CLONING AND EXPRESSION OF beta APP-C100 RECEPTOR

Abstract: The present invention relates to the cloning of beta APP-C100 receptor (C100-R), and genetically engineered host cells which express the C100-R. Such engineered cells may be used to evaluate and screen drugs and analogs of beta -APP involved in Alzheimer's Disease.

Description: CLONING AND EXPRESSION OF /3APP-C100 RECEPTOR 1. INTRODUCTION The present invention relates to the cloning of the receptor for the carboxy-terminus of the /3-amyloid precursor protein (including the amyloid domain) , referred to herein as C100-R, and genetically engineered host cells which expresε the C100-R. Such engineered cells may be used to evaluate and screen drugs and analogε of the β-amyloid precurεor protein (β-APP) which may be used for the diagnosiε and/or treatment of Alzheimer'ε Diεease. 2. BACKGROUND OF THE INVENTION Alzheimer's diseaεe is a neurodegenerative disorder that is the most frequent cause of dementia among aged individuals. The disease iε characterized by the accumulation of amyloid-containing plaques in the brain, particularly in the temporal cortex and hippocampus and along the wallε of the cerebral vascu- lature (Roch et al., 1966, Nature 209:109-110; Terry et al., 1981, Ann. Neurol. 10:184-192; Glenner G.G., 1983, Arch. Pathol. Lab. Med. 107:281-282; Katzman, R. , 1983, Banbury Report 15, Cold Spring Harbor Lab., Cold Spring, N.Y.). The amyloid peptide (j8A4) , found in the plaqueε of the brain derives from a protein referred to as the amyloid (or beta-amyloid) precursor protein (/3APP) . jSAPP is normally cleaved within the amyloid domain, which lies near its C-terminus, εo that no intact amyloid iε produced. An alternative proceεεing pathway reεultε in cleavage of APP N-terminal to the amyloid domain, releaεing the entire C-terminal from which intact amyloid peptide (j3A4) of the amyloid protein precursor may be produced (Glenner and Wong, 1984, Biochem Biophys Res. Commun. 120:885-890; Maεters et al., 1985, Proc. Natl Acad. Sci. USA 82:4245-4249) . Recent evidence suggests that aberrant processing of β-APP underlies the neuronal degeneration that occurs in Alzheimer'ε Diεease. Neve and coworkers have propoεed that the primary ..APP proceεεing event in Alzheimer's Disease is cleavage of the amino terminus of the 0/A4 sequence, producing a carboxy- terminal βAPP fragment of 100 amino acid residueε (jδAPP-ClOO) which waε expreεεed from a cDNA εequence encoding the carboxyterminal 104 amino acidε of βAPP (Yankner et al., 1989, Science 245:417-420) . Hereinafter, thiε fragment of βAPP will be referred to as jSAPP-ClOO regardlesε of whether it iε expreεsed from a cDNA encoding the carboxyterminal 100 or 104 amino acid residues of /3APP. Expresεion of the /3APP- C100 peptide in primate cellε haε been shown to lead to production of a protein that aggregates and accumu¬ lates into deposit-like structures that result in formation of amyloid-like fibrils (Wolf et al., 1990 EMBO J. 9:2079-2084). A retroviral reco binant which directs the expresεion of / SAPP-CIOO haε alεo been εhown to be neurotoxic when tranεfected into PC-12 cells that have been induced to differentiate by addition of NGF (nerve growth factor) . Furthermore, the conditioned media from theεe tranεfected cellε iε toxic to differentiated neuroblastoma and neural cells, and the neurotoxicity can be removed from the medium by immunoabsorption with an antibody to / SAPP- C100 suggesting that βAPP-ClOO is secreted by transfected cells and is neurotoxic (Yankner et al., 1989, Science 245:417-420). The toxicity of / SAPP-CIOO has been further demonstrated by tranεplantation of cells expresεing the peptide into the brainε of newborn mice and by creation of mice transgenic for human /3APP-C100 (Neve et al., 1992, Proc. Natl. Acad. Sci. USA 89:3448-3452). Taken together, evidence indicates a role for 3APP-C100 in development of the _ neurodegeneration in Alzheimer's diεease. Despite the intense interest in the / SAPP-CIOO peptide, and its biological role in development of Alzheimer's Disease, very little is known about the proteins, receptors or other tissue elements with which the / SAPP-CIOO peptide interacts to produce neurotoxicity. Very recently, the high affinity binding of / SAPP-CIOO to the εurface of differentiated PC-12 cellε haε