCA CCC ACC ATC ACA GAG TCT 727 Gin Val Lys Val Thr Val Asn Tyr Pro Pro Thr He Thr Glu Ser 210 215 220

AAG AGC AAT GAA GCC ACC ACA GGA CGA CAA GCT TCC CTC AAA TGT 772 Lys Ser Asn Glu Ala Thr Thr Gly Arg Gin Ala Ser Leu Lys Cys 225 230 235

GAA GCC TCA GCG GTG CCT GCA CCT GAC TTT GAG TGG TAC CGG GAT 817 Glu Ala Ser Ala Val Pro Ala Pro Asp Phe Glu Trp Tyr Arg Asp 240 245 250

GAC ACC AGG ATA AAC AGT GCA AAC GGC CTT GAG ATT AAG AGC ACT 862 Asp Thr Arg He Asn Ser Ala Asn Gly Leu Glu He Lys Ser Thr 255 260 265

GAG GGC CAG TCC TCC CTG ACG GTG ACC AAC GTC ACT GAG GAA CAC 907 Glu Gly Gin Ser Ser Leu Thr Val Thr Asn Val Thr Glu Glu His 270 275 280

TAC GGC AAC TAT ACC TGT GTG GCT GCC AAC AAG CTC GGC GTC ACC 952 Tyr Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly Val Thr 285 290 295

AAT GCC AGC CTA GTC CTT TTC AGA CCC GGG TCG GTG AGA GGA ATC 997 Asn Ala Ser Leu Val Leu Phe Arg Pro Gly Ser Val Arg Gly He 300 305 310

AAC GGA TCC ATC AGT CTG GCC GTA CCA CTG TGG CTG CTG GCA GCG 1042 Asn Gly Ser He Ser Leu Ala Val Pro Leu Trp Leu Leu Ala Ala 315 320 325

TCC CTG TTC TGC CTT CTC AGC AAA TGT TAATAGAATA AA 1081

Ser Leu Phe Cys Leu Leu Ser Lys Cys 330 335

AATTTAAAAA TAATTACAAA ACACACAAAA ATGCGTCACA CAGATACAGA 1131

GAGAGAGAGA GAGAGAGAGA GAAAGTACAA GATGGGGGGA GACTATTGTT 1181

TCACAAGATT GTGTGTTTAT AAATGAAGGG GGGATATGAA AAAAATGAAG 1231

AAAATAC 1238

(2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1014 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: CDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:4:FROM 1 TO 1014 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

ATG GTC GGG AGA GTT CAA CCT GAT CGG AAA CAG TTG CCA CTG 42 Met Val Gly Arg Val Gin Pro Asp Arg Lys Gin Leu Pro Leu 1 5 10

GTC CTA CTG AGA CTG CTC TGC CTT CTT CCC ACA GGA CTG CCC GTT 87 Val Leu Leu Arg Leu Leu Cys Leu Leu Pro Thr Gly Leu Pro Val 15 20 25

CGC AGC GTG GAT TTT AAC CGA GGC ACG GAC AAC ATC ACC GTG AGG 132 Arg Ser Val Asp Phe Asn Arg Gly Thr Asp Asn He Thr Val Arg 30 35 40

CAG GGG GAC ACG GCC ATC CTC AGG TGT GTG GTA GAA GAC AAG AAC 177 Gin Gly Asp Thr Ala He Leu Arg Cys Val Val Glu Asp Lys Asn 45 50 55

TCG AAA GTG GCC TGG TTG AAC CGC TCT GGC ATC ATC TTC GCT GGA 222 Ser Lys Val Ala Trp Leu Asn Arg Ser Gly He He Phe Ala Gly 60 65 70

CAC GAC AAG TGG TCT CTG GAC CCT CGG GTT GAG CTG GAG AAA CGC 267 His Asp Lys Trp Ser Leu Asp Pro Arg Val Glu Leu Glu Lys Arg 75 80 85

CAT GCT CTG GAA TAC AGC CTC CGA ATC CAG AAG GTG GAT GTC TAT 312 His Ala Leu Glu Tyr Ser Leu Arg He Gin Lys Val Asp Val Tyr 90 95 100

GAT GAA GGA TCC TAC ACA TGC TCA GTT CAG ACA CAG CAT GAG CCC 357 Asp Glu Gly Ser Tyr Thr Cys Ser Val Gin Thr Gin His Glu Pro 105 110 115

AAG ACC TCT CAA GTT TAC TTG ATT GTA CAA GTT CCA CCA AAG ATC 402 Lys Thr Ser Gin Val Tyr Leu He Val Gin Val Pro Pro Lys He 120 125 130

TCC AAC ATC TCC TCG GAT GTC ACT GTG AAT GAG GGC AGC AAT GTA 447 Ser Asn He Ser Ser Asp Val Thr Val Asn Glu Gly Ser Asn Val 135 140 145

ACC CTG GTC TGC ATG GCC AAT GGG CGC CCT GAA CCT GTT ATC ACC 492 Thr Leu Val Cys Met Ala Asn Gly Arg Pro Glu Pro Val He Thr 150 155 160

TGG AGA CAC CTT ACA CCA CTT GGA AGA GAA TTT GAA GGA GAA GAA 537 Trp Arg His Leu Thr Pro Leu Gly Arg Glu Phe Glu Gly Glu Glu 165 170 175

GAA TAT CTG GAG ATC CTA GGC ATC ACC AGG GAA CAG TCA GGC AAA 582 Glu Tyr Leu Glu He Leu Gly He Thr Arg Glu Gin Ser Gly Lys 180 185 190

TAT GAG TGC AAG GCT GCC AAC GAG GTC TCC TCC GCG GAT GTC AAA 627 Tyr Glu Cys Lys Ala Ala Asn Glu Val Ser Ser Ala Asp Val Lys 195 200 205

CAA GTC AAG GTC ACT GTG AAC TAT CCA CCC ACC ATC ACA GAG TCT 672 Gin Val Lys Val Thr Val Asn Tyr Pro Pro Thr He Thr Glu Ser 210 215 220

AAG AGC AAT GAA GCC ACC ACA GGA CGA CAA GCT TCC CTC AAA TGT 717 Lys Ser Asn Glu Ala Thr Thr Gly Arg Gin Ala Ser Leu Lys Cys 225 230 235

GAA GCC TCA GCG GTG CCT GCA CCT GAC TTT GAG TGG TAC CGG GAT 762 Glu Ala Ser Ala Val Pro Ala Pro Asp Phe Glu Trp Tyr Arg Asp 240 245 250

GAC ACC AGG ATA AAC AGT GCA AAC GGC CTT GAG ATT AAG AGC ACT 807 Asp Thr Arg He Asn Ser Ala Asn Gly Leu Glu He Lys Ser Thr 255 260 265

GAG GGC CAG TCC TCC CTG ACG GTG ACC AAC GTC ACT GAG GAA CAC 852 Glu Gly Gin Ser Ser Leu Thr Val Thr Asn Val Thr Glu Glu His 270 275 280

TAC GGC AAC TAT ACC TGT GTG GCT GCC AAC AAG CTC GGC GTC ACC 897 Tyr Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly Val Thr 285 290 295

AAT GCC AGC CTA GTC CTT TTC AGA CCC GGG TCG GTG AGA GGA ATC 942 Asn Ala Ser Leu Val Leu Phe Arg Pro Gly Ser Val Arg Gly He 300 305 310

AAC GGA TCC ATC AGT CTG GCC GTA CCA CTG TGG CTG CTG GCA GCG 987 Asn Gly Ser He Ser Leu Ala Val Pro Leu Trp Leu Leu Ala Ala 315 320 325

TCC CTG TTC TGC CTT CTC AGC AAA TGT 1014

Ser Leu Phe Cys Leu Leu Ser Lys Cys 330 335

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 912 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:5:FROM 1 TO 912 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GTT CGC AGC GTG GAT TTT AAC CGA GGC 27

Val Arg Ser Val Asp Phe Asn Arg Gly 30 35

ACG GAC AAC ATC ACC GTG AGG CAG GGG GAC ACA GCC ATC CTC AGG 72 Thr Asp Asn He Thr Val Arg Gin Gly Asp Thr Ala He Leu Arg 40 45 50

TGC GTT CTA GAA GAC AAG AAC TCA AAG GTG GCC TGG TTG AAC CGT 117 Cys Val Leu Glu Asp Lys Asn Ser Lys Val Ala Trp Leu Asn Arg 55 60 65

TCT GGC ATC ATT TTT GCT GGA CAT GAC AAG TGG TCT CTG GAC CCA 162 Ser Gly He He Phe Ala Gly His Asp Lys Trp Ser Leu Asp Pro 70 75 80

CGG GTT GAG CTG GAG AAA CGC CAT TCT CTG GAA TAC AGC CTC CGA 207 Arg Val Glu Leu Glu Lys Arg His Ser Leu Glu Tyr Ser Leu Arg 85 90 95

ATC CAG AAG GTG GAT GTC TAT GAT GAG GGT TCC TAC ACT TGC TCA 252 He Gin Lys Val Asp Val Tyr Asp Glu Gly Ser Tyr Thr Cys Ser 100 105 110

GTT CAG ACA CAG CAT GAG CCC AAG ACC TCC CAA GTT TAC TTG ATC 297 Val Gin Thr Gin His Glu Pro Lys Thr Ser Gin Val Tyr Leu He 115 120 125

GTA CAA GTC CCA CCA AAG ATC TCC AAT ATC TCC TCG GAT GTC ACT 342 Val Gin Val Pro Pro Lys He Ser Asn He Ser Ser Asp Val Thr 130 135 140

GTG AAT GAG GGC AGC AAC GTG ACT CTG GTC TGC ATG GCC AAT GGC 387 Val Asn Glu Gly Ser Asn Val Thr Leu Val Cys Met Ala Asn Gly 145 150 155

CGT CCT GAA CCT GTT ATC ACC TGG AGA CAC CTT ACA CCA ACT GGA 432 Arg Pro Glu Pro Val He Thr Trp Arg His Leu Thr Pro Thr Gly 160 165 170

AGG GAA TTT GAA GGA GAA GAA GAA TAT CTG GAG ATC CTT GGC ATC 477 Arg Glu Phe Glu Gly Glu Glu Glu Tyr Leu Glu He Leu Gly He 175 180 185

ACC AGG GAG CAG TCA GGC AAA TAT GAG TGC AAA GCT GCC AAC GAG 522 Thr Arg Glu Gin Ser Gly Lys Tyr Glu Cys Lys Ala Ala Asn Glu 190 195 200

GTC TCC TCG GCG GAT GTC AAA CAA GTC AAG GTC ACT GTG AAC TAT 567 Val Ser Ser Ala Asp Val Lys Gin Val Lys Val Thr Val Asn Tyr 205 210 215

CCT CCC ACT ATC ACA GAA TCC AAG AGC AAT GAA GCC ACC ACA GGA 612 Pro Pro Thr He Thr Glu Ser Lys Ser Asn Glu Ala Thr Thr Gly 220 225 230

CGA CAA GCT TCA CTC AAA TGT GAG GCC TCG GCA GTG CCT GCA CCT 657 Arg Gin Ala Ser Leu Lys Cys Glu Ala Ser Ala Val Pro Ala Pro 235 240 245

GAC TTT GAG TGG TAC CGG GAT GAC ACT AGG ATA AAT AGT GCC AAT 702 Asp Phe Glu Trp Tyr Arg Asp Asp Thr Arg He Asn Ser Ala Asn 250 255 260

GGC CTT GAG ATT AAG AGC ACG GAG GGC CAG TCT TCC CTG ACG GTG 747 Gly Leu Glu He Lys Ser Thr Glu Gly Gin Ser Ser Leu Thr Val 265 270 275

ACC AAC GTC ACT GAG GAG CAC TAC GGC AAC TAC ACC TGT GTG GCT 792 Thr Asn Val Thr Glu Glu His Tyr Gly Asn Tyr Thr Cys Val Ala 280 285 290

GCC AAC AAG CTG GGG GTC ACC AAT GCC AGC CTA GTC CTT TTC AGA 837 Ala Asn Lys Leu Gly Val Thr Asn Ala Ser Leu Val Leu Phe Arg 295 300 305

CCT GGG TCG GTG AGA GGA ATA AAT GGA TCC ATC AGT CTG GCC GTA 882 Pro Gly Ser Val Arg Gly He Asn Gly Ser He Ser Leu Ala Val 310 315 320

CCA CTG TGG CTG CTG GCA GCA TCT CTG CTC 912

Pro Leu Trp Leu Leu Ala Ala Ser Leu Leu 325 330

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 930 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO: 6 :FROM 1 TO 930 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

GTT Val

CGC AGC GTG GAT TTT AAC CGA GGC ACG GAC AAC ATC ACC GTG AGG 48 Arg Ser Val Asp Phe Asn Arg Gly Thr Asp Asn He Thr Val Arg 30 35 40

CAG GGG GAC ACG GCC ATC CTC AGG TGT GTG GTA GAA GAC AAG AAC 93 Gin Gly Asp Thr Ala He Leu Arg Cys Val Val Glu Asp Lys Asn 45 50 55

TCG AAA GTG GCC TGG TTG AAC CGC TCT GGC ATC ATC TTC GCT GGA 138 Ser Lys Val Ala Trp Leu Asn Arg Ser Gly He He Phe Ala Gly 60 65 70

CAC GAC AAG TGG TCT CTG GAC CCT CGG GTT GAG CTG GAG AAA CGC 183 His Asp Lys Trp Ser Leu Asp Pro Arg Val Glu Leu Glu Lys Arg 75 80 85

CAT GCT CTG GAA TAC AGC CTC CGA ATC CAG AAG GTG GAT GTC TAT 228 His Ala Leu Glu Tyr Ser Leu Arg He Gin Lys Val Asp Val Tyr 90 95 100

GAT GAA GGA TCC TAC ACA TGC TCA GTT CAG ACA CAG CAT GAG CCC 273 Asp Glu Gly Ser Tyr Thr Cys Ser Val Gin Thr Gin His Glu Pro 105 110 115

AAG ACC TCT CAA GTT TAC TTG ATT GTA CAA GTT CCA CCA AAG ATC 318 Lys Thr Ser Gin Val Tyr Leu He Val Gin Val Pro Pro Lys He 120 125 130

TCC AAC ATC TCC TCG GAT GTC ACT GTG AAT GAG GGC AGC AAT GTA 363 Ser Asn He Ser Ser Asp Val Thr Val Asn Glu Gly Ser Asn Val 135 140 145

ACC CTG GTC TGC ATG GCC AAT GGG CGC CCT GAA CCT GTT ATC ACC 408 Thr Leu Val Cys Met Ala Asn Gly Arg Pro Glu Pro Val He Thr 150 155 160

TGG AGA CAC CTT ACA CCA CTT GGA AGA GAA TTT GAA GGA GAA GAA 453 Trp Arg His Leu Thr Pro Leu Gly Arg Glu Phe Glu Gly Glu Glu 165 170 175