been demonstrated and correlated with neurotoxicity (Kozlowski et al., 1992, J. of Neuroεcience 12:1679-1687). Both the binding interaction and the occurrence of the neurotoxic reεponse have the same pH dependence. In addition, in PC-12 cells, both the binding and the susceptibility to neurotoxicity develop with similar time courses during NGF-induced differentiation. Furthermore, a single amino-acid change in the / SAPP-CIOO peptide (Tyr 687 to Phe) , which eliminates its neurotoxic effect, also produces a losε of binding potency. However, the molecular εpecieε reεponεible for binding haε not been identified or characterized. The isolation of a cDNA clone coding for the / SAPP-CIOO binding site or receptor would facilitate studieε aimed at determining the biological function of C100-R and itε role in development of Alzheimer's Diseaεe. However, thiε haε not, heretofore, been accomplished. 3. SUMMARY OF THE INVENTION The preεent invention relateε to the C100-R geneε and proteinε. The neurotoxic effectε, reεulting from the interaction between / SAPP-CIOO and the C100-R suggest that therapeutic applications designed to - A - block this particular receptor/ligand interaction may be useful for treatment of Alzheimer's Disease. The DNA sequences disclosed herein may be engineered into expression systems designed for the production of C100-R and/or cell lines which expreεε the C100-R. Such cell lineε may advantageouεly be used for screening and identifying β-APP analogs, including agonists and antagonists. In accordance with another aspect of the invention, the C100-R DNA, antisense oligonucleotide sequences, or antagonists including antibodies to C100-R may be used in the diagnosis and therapy of Alzheimer's Diseaεe. Transgenic animals containing the C100-R transgene may be uεed aε animal modelε for the evaluation of B-APP-CIOO analogε in vivo. The invention iε baεed, in part, on the diεcovery, identification and cloning of a /SAPP-CIOO binding εite that iε expreεεed on the cell εurface of neuronally derived cellε. The binding εite, referred to herein aε C100-R, haε characteristics indicating that it is involved in mediating the neurotoxic effects of the peptide /SAPP-CIOO. The invention is also based on the isolation and characterization of a cDNA clone coding for the C100-R which may be of therapeutic value for the diagnoseε and deεign of the drugε for treatment of Alzheimer'ε Diεeaεe. 4. BRIEF DESCRIPTION OF THE FIGURES FIG. 1. - Representative curve showing the inhibition of 35 S- / SAPP-C100 binding to (A) NGF-treated (5d) PC12 cells and (B) SK-N-MC cellε, by βAPP-ClOO. FIG. 2. - Repreεentative saturation isotherm (top panel) and Scatchard plot (bottom panel) of 35 S-/8APP- C100 binding to NGF-treated (6d) PC12 cellε. The lines were computer generated and represent the beεt interpretation of the data. FIG. 3. - Screening of λgtll rat cDNA libraries using the Direct Binding method. FIG. 4. - Nucleotide Sequence of cDNA inεert of rat C100-R clone, AB1R. Underlined region repreεentε SER/THR Kinase "signature" or conserved catalytic core sequence. FIG. 5. - Sequence homology between C100-R and (A) "B" type calcium channelε, (B) calreticulin, and (C) Ryanodine calcium channel. FIG. 6. - Southern blot analyεiε of the AB1R cDNA. Lane A is EcoRI digested human DNA. Lane B is EcoRI digested mouse DNA. Lane C is Hindlll digested mouse DNA. Lane D is Hindlll digested human DNA. λ/Hindlll markers were run to establish molecular weights. FIG. 7. - Representative autoradiographε of specific binding to rat brain sections at (A) anterior and (B) middle to poεterior levelε. The images were digitized and enhanced so that areas with higher levels of binding are lighter. Note that certain areas of the olfactory tubercle (lower part of sectionε in (A) and hippocampuε (mid-lateral part of section in (B) stand out againεt the relatively uniform labelling of the reεt of the grey matter. FIG. 8. - Representative autoradiographs of the in situ hybridization of the pABlR antisenεe probe, section locations and method of reproduction are as in FIG. 7. Note the areas of high labelling (lightest areas) in the olfactory tubercle and hippocampus in agreement with the resultε from the binding autoradiography (FIG. 7) . FIG. 9. - Nucleotide εequence of cDNA insert of rat C100-R clone, AB2R. The