GAA TAT CTG GAG ATC CTA GGC ATC ACC AGG GAA CAG TCA GGC AAA 498 Glu Tyr Leu Glu He Leu Gly He Thr Arg Glu Gin Ser Gly Lys 180 185 190

TAT GAG TGC AAG GCT GCC AAC GAG GTC TCC TCC GCG GAT GTC AAA 543 Tyr Glu Cys Lys Ala Ala Asn Glu Val Ser Ser Ala Asp Val Lys 195 200 205

CAA GTC AAG GTC ACT GTG AAC TAT CCA CCC ACC ATC ACA GAG TCT 588 Gin Val Lys Val Thr Val Asn Tyr Pro Pro Thr He Thr Glu Ser 210 215 220

AAG AGC AAT GAA GCC ACC ACA GGA CGA CAA GCT TCC CTC AAA TGT 633 Lys Ser Asn Glu Ala Thr Thr Gly Arg Gin Ala Ser Leu Lys Cys 225 230 235

GAA GCC TCA GCG GTG CCT GCA CCT GAC TTT GAG TGG TAC CGG GAT 678 Glu Ala Ser Ala Val Pro Ala Pro Asp Phe Glu Trp Tyr Arg Asp 240 245 250

GAC ACC AGG ATA AAC AGT GCA AAC GGC CTT GAG ATT AAG AGC ACT 723 Asp Thr Arg He Asn Ser Ala Asn Gly Leu Glu He Lys Ser Thr 255 260 265

GAG GGC CAG TCC TCC CTG ACG GTG ACC AAC GTC ACT GAG GAA CAC 768 Glu Gly Gin Ser Ser Leu Thr Val Thr Asn Val Thr Glu Glu His 270 275 280

TAC GGC AAC TAT ACC TGT GTG GCT GCC AAC AAG CTC GGC GTC ACC 813 Tyr Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly Val Thr 285 290 295

AAT GCC AGC CTA GTC CTT TTC AGA CCC GGG TCG GTG AGA GGA ATC 858 Asn Ala Ser Leu Val Leu Phe Arg Pro Gly Ser Val Arg Gly He 300 305 310

AAC GGA TCC ATC AGT CTG GCC GTA CCA CTG TGG CTG CTG GCA GCG 903 Asn Gly Ser He Ser Leu Ala Val Pro Leu Trp Leu Leu Ala Ala 315 320 325

TCC CTG TTC TGC CTT CTC AGC AAA TGT 930

Ser Leu Phe Cys Leu Leu Ser Lys Cys 330 335

(2) INFORMATION FOR SEQ ID NO: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 924 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no

(iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

<F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:7:FROM 1 TO 924 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 :

GAT CGG AAA CAG TTG CCA CTG GTC CTA CTG AGA TTG CTC TGC CTT 45 Asp Arg Lys Gin Leu Pro Leu Val Leu Leu Arg Leu Leu Cys Leu 10 15 20

CTT CCC ACA GGA CTG CCT GTT CGC AGC GTG GAT TTT AAC CGA GGC 90 Leu Pro Thr Gly Leu Pro Val Arg Ser Val Asp Phe Asn Arg Gly 25 30 35

ACG GAC AAC ATC ACC GTG AGG CAG GGG GAC ACA GCC ATC CTC AGG 135

Thr Asp Asn He Thr Val Arg Gin Gly Asp Thr Ala He Leu Arg 40 45 50

TGC GTT CTA GAA GAC AAG AAC TCA AAG GTG GCC TGG TTG AAC CGT 180 Cys Val Leu Glu Asp Lys Asn Ser Lys Val Ala Trp Leu Asn Arg 55 60 65

TCT GGC ATC ATT TTT GCT GGA CAT GAC AAG TGG TCT CTG GAC CCA 225 Ser Gly He He Phe Ala Gly His Asp Lys Trp Ser Leu Asp Pro 70 75 80

CGG GTT GAG CTG GAG AAA CGC CAT TCT CTG GAA TAC AGC CTC CGA 270 Arg Val Glu Leu Glu Lys Arg His Ser Leu Glu Tyr Ser Leu Arg 85 90 95

ATC CAG A GTG GAT GTC TAT GAT GAG GGT TCC TAC ACT TGC TCA 315 He Gin Lys Val Asp Val Tyr Asp Glu Gly Ser Tyr Thr Cys Ser 100 105 110

GTT CAG ACA CAG CAT GAG CCC AAG ACC TCC CAA GTT TAC TTG ATC 360 Val Gin Thr Gin His Glu Pro Lys Thr Ser Gin Val Tyr Leu He 115 120 125

GTA CAA GTC CCA CCA AAG ATC TCC AAT ATC TCC TCG GAT GTC ACT 405 Val Gin Val Pro Pro Lys He Ser Asn He Ser Ser Asp Val Thr 130 135 140

GTG AAT GAG GGC AGC AAC GTG ACT CTG GTC TGC ATG GCC AAT GGC 450 Val Asn Glu Gly Ser Asn Val Thr Leu Val Cys Met Ala Asn Gly 145 150 155

CGT CCT GAA CCT GTT ATC ACC TGG AGA CAC CTT ACA CCA ACT GGA 495 Arg Pro Glu Pro Val He Thr Trp Arg His Leu Thr Pro Thr Gly 160 165 170

AGG GAA TTT GAA GGA GAA GAA GAA TAT CTG GAG ATC CTT GGC ATC 540 Arg Glu Phe Glu Gly Glu Glu Glu Tyr Leu Glu He Leu Gly He 175 180 185

ACC AGG GAG CAG TCA GGC AAA TAT GAG TGC AAA GCT GCC AAC GAG 585 Thr Arg Glu Gin Ser Gly Lys Tyr Glu Cys Lys Ala Ala Asn Glu 190 195 200

GTC TCC TCG GCG GAT GTC AAA CAA GTC AAG GTC ACT GTG AAC TAT 630 Val Ser Ser Ala Asp Val Lys Gin Val Lys Val Thr Val Asn Tyr 205 210 215

CCT CCC ACT ATC ACA GAA TCC AAG AGC AAT GAA GCC ACC ACA GGA 675 Pro Pro Thr He Thr Glu Ser Lys Ser Asn Glu Ala Thr Thr Gly 220 225 230

CGA CAA GCT TCA CTC AAA TGT GAG GCC TCG GCA GTG CCT GCA CCT 720 Arg Gin Ala Ser Leu Lys Cys Glu Ala Ser Ala Val Pro Ala Pro 235 240 245

GAC TTT GAG TGG TAC CGG GAT GAC ACT AGG ATA AAT AGT GCC AAT 765 Asp Phe Glu Trp Tyr Arg Asp Asp Thr Arg He Asn Ser Ala Asn 250 255 260

GGC CTT GAG ATT AAG AGC ACG GAG GGC CAG TCT TCC CTG ACG GTG 810 Gly Leu Glu He Lys Ser Thr Glu Gly Gin Ser Ser Leu Thr Val 265 270 275

ACC AAC GTC ACT GAG GAG CAC TAC GGC AAC TAC ACC TGT GTG GCT 855 Thr Asn Val Thr Glu Glu His Tyr Gly Asn Tyr Thr Cys Val Ala 280 285 290

GCC AAC AAG CTG GGG GTC ACC AAT GCC AGC CTA GTC CTT TTC AGA 900 Ala Asn Lys Leu Gly Val Thr Asn Ala Ser Leu Val Leu Phe Arg 295 300 305

CCT GGG TCG GTG AGA GGA ATA AAT 924

Pro Gly Ser Val Arg Gly He Asn

310 315

(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 945 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:8 :FROM 1 TO 945 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

ATG GTC GGG AGA GTT CAA CCT GAT CGG AAA CAG TTG CCA CTG 42 Met Val Gly Arg Val Gin Pro Asp Arg Lys Gin Leu Pro Leu 1 5 10

GTC CTA CTG AGA CTG CTC TGC CTT CTT CCC ACA GGA CTG CCC GTT 87 Val Leu Leu Arg Leu Leu Cys Leu Leu Pro Thr Gly Leu Pro Val 15 20 25

CGC AGC GTG GAT TTT AAC CGA GGC ACG GAC AAC ATC ACC GTG AGG 132 Arg Ser Val Asp Phe Asn Arg Gly Thr Asp Asn He Thr Val Arg 30 35 40

CAG GGG GAC ACG GCC ATC CTC AGG TGT GTG GTA GAA GAC AAG AAC 177 Gin Gly Asp Thr Ala He Leu Arg Cys Val Val Glu Asp Lys Asn 45 50 55

TCG AAA GTG GCC TGG TTG AAC CGC TCT GGC ATC ATC TTC GCT GGA 222 Ser Lys Val Ala Trp Leu Asn Arg Ser Gly He He Phe Ala Gly 60 65 70

CAC GAC AAG TGG TCT CTG GAC CCT CGG GTT GAG CTG GAG AAA CGC 267 His Asp Lys Trp Ser Leu Asp Pro Arg Val Glu Leu Glu Lys Arg 75 80 85

CAT GCT CTG GAA TAC AGC CTC CGA ATC CAG AAG GTG GAT GTC TAT 312 His Ala Leu Glu Tyr Ser Leu Arg He Gin Lys Val Asp Val Tyr 90 95 100

GAT GAA GGA TCC TAC ACA TGC TCA GTT CAG ACA CAG CAT GAG CCC 357 Asp Glu Gly Ser Tyr Thr Cys Ser Val Gin Thr Gin His Glu Pro 105 110 115

AAG ACC TCT CAA GTT TAC TTG ATT GTA CAA GTT CCA CCA AAG ATC 402 Lys Thr Ser Gin Val Tyr Leu He Val Gin Val Pro Pro Lys He 120 125 130

TCC AAC ATC TCC TCG GAT GTC ACT GTG AAT GAG GGC AGC AAT GTA 447 Ser Asn He Ser Ser Asp Val Thr Val Asn Glu Gly Ser Asn Val 135 140 145

ACC CTG GTC TGC ATG GCC AAT GGG CGC CCT GAA CCT GTT ATC ACC 492 Thr Leu Val Cys Met Ala Asn Gly Arg Pro Glu Pro Val He Thr 150 155 160

TGG AGA CAC CTT ACA CCA CTT GGA AGA GAA TTT GAA GGA GAA GAA 537 Trp Arg His Leu Thr Pro Leu Gly Arg Glu Phe Glu Gly Glu Glu 165 170 175

GAA TAT CTG GAG ATC CTA GGC ATC ACC AGG GAA CAG TCA GGC AAA 582 Glu Tyr Leu Glu He Leu Gly He Thr Arg Glu Gin Ser Gly Lys 180 185 190

TAT GAG TGC AAG GCT GCC AAC GAG GTC TCC TCC GCG GAT GTC AAA 627 Tyr Glu Cys Lys Ala Ala Asn Glu Val Ser Ser Ala Asp Val Lys 195 200 205

CAA GTC AAG GTC ACT GTG AAC TAT CCA CCC ACC ATC ACA GAG TCT 672 Gin Val Lys Val Thr Val Asn Tyr Pro Pro Thr He Thr Glu Ser 210 215 220

AAG AGC AAT GAA GCC ACC ACA GGA CGA CAA GCT TCC CTC AAA TGT 717 Lys Ser Asn Glu Ala Thr Thr Gly Arg Gin Ala Ser Leu Lys Cys 225 230 235

GAA GCC TCA GCG GTG CCT GCA CCT GAC TTT GAG TGG TAC CGG GAT 762 Glu Ala Ser Ala Val Pro Ala Pro Asp Phe Glu Trp Tyr Arg Asp 240 245 250

GAC ACC AGG ATA AAC AGT GCA AAC GGC CTT GAG ATT AAG AGC ACT 807 Asp Thr Arg He Asn Ser Ala Asn Gly Leu Glu He Lys Ser Thr 255 260 265

GAG GGC CAG TCC TCC CTG ACG GTG ACC AAC GTC ACT GAG GAA CAC 852 Glu Gly Gin Ser Ser Leu Thr Val Thr Asn Val Thr Glu Glu His 270 275 280

TAC GGC AAC TAT ACC TGT GTG GCT GCC AAC AAG CTC GGC GTC ACC 897 Tyr Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly Val Thr 285 290 295

AAT GCC AGC CTA GTC CTT TTC AGA CCC GGG TCG GTG AGA GGA ATC 942 Asn Ala Ser Leu Val Leu Phe Arg Pro Gly Ser Val Arg Gly He 300 305 310

AAC 945

Asn

315

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 861 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LI3RARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO: 9:FROM 1 TO 861 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 :

GTT CGC AGC GTG GAT TTT AAC CGA GGC 27

Val Arg Ser Val Asp Phe Asn Arg Gly 30 35

ACG GAC AAC ATC ACC GTG AGG CAG GGG GAC ACA GCC ATC CTC AGG 72 Thr Asp Asn He Thr Val Arg Gin Gly Asp Thr Ala He Leu Arg 40 45 50

- 55 -

TGC GTT CTA GAA GAC AAG AAC TCA AAG GTG GCC TGG TTG AAC CGT 117 Cys Val Leu Glu Asp Lys Asn Ser Lys Val Ala Trp Leu Asn Arg 55 60 65

TCT GGC ATC ATT TTT GCT GGA CAT GAC AAG TGG TCT CTG GAC CCA 162 Ser Gly He He Phe Ala Gly His Asp Lys Trp Ser Leu Asp Pro 70 75 80

CGG GTT GAG CTG GAG AAA CGC CAT TCT CTG GAA TAC AGC CTC CGA 207 Arg Val Glu Leu Glu Lys Arg His Ser Leu Glu Tyr Ser Leu Arg 85 90 95

ATC CAG AAG GTG GAT GTC TAT GAT GAG GGT TCC TAC ACT TGC TCA 252 He Gin Lys Val Asp Val Tyr Asp Glu Gly Ser Tyr Thr Cys Ser 100 105 110

GTT CAG ACA CAG CAT GAG CCC AAG ACC TCC CAA GTT TAC TTG ATC 297 Val Gin Thr Gin His Glu Pro Lys Thr Ser Gin Val Tyr Leu He 115 120 125

GTA CAA GTC CCA CCA AAG ATC TCC AAT ATC TCC TCG GAT GTC ACT 342 Val Gin Val Pro Pro Lys He Ser Asn He Ser Ser Asp Val Thr 130 135 140

GTG AAT GAG GGC AGC AAC GTG ACT CTG GTC TGC ATG GCC AAT GGC 387 Val Asn Glu Gly Ser Asn Val Thr Leu Val Cys Met Ala Asn Gly 145 150 155

CGT CCT GAA CCT GTT ATC ACC TGG AGA CAC CTT ACA CCA ACT GGA 432 Arg Pro Glu Pro Val He Thr Trp Arg His Leu Thr Pro Thr Gly 160 165 170

AGG GAA TTT GAA GGA GAA GAA GAA TAT CTG GAG ATC CTT GGC ATC 477 Arg Glu Phe Glu Gly Glu Glu Glu Tyr Leu Glu He Leu Gly He 175 180 185