EcoRI fragment from nucleotide 31 through nucleotide 409 was used to screen a human hippocampal cDNA library. FIG. 10. - Nucleotide sequence of rat C100-R clone. The disclosed sequence is the result of cloning and sequencing of a number of overlapping rat cDNA clones. The * above nucleotide 686 representε the junction with the rat C100-R nucleotide sequence depicted in Figure 4 (nucleotide 40) . FIG. 11. - Nucleotide εequence of cDNA inεerts of human C100-R clone. The nucleotide designated M is either A or C and the nucleotide deεignated K iε either T or G. FIG. 12A. - Sequence homology between human C100- R and rat C100-R. Human #4.t3 sequence is aligned with the rat C100-R sequence depicted in Figure 9 starting at nucleotide 544. FIG. 12B. - Sequence homology between human C100- R and rat C100-R. Human clone HABIR #1#4 is aligned with the rat C100-R sequence depicted in Figure 4, starting at nucleotide 182. FIG. 12C. - Sequence homology between human C100- R and rat C100-R. Human clone #1#1T3 is aligned with the rat C100-R sequence depicted in Figure 4 starting at nucleotide 1868. 5. DETAILED DESCRIPTION OF THE INVENTION The preεent invention relateε to the cloning and expression of the C100-R. The C100-R was initially characterized as a binding site, present on the surface of neuronally derived cells, that bound εpecifically to the amyloid peptide / 8APP-C100. Expreεsion of the / 8APP-C100 amyloid peptide has been shown in animal models to correlate with the εpecific type of neuronal degeneration that occurs in Alzheimer's Disease. The C100-R produced herein may be used to evaluate and screen drugs and analogs of β- APP that may be used in the diagnoεiε and/or treatment of Alzheimer's Disease. 5.1. THE C100-R CODING SEQUENCE The initial rat C100-R cDNA clone, designated λABlR, was obtained by screening a λgtll rat brain expresεion library with /3-APP-C100 ligand labeled with 35 S-methionine or a peptide epitope "flag" sequence as described in Section 6, infra. The initial clone, designated pABIR-rat, was found to contain a 1970 bp insert comprised of the nucleotide coding sequence and deduced amino acid sequence depicted in Figure 4. A corresponding cDNA clone has been deposited with the ATCC and asεigned numberε deεcribed in Section 7, infra. Sequence analyεis reveals that the clone encompasεeε the carboxy-terminal εection of the gene and containε 1395 baεe pairs of coding εequence and 575 bp of 3' untranεlated εequence (FIG. 4). Using RNA prepared from rat brain and a primer specific for the most 5' end of the ABIR cDNA insert, first εtrand εynthesis waε performed in a reverεe tranεcriptaεe reaction. Following εecond εtrand synthesiε, the cDNA waε inεerted into the bacterial Blueεcript plaεmid (Stratagene) . Additional rat C100- R cDNA cloneε were isolated and sequenced (FIG. 9 and FIG. 10) . The new εequences overlap with those rat sequences shown in Figure 4 and extend the coding region towardε the amino-terminal end of the C100-R protein (FIG. 10) . The sequence disclosed in Figure 9 diverges upstream of nucleotide 106 from the rat sequence shown in Figure 10, indicating that alternatively εpliced mRNAs may exist for the C100-R clone. A search of the Genebank nucleotide sequence data base for homologies to the C100-R reveals a number of matches with the "B" type calcium channels, the Ryanodine calcium channel, and the high affinity calcium binding protein, calreticulin (FIG 5) . The homology shared between C100-R and calcium channels is consiεtent with the unusual pH dependence of binding and the observation that disruption of calcium levels in neurons, particularly in cortex and hippocampus, reεults in neurotoxicity. The cDNA sequence of C100-R also contains homologieε to the SER/THR Kinase "signature" or conserved catalytic core sequence described by Hankε et al. (1988, Science 241:42-52) FIG. 4. Aε indicated by the detection of an 11 Kb C100-R mRNA on Northern blots, the nucleotide sequence depicted in Figure 4 is only a partial representation of the C100-R gene. Full length rat C100-R cDNA sequence may be obtained using a number of different methods. For instance, probeε specific for the most 5' sequence of the rat clone may be uεed to reεcreen the rat cDNA librarieε. With each εucceεεful round of screening new 5' probes can be