ACC AGG GAG CAG TCA GGC AAA TAT GAG TGC AAA GCT GCC AAC GAG 522 Thr Arg Glu Gin Ser Gly Lys Tyr Glu Cys Lys Ala Ala Asn Glu 190 195 200

GTC TCC TCG GCG GAT GTC AAA CAA GTC AAG GTC ACT GTG AAC TAT 567 Val Ser Ser Ala Asp Val Lys Gin Val Lys Val Thr Val Asn Tyr 205 210 215

CCT CCC ACT ATC ACA GAA TCC AAG AGC AAT GAA GCC ACC ACA GGA 612 Pro Pro Thr He Thr Glu Ser Lys Ser Asn Glu Ala Thr Thr Gly 220 225 230

CGA CAA GCT TCA CTC AAA TGT GAG GCC TCG GCA GTG CCT GCA CCT 657 Arg Gin Ala Ser Leu Lys Cys Glu Ala Ser Ala Val Pro Ala Pro 235 240 245

GAC TTT GAG TGG TAC CGG GAT GAC ACT AGG ATA AAT AGT GCC AAT 702 Asp Phe Glu Trp Tyr Arg Asp Asp Thr Arg He Asn Ser Ala Asn 250 255 260

GGC CTT GAG ATT AAG AGC ACG GAG GGC CAG TCT TCC CTG ACG GTG 747 Gly Leu Glu He Lys Ser Thr Glu Gly Gin Ser Ser Leu Thr Val 265 270 275

ACC AAC GTC ACT GAG GAG CAC TAC GGC AAC TAC ACC TGT GTG GCT 792 Thr Asn Val Thr Glu Glu His Tyr Gly Asn Tyr Thr Cys Val Ala 280 285 290

GCC AAC AAG CTG GGG GTC ACC AAT GCC AGC CTA GTC CTT TTC AGA 837 Ala Asn Lys Leu Gly Val Thr Asn Ala Ser Leu Val Leu Phe Arg 295 300 305

CCT GGG TCG GTG AGA GGA ATA AAT 861

Pro Gly Ser Val Arg Gly He Asn 310 315

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 861 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO: 10: FROM 1 TO 861 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GTT Val

CGC AGC GTG GAT TTT AAC CGA GGC ACG GAC AAC ATC ACC GTG AGG 48 Arg Ser Val Asp Phe Asn Arg Gly Thr Asp Asn He Thr Val Arg 30 35 40

CAG GGG GAC ACG GCC ATC CTC AGG TGT GTG GTA GAA GAC AAG AAC 93 Gin Gly Asp Thr Ala He Leu Arg Cys Val Val Glu Asp Lys Asn 45 50 55

TCG AAA GTG GCC TGG TTG AAC CGC TCT GGC ATC ATC TTC GCT GGA 138 Ser Lys Val Ala Trp Leu Asn Arg Ser Gly He He Phe Ala Gly 60 65 70

CAC GAC AAG TGG TCT CTG GAC CCT CGG GTT GAG CTG GAG AAA CGC 183 His Asp Lys Trp Ser Leu Asp Pro Arg Val Glu Leu Glu Lys Arg 75 80 85

CAT GCT CTG GAA TAC AGC CTC CGA ATC CAG AAG GTG GAT GTC TAT 228 His Ala Leu Glu Tyr Ser Leu Arg He Gin Lys Val Asp Val Tyr 90 95 100

GAT GAA GGA TCC TAC ACA TGC TCA GTT CAG ACA CAG CAT GAG CCC 273 Asp Glu Gly Ser Tyr Thr Cys Ser Val Gin Thr Gin His Glu Pro 105 110 115

AAG ACC TCT CAA GTT TAC TTG ATT GTA CAA GTT CCA CCA AAG ATC 318 Lys Thr Ser Gin Val Tyr Leu He Val Gin Val Pro Pro Lys He 120 125 130

TCC AAC ATC TCC TCG GAT GTC ACT GTG AAT GAG GGC AGC AAT GTA 363 Ser Asn He Ser Ser Asp Val Thr Val Asn Glu Gly Ser Asn Val 135 140 145

ACC CTG GTC TGC ATG GCC AAT GGG CGC CCT GAA CCT GTT ATC ACC 408 Thr Leu Val Cys Met Ala Asn Gly Arg Pro Glu Pro Val He Thr 150 155 160

TGG AGA CAC CTT ACA CCA CTT GGA AGA GAA TTT GAA GGA GAA GAA 453 Trp Arg His Leu Thr Pro Leu Gly Arg Glu Phe Glu Gly Glu Glu 165 170 175

GAA TAT CTG GAG ATC CTA GGC ATC ACC AGG GAA CAG TCA GGC AAA 498 Glu Tyr Leu Glu He Leu Gly He Thr Arg Glu Gin Ser Gly Lys 180 185 190

TAT GAG TGC AAG GCT GCC AAC GAG GTC TCC TCC GCG GAT GTC AAA 543 Tyr Glu Cys Lys Ala Ala Asn Glu Val Ser Ser Ala Asp Val Lys 195 200 205

CAA GTC AAG GTC ACT GTG AAC TAT CCA CCC ACC ATC ACA GAG TCT 588 Gin Val Lys Val Thr Val Asn Tyr Pro Pro Thr He Thr Glu Ser 210 215 220

AAG AGC AAT GAA GCC ACC ACA GGA CGA CAA GCT TCC CTC AAA TGT 633 Lys Ser Asn Glu Ala Thr Thr Gly Arg Gin Ala Ser Leu Lys Cys 225 230 235

GAA GCC TCA GCG GTG CCT GCA CCT GAC TTT GAG TGG TAC CGG GAT 678 Glu Ala Ser Ala Val Pro Ala Pro Asp Phe Glu Trp Tyr Arg Asp 240 245 250

GAC ACC AGG ATA AAC AGT GCA AAC GGC CTT GAG ATT AAG AGC ACT 723 Asp Thr Arg He Asn Ser Ala Asn Gly Leu Glu He Lys Ser Thr 255 260 265

GAG GGC CAG TCC TCC CTG ACG GTG ACC AAC GTC ACT GAG GAA CAC 768 Glu Gly Gin Ser Ser Leu Thr Val Thr Asn Val Thr Glu Glu His 270 275 280

TAC GGC AAC TAT ACC TGT GTG GCT GCC AAC AAG CTC GGC GTC ACC 813 Tyr Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly Val Thr 285 290 295

AAT GCC AGC CTA GTC CTT TTC AGA CCC GGG TCG GTG AGA GGA ATC 858 Asn Ala Ser Leu Val Leu Phe Arg Pro Gly Ser Val Arg Gly He 300 305 310

AAC 861

Asn

315

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 219 base pairs

(3) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:11:FROM 1 TO 219 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

GGG GAC ACA GCC ATC CTC AGG 21

Gly Asp Thr Ala He Leu Arg 50

TGC GTT CTA GAA GAC AAG AAC TCA AAG GTG GCC TGG TTG AAC CGT 66 Cys Val Leu Glu Asp Lys Asn Ser Lys Val Ala Trp Leu Asn Arg 55 60 65

TCT GGC ATC ATT TTT GCT GGA CAT GAC AAG TGG TCT CTG GAC CCA 111 Ser Gly He He Phe Ala Gly His Asp Lys Trp Ser Leu Asp Pro 70 75 80

CGG GTT GAG CTG GAG AAA CGC CAT TCT CTG GAA TAC AGC CTC CGA 156 Arg Val Glu Leu Glu Lys Arg His Ser Leu Glu Tyr Ser Leu Arg 85 90 95

ATC CAG AAG GTG GAT GTC TAT GAT GAG GGT TCC TAC ACT TGC TCA 201 He Gin Lys Val Asp Val Tyr Asp Glu Gly Ser Tyr Thr Cys Ser 100 105 110

GTT CAG ACA CAG CAT GAG 219

Val Gin Thr Gin His Glu 115

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 219 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:12:FROM 1 TO 219 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GGG GAC ACG GCC ATC CTC AGG TGT GTG GTA GAA GAC AAG AAC 42 Gly Asp Thr Ala He Leu Arg Cys Val Val Glu Asp Lys Asn 50 55

TCG AAA GTG GCC TGG TTG AAC CGC TCT GGC ATC ATC TTC GCT GGA 87 Ser Lys Val Ala Trp Leu Asn Arg Ser Gly He He Phe Ala Gly 60 65 70

CAC GAC AAG TGG TCT CTG GAC CCT CGG GTT GAG CTG GAG AAA CGC 132 His Asp Lys Trp Ser Leu Asp Pro Arg Val Glu Leu Glu Lys Arg 75 80 85

CAT GCT CTG GAA TAC AGC CTC CGA ATC CAG AAG GTG GAT GTC TAT 177 His Ala Leu Glu Tyr Ser Leu Arg He Gin Lys Val Asp Val Tyr 90 95 100

GAT GAA GGA TCC TAC ACA TGC TCA GTT CAG ACA CAG CAT GAG 219 Asp Glu Gly Ser Tyr Thr Cys Ser Val Gin Thr Gin His Glu 105 110 115

(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 177 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:13:FROM 1 TO 177 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

GGC AGC AAC GTG ACT CTG GTC TGC ATG GCC AAT GGC 36

Gly Ser Asn Val Thr Leu Val Cys Met Ala Asn Gly 150 155

CGT CCT GAA CCT GTT ATC ACC TGG AGA CAC CTT ACA CCA ACT GGA 81 Arg Pro Glu Pro Val He Thr Trp Arg His Leu Thr Pro Thr Gly 160 165 170

AGG GAA TTT GAA GGA GAA GAA GAA TAT CTG GAG ATC CTT GGC ATC 126 Arg Glu Phe Glu Gly Glu Glu Glu Tyr Leu Glu He Leu Gly He 175 180 185

ACC AGG GAG CAG TCA GGC AAA TAT GAG TGC AAA GCT GCC AAC GAG 171 Thr Arg Glu Gin Ser Gly Lys Tyr Glu Cys Lys Ala Ala Asn Glu 190 195 200

GTC TCC 177

Val Ser

(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 177 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:14:FROM 1 TO 177 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

GGC AGC AAT GTA 12

Gly Ser Asn Val

ACC CTG GTC G ATG GCC AAT GGG CGC CCT GAA CCT GTT ATC ACC 57 Thr Leu Val Cys Met Ala Asn Gly Arg Pro Glu Pro Val He Thr 150 155 160

TGG AGA CAC CTT ACA CCA CTT GGA AGA GAA TTT GAA GGA GAA GAA 102 Trp Arg His Leu Thr Pro Leu Gly Arg Glu Phe Glu Gly Glu Glu 165 170 175

GAA TAT CTG GAG ATC CTA GGC ATC ACC AGG GAA CAG TCA GGC AAA 147 Glu Tyr Leu Glu He Leu Gly He Thr Arg Glu Gin Ser Gly Lys 180 185 190

TAT GAG TGC AAG GCT GCC AAC GAG GTC TCC 177

Tyr Glu Cys Lys Ala Ala Asn Glu Val Ser 195 200

(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 198 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LIBRARY: CDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:15:FROM 1 TO 198 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:IS:

CGA 3

Gly

CGA CAA GCT TCA CTC AAA TGT GAG GCC TCG GCA GTG CCT GCA CCT 48 Arg Gin Ala Ser Leu Lys Cys Glu Ala Ser Ala Val Pro Ala Pro 235 240 245

GAC TTT GAG TGG TAC CGG GAT GAC ACT AGG ATA AAT AGT GCC AAT 93 Asp Phe Glu Trp Tyr Arg Asp Asp Thr Arg He Asn Ser Ala Asn 250 255 260

GGC CTT GAG ATT AAG AGC ACG GAG GGC CAG TCT TCC CTG ACG GTG 138 Gly Leu Glu He Lys Ser Thr Glu Gly Gin Ser Ser Leu Thr Val 265 270 275

ACC AAC GTC ACT GAG GAG CAC TAC GGC AAC TAC ACC TGT GTG GCT 183 Thr Asn Val Thr Glu Glu His Tyr Gly Asn Tyr Thr Cys Val Ala 280 285 290

GCC AAC AAG CTG GGG 198

Ala Asn Lys Leu Gly 295

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 198 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: CDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:16:FROM 1 TO 198 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

GGA CGA CAA GCT TCC CTC AAA TGT 24

Gly Arg Gin Ala Ser Leu Lys Cys 235

GAA GCC TCA GCG GTG CCT GCA CCT GAC TTT GAG TGG TAC CGG GAT 69 Glu Ala Ser Ala Val Pro Ala Pro Asp Phe Glu Trp Tyr Arg Asp 240 245 250

GAC ACC AGG ATA AAC AGT GCA AAC GGC CTT GAG ATT AAG AGC ACT 114 Asp Thr Arg He Asn Ser Ala Asn Gly Leu Glu He Lys Ser Thr 255 260 265

GAG GGC CAG TCC TCC CTG ACG GTG ACC AAC GTC ACT GAG GAA CAC 159 Glu Gly Gin Ser Ser Leu Thr Val Thr Asn Val Thr Glu Glu His 270 275 280

TAC GGC AAC TAT ACC TGT GTG GCT GCC AAC AAG CTC GGC 198

Tyr Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly 285 290 295

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 756 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(F) TISSUE TYPE: cerebral cortex (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:17:FROM 1 TO 756 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GGG GAC ACA GCC ATC CTC AGG 21

Gly Asp Thr Ala He Leu Arg 50

TGC GTT CTA GAA GAC AAG AAC TCA AAG GTG GCC TGG TTG AAC CGT 66 Cys Val Leu Glu Asp Lys Asn Ser Lys Val Ala Trp Leu Asn Arg 55 60 65

TCT GGC ATC ATT TTT GCT GGA CAT GAC AAG TGG TCT CTG GAC CCA 111 Ser Gly He He Phe Ala Gly His Asp Lys Trp Ser Leu Asp Pro 70 75 80

CGG GTT GAG CTG GAG AAA CGC CAT TCT CTG GAA TAC AGC CTC CGA 156 Arg Val Glu Leu Glu Lys Arg His Ser Leu Glu Tyr Ser Leu Arg 85 90 95

ATC CAG AAG GTG GAT GTC TAT GAT GAG GGT TCC TAC ACT TGC TCA 201 He Gin Lys Val Asp Val Tyr Asp Glu Gly Ser Tyr Thr Cys Ser 100 105 110

GTT CAG ACA CAG CAT GAG CCC AAG ACC TCC CAA GTT TAC TTG ATC 246 Val Gin Thr Gin His Glu Pro Lys Thr Ser Gin Val Tyr Leu He 115 120 125

GTA CAA GTC CCA CCA AAG ATC TCC AAT ATC TCC TCG GAT GTC ACT 291 Val Gin Val Pro Pro Lys He Ser Asn He Ser Ser Asp Val Thr 130 135 140

GTG AAT GAG GGC AGC AAC GTG ACT CTG GTC TGC ATG GCC AAT GGC 336 Val Asn Glu Gly Ser Asn Val Thr Leu Val Cys Met Ala Asn Gly 145 150 155