designed and used to reprobe libraries until the entire sequence has been obtained. PCR technology may also be used to isolate the full-length rat C100-R gene. Using RNA prepared from an appropriate rat source, a primer specific for the most 5 ' end of the rat cDNA inεert can be uεed to prime firεt strand syntheεiε in a reverεe tranεcriptaεe reaction. The RNA/DNA hybrid can then be "tailed" with guanineε uεing terminal tranεferase, digested with RNAase H, followed by εecond εtrand synthesiε primed with a poly-C primer. By using primers with specific restriction εites incorporated into them, these "clone-specific" cDNAε can be eaεily inεerted into an appropriate plasmid vector and sequenced. The invention also relateε to C100-R geneε iεo- lated from other εpecies, including humans, in which C100-R activity exiεts. Members of the C100-R family are defined herein aε thoεe receptorε that bind 3APP- C100 or fragmentε of the peptide. Such receptorε may demonεtrate about 80% homology at the nucleotide level, and even 90% homology at the amino acid level in εubstantial stretches of DNA sequence. A bacteriophage cDNA library may be screened, under conditions of reduced stringency, using a radioactively labeled fragment of the rat C100-R clone. Alternatively the rat C100-R sequence can be used to design degenerate or fully degenerate oligonucleotide probes which can be used aε PCR probeε or to εcreen bacteriophage cDNA librarieε. A poly- meraεe chain reaction (PCR) baεed εtrategy may be uεed to clone human C100-R. Two poolε of degenerate oligo- nucleotides, corresponding to conserved motifs between the rat C100-R and calcium channels, may be designed to serve as primers in a PCR reaction. The template for the reaction is cDNA obtained by reverse transcription of mRNA prepared from cell lines or tisεue known to expreεε human APP-R. The PCR product may be εubcloned and εequenced to inεure that the amplified sequences represent the C100-R sequenceε. The PCR fragment may be uεed to iεolate a full length C100-R cDNA clone by radioactively labeling the amplified fragment and εcreening a bacteriophage cDNA library. Alternatively, the labeled fragment may be uεed to εcreen a genomic library. For a review of cloning strategieε which may be uεed, see E.g.. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Cold Springε Harbor Preεε, N.Y.; and Auεubel et al., 1989, Current Protocols in Molecular Biology, (Green Publishing Asεociates and Wiley Interscience, N.Y.) A cDNA library may also be constructed in a mammalian expression vector such as pcDNAl, that contains SV40 origin of replication sequences which permit high copy number expreεεion of plaεmidε when tranεferred into COS cells. The expression of C100-R on the surface of tranεfected COS cellε may be detected in a number of ways, including the use of a labeled ligand such as / SAPP-CIOO or a βAPP-ClOO agonist labeled with a radiolabel, fluoreεcent label or an enzyme. Cellε expressing the human C100-R may be enriched by subjecting tranεfected cellε to a FACS (fluorescent activated cell sorter) sort. In a specific embodiment described herein, an adult hippocampal cDNA library was screened with a labeled nucleotide fragment from the rat C100-R clone. The large EcoRI fragment, extending from nucleotide 31 through nucleotide 409 of the AB2R rat clone (FIG. 9) waε used to probe the human cDNA library. Several clones were obtained and the cDNA insertε were sequenced. Human C100-R sequence is shown in Figure 11, and regions of homology between the rat and human C100-R sequences are shown in Figure 12. Any of the methods described above for iεolation of full length rat C100-R cloneε may be uεed equally well for isolation of full length human C100-R clones. For instance, probeε εpecific for the moεt 5' εequence of the human clone may be uεed to reεcreen the human cDNA library. With each successful round of sequencing new 5' probes may be designed and used to reprobe libraries until the entire sequence has been obtained. In accordance with the invention, nucleotide sequences which encode C100-R, fragments, fusion proteins or functional equivalents thereof, may be used to generate recombinant DNA moleculeε that direct the expression of the C100-R, or a functionally active peptide, fusion protein or functional equivalent thereof, in