CGT CCT GAA CCT GTT ATC ACC TGG AGA CAC CTT ACA CCA ACT GGA 381 Arg Pro Glu Pro Val He Thr Trp Arg His Leu Thr Pro Thr Gly 160 165 170

AGG GAA TTT GAA GGA GAA GAA GAA TAT CTG GAG ATC CTT GGC ATC 426 Arg Glu Phe Glu Gly Glu Glu Glu Tyr Leu Glu He Leu Gly He 175 180 185

ACC AGG GAG CAG TCA GGC AAA TAT GAG TGC AAA GCT GCC AAC GAG 4 ^* 71 Thr Arg Glu Gin Ser Gly Lys Tyr Glu Cys Lys Ala Ala Asn Glu 190 195 200

GTC TCC TCG GCG GAT GTC AAA CAA GTC AAG GTC ACT GTG AAC TAT 516 Val Ser Ser Ala Asp Val Lys Gin Val Lys Val Thr Val Asn Tyr 205 210 215

CCT CCC ACT ATC ACA GAA TCC AAG AGC AAT GAA GCC ACC ACA GGA 561 Pro Pro Thr He Thr Glu Ser Lys Ser Asn Glu Ala Thr Thr Gly 220 225 230

CGA CAA GCT TCA CTC AAA TGT GAG GCC TCG GCA GTG CCT GCA CCT 606 Arg Gin Ala Ser Leu Lys Cys Glu Ala Ser Ala Val Pro Ala Pro 235 240 245

GAC TTT GAG TGG TAC CGG GAT GAC ACT AGG ATA AAT AGT GCC AAT 651 Asp Phe Glu Trp Tyr Arg Asp Asp Thr Arg He Asn Ser Ala Asn 250 255 260

GGC CTT GAG ATT AAG AGC ACG GAG GGC CAG TCT TCC CTG ACG GTG 696 Gly Leu Glu He Lys Ser Thr Glu Gly Gin Ser Ser Leu Thr Val 265 270 275

ACC AAC GTC ACT GAG GAG CAC TAC GGC AAC TAC ACC TGT GTG GCT 7 1 Thr Asn Val Thr Glu Glu His Tyr Gly Asn Tyr Thr Cys Val Ala 280 285 290

GCC AAC AAG CTG GGG 756

Ala Asn Lys Leu Gly 295

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 756 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat

(F) TISSUE TYPE: hippocampus (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(K) RELEVANT RESIDUES IN SEQ ID NO:18:FROM 1 TO 756 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

GGG GAC ACG GCC ATC CTC AGG TGT GTG GTA GAA GAC AAG AAC 42 Gly Asp Thr Ala He Leu Arg Cys Val Val Glu Asp Lys Asn 50 55

TCG AAA GTG GCC TGG TTG AAC CGC TCT GGC ATC ATC TTC GCT GGA 87 Ser Lys Val Ala Trp Leu Asn Arg Ser Gly He He Phe Ala Gly 60 65 70

CAC GAC AAG TGG TCT CTG GAC CCT CGG GTT GAG CTG GAG AAA CGC 132 His Asp Lys Trp Ser Leu Asp Pro Arg Val Glu Leu Glu Lys Arg 75 80 85

CAT GCT CTG GAA TAC AGC CTC CGA ATC CAG AAG GTG GAT GTC TAT 177 His Ala Leu Glu Tyr Ser Leu Arg He Gin Lys Val Asp Val Tyr 90 95 100

GAT GAA GGA TCC TAC ACA TGC TCA GTT CAG ACA CAG CAT GAG CCC 222 Asp Glu Gly Ser Tyr Thr Cys Ser Val Gin Thr Gin His Glu Pro 105 110 115

AAG ACC TCT CAA GTT TAC TTG ATT GTA CAA GTT CCA CCA AAG ATC 267 Lys Thr Ser Gin Val Tyr Leu He Val Gin Val Pro Pro Lys He 120 125 130

TCC AAC ATC TCC TCG GAT GTC ACT GTG AAT GAG GGC AGC AAT GTA 312 Ser Asn He Ser Ser Asp Val Thr Val Asn Glu Gly Ser Asn Val 135 140 145

ACC CTG GTC TGC ATG GCC AAT GGG CGC CCT GAA CCT GTT ATC ACC 357

Thr Leu Val Cys Met Ala Asn Gly Arg Pro Glu Pro Val He Thr 150 155 160

TGG AGA CAC CTT ACA CCA CTT GGA AGA GAA TTT GAA GGA GAA GAA 402 Trp Arg His Leu Thr Pro Leu Gly Arg Glu Phe Glu Gly Glu Glu 165 170 175

GAA TAT CTG GAG ATC CTA GGC ATC ACC AGG GAA CAG TCA GGC AAA 447 Glu Tyr Leu Glu He Leu Gly He Thr Arg Glu Gin Ser Gly Lys 180 185 190

TAT GAG TGC AAG GCT GCC AAC GAG GTC TCC TCC GCG GAT GTC AAA 492 Tyr Glu Cys Lys Ala Ala Asn Glu Val Ser Ser Ala Asp Val Lys 195 200 205

CAA GTC AAG GTC ACT GTG AAC TAT CCA CCC ACC ATC ACA GAG TCT 537 Gin Val Lys Val Thr Val Asn Tyr Pro Pro Thr He Thr Glu Ser 210 215 220

AAG AGC AAT GAA GCC ACC ACA GGA CGA CAA GCT TCC CTC AAA TGT 582 Lys Ser Asn Glu Ala Thr Thr Gly Arg Gin Ala Ser Leu Lys Cys 225 230 235

GAA GCC TCA GCG GTG CCT GCA CCT GAC TTT GAG TGG TAC CGG GAT 627 Glu Ala Ser Ala Val Pro Ala Pro Asp Phe Glu Trp Tyr Arg Asp 240 245 250

GAC ACC AGG ATA AAC AGT GCA AAC GGC CTT GAG ATT AAG AGC ACT 672 Asp Thr Arg He Asn Ser Ala Asn Gly Leu Glu He Lys Ser Thr 255 260 265

GAG GGC CAG TCC TCC CTG ACG GTG ACC AAC GTC ACT GAG GAA CAC 717 Glu Gly Gin Ser Ser Leu Thr Val Thr Asn Val Thr Glu Glu His 270 275 280

TAC GGC AAC TAT ACC TGT GTG GCT GCC AAC AAG CTC GGC 756

Tyr Gly Asn Tyr Thr Cys Val Ala Ala Asn Lys Leu Gly 285 290 295

(2) INFORMATION FOR SEQ ID NO: 19 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1033 base pairs

(3) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Mouse

(F) TISSUE TYPE: (vii) IMMEDIATE SOURCE:

(A) LIBRARY: genomic

(K) RELEVANT RESIDUES IN SEQ ID NO:19: FROM 1 TO 1033 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