appropriate hoεt cellε. Alternatively, nucleotide εequenceε which hybridize to portionε of the C100-R εequence may alεo be uεed in nucleic acid hybridization aεsays, Southern and Northern blot analyεeε, etc. Due to the degeneracy of the genetic code, other DNA εequenceε which encode substantially the C100-R amino acid sequence, e.g.. such as the rat sequence depicted in Figure 4 or a functional equivalent may be used in the practice of the present invention for the cloning and expresεion of the C100-R. Such DNA sequences include those which are capable of hybridizing to the rat or human C100-R εequence under εtringent conditionε, or which would be capable of hybridizing under εtringent conditionε but for the degeneracy of the genetic code. The εtringency conditionε may be adjuεted in a number of wayε. For example, when performing polymeraεe chain reactionε (PCR) , the temperature at which annealing of pri erε to template takeε place or the concentration of MgCl 2 in the reaction buffer may be adjuεted. When uεing radioactively labeled DNA fragmentε or oligonucleotideε to probe filterε, the εtringency may be adjuεted by changeε in the ionic strength of the wash solutions or by careful control of the temperature at which the filter washeε are carried out. Altered DNA sequences which may be used in accor¬ dance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within the C100-R sequence, which result in a silent change thus produ¬ cing a functionally equivalent C100-R. Such amino acid substitutions may be made on the basiε of εimilarity in polarity, charge, εolubility, hydrophobicity, hydrophilicity, and/or the amphipatic nature of the reεidueε involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; poεitively charged amino acidε include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, aniline; aεparagine, glutamine; serine, threonine; phenylalanine, tyrosine. Aε uεed herein, a functionally equivalent C100-R referε to a receptor which bindε to β-APP-ClOO or fragmentε, but not neceεsarily with the εame binding affinity of its counterpart native C100-R. The DNA sequenceε of the invention may be engi¬ neered in order to alter the C100-R coding εequence for a variety of endε including but not limited to alterationε which modify proceεεing and expreεεion of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g. site-directed mutagenesiε, to inεert new reεtriction εiteε, to alter glycosylation patterns, phosphorylation, etc. For example, in certain expreεεion systemε such as yeast, hoεt cellε may over glycoεylate the gene product. When uεing εuch expres- sion systems it may be preferable to alter the C100-R coding sequence to eliminate any N-linked glycosyla- tion εite. In another embodiment of the invention, the C100-R or a modified C100-R εequence may be ligated to a heterologous sequence to encode a fusion protein. The fusion protein may be engineered to contain a cleavage site located between the C100-R εequence and the heterologouε protein sequence, so that the C100-R can be cleaved away from the heterologouε moiety. In an alternate embodiment of the invention, the coding sequence of C100-R could be syntheεized in whole or in part, uεing chemical methodε well known in the art. See, for example, Carutherε, et al., 1980, Nuc. Acidε Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nuc. Acidε Res. 9(12) :2807-2817. Alternatively, the protein itself could be produced uεing chemical methodε to synthesize the C100-R amino acid sequence in whole or in part. For example, peptides can be syntheεized by solid phase techniques, cleaved from the reεin, and purified by preparative high performance liquid chromatography. (E.g.. see Creighton, 1983, Proteins Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). The composition of the εynthetic peptideε may be confirmed by amino acid analysis or sequencing (e.g. _ the Ed an degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principleε, W.H. Freeman and Co., N.Y., pp. 34-49. 5.2. EXPRESSION OF THE C100-R In order to express a biologically active C100-R, the nucleotide sequence coding for C100-R, or a func¬ tional equivalent as deεcribed in Section 5.1 supra. is inεerted into an appropriate expreεεion vector, i.e.. a vector which containε the neceεsary elements for the tranεcription and tranεlation of the