CTGCAGTATG CCTTCCTATC CATGTGTATG TAACTCCATT TGTCAAGGTT 50

TGCATTTCTT TCTTGCCATG CCTTCCCTCT CCTTTCCTGG ACCCTTTCTC 100

CTGTCCTTTT ACAGCTTAAG TCCACCTCCC CTTCCCTGTC TTAACAGTAC 150

CTCTGAGCCC CATCCCCTCT TCTTAAGGAA ATCAGCCAGG CCTCAGATGG 200

AGCTGTTGTC CTGACAACCC AAAGGCGCAT CAGGCTTTCA TTGAGGCTGG 250

CTGCTGGTGA AGAGGGCAGT TGTGCAAAGC AAGGGGCCAC GCTGAGGGGT 300

GGGAGGAGGG GATGACGTGG TGGGGCTGTT GAAAACCAGC AGGGTAGGGG 350

GGAGGTGCTG AGTAGAGAGA GAACAGGGAC TGGAGGGAGA AACAAGAAAG 400

AGGAGGGGGA GAGAGCTCCT GGGTTGCTGC CGCTACTGCT GCTGCTGCTG 450

CAAGAGGCTG TTTCTTTACT CTCCCTGGCA GGCTCTCCTG CTGCCTGGGA 500

AAGTGGGTTA CAGAGGGAAG CAGCTCAGCC CAGACGCTGG CAGAGAAGCA 550

GCCAGCTACA GAGAGTCTAA GGAAGCACCC CTGCCATTGA CAGTCGCCTC 600

CTCATCATTA AAGCATTTTA TATTTGCACT CTTCCTTCGG AAAATTTGTT 650

CCTCCACTTT CTCCCCGACT CCTGCTTGGA TTTGATGAGG GCTTTGTTAA 700

ATCCCAGAGG AAAAGAGACT AAGCGAGGGA AAGAGCAAGG CAAAGTGGAA 750

GGGAGTGCGC GCTGGACCCG CCCGAGCAGC CTTGGCAGTG GCTGCGAGCC 800

CCGCGCGCTA GAGCCCCTCT CCGTGTCCAG CAGCGCGCAC ACGCAGTCCA 850

CCGCGGACCA ACTCGCCGAG GCCACC ATG GTC GGG AGA GTT CAG 894

Met Val Gly Arg Val Gin 1 5

CCC GAT CGG AAA CAG TTG CCG CTG GTC CTA CTG AGA TCT TCC CAC 939 Pro Asp Arg Lys Gin Leu Pro Leu Val Leu Leu Arg Leu Ser His 10 15 20

CTT CTT CCC ACA GGA CTG CCC GTT CGC AGC GTG GAT TTT AAC CGA 984 Leu Leu Pro Thr Gly Leu Pro Val Arg Ser Val Asp Phe Asn Arg 25 30 35

GGC ACG GAC AAC ATC ACC GTG AGA CAG GGG GAC ACG GCC ATC CTC 1029 Gly Thr Asp Asn He Thr Val Arg Gin Gly Asp Thr Ala He Leu

40 45 50

AGG T 1033

Arg

(2) INFORMATION FOR SEQ ID NO: 20 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1851 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomic (iii) HYPOTHETICAL: no

(iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Mouse

(F) TISSUE TYPE: (vii) IMMEDIATE SOURCE:

(A) LI3RARY: genomic

(K) RELEVANT RESIDUES IN SEQ ID NO:20: FROM 1 TO 1851 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

CTGCAGTATG CCTTCCTATC CATGTGTATG TAACTCCATT TGTCAAGGTT 50

TGCATTTCTT TCTTGCCATG CCTTCCCTCT CCTTTCCTGG ACCCTTTCTC 100

CTGTCCTTTT ACAGCTTAAG TCCACCTCCC CTTCCCTGTC TTAACAGTAC 150

CTCTGAGCCC CATCCCCTCT TCTTAAGGAA ATCAGCCAGG CCTCAGATGG 200

AGCTGTTGTC CTGACAACCC AAAGGCGCAT CAGGCTTTCA TTGAGGCTGG 250

CTGCTGGTGA AGAGGGCAGT TGTGCAAAGC AAGGGGCCAC GCTGAGGGGT 300

GGGAGGAGGG GATGACGTGG TGGGGCTGTT GAAAACCAGC AGGGTAGGGG 350

GGAGGTGCTG AGTAGAGAGA GAACAGGGAC TGGAGGGAGA AACAAGAAAG 400

AGGAGGGGGA GAGAGCTCCT GGGTTGCTGC CGCTACTGCT GCTGCTGCTG 450

CAAGAGGCTG TTTCTTTACT CTCCCTGGCA GGCTCTCCTG CTGCCTGGGA 500

AAGTGGGTTA CAGAGGGAAG CAGCTCAGCC CAGACGCTGG CAGAGAAGCA 550

GCCAGCTACA GAGAGTCTAA GGAAGCACCC CTGCCATTGA CAGTCGCCTC 600

CTCATCATTA AAGCATTTTA TATTTGCACT CTTCCTTCGG AAAATTTGTT 650

CCTCCACTTT CTCCCCGACT CCTGCTTGGA TTTGATGAGG GCTTTGTTAA 700

ATCCCAGAGG AAAAGAGACT AAGCGAGGGA AAGAGCAAGG CAAAGTGGAA 750

GGGAGTGCGC GCTGGACCCG CCCGAGCAGC CTTGGCAGTG GCTGCGAGCC 800

CCGCGCGCTA GAGCCCCTCT CCGTGTCCAG CAGCGCGCAC ACGCAGTCCA 850

CCGCGGACCA ACTCGCCGAG GCCACC ATG GTC GGG AGA GTT CAG 894

Met Val Gly Arg Val Gin 1 5

CCC GAT CGG AAA CAG TTG CCG CTG GTC CTA CTG AGA TCT TCC CAC 939 Pro Asp Arg Lys Gin Leu Pro Leu Val Leu Leu Arg Leu Ser His 10 15 20

CTT CTT CCC ACA GGA CTG CCC GTT CGC AGC GTG GAT TTT AAC CGA 984 Leu Leu Pro Thr Gly Leu Pro Val Arg Ser Val Asp Phe Asn Arg 25 30 35

GGC ACG GAC AAC ATC ACC GTG AGA CAG GGG GAC ACG GCC ATC CTC 1029 Gly Thr Asp Asn He Thr Val Arg Gin Gly Asp Thr Ala He Leu

40 45 50

AGG TAGGGCTT GCGAGCAACT 1050

Arg

TTTCTGGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT 1100

GTGTAATAGT GAACTCCAGC TGCCCTGGGT TAGTGGGCGT GTGTGTGTGT 1150

GTGTGTGTGT GTGTGTGTCC CTTACGTTAC TCGACTTGAA GATTTAGCCA 1200

GGAACAAAAT TTAAGGCGAG TCTGGTCCCT GTCAAGAGCC AAGGGTGCTT 1250

TTGGAATGTT GTTCCGTTCT TTGAATGTTG TTTTCTCTAG TCAAGAAAGC 1300

CGAACTTTAT CTATGGCATT AGTGGCATTG GGCTGTATCA TGCTGTGGTA 1350

ATTGCTCACG CTTGGCACTT AGACTTTTGT TGAGATTCTT CTATTCAGAC 1400

ACAAGAGTTG TTGAGTTATG GCTTTCAAAA CGTGGTACGC AAGGCTGCAT 1450

TCTCTTGTTC GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT 1500

GTGTTGCTCA GCAAGGCTCA GTCTGCCCTA GCAGTAGTTC CTGATAGAAG 1550

ACTTTCTGTA AAGATCTCTG AATTGACATC ATAGGCAATA AATCAATCTT 1600

ACAACTTTGG CATGATTACT GAGGCTTTTT GGGAATGTGG ACAGAAATCA 1650

ACACGAGAAT GAGAGAACGG AAGGAAAGGA TCCAGCCTAA TGGCAGGCCG 1700

TTAAGAATAG AAAACTTAAA CAGAGGAGGA GAAGGCATTA ACCTGATATT 1750

ACATTAGATA CTACAAATTG ATCATTGAGT TCAAAGTCTT ATGCTTATGC 1800

AGCTCTGCCA ACGTCCGCAA TATAATTTGG GATGGAAATT TGGAAAAGCT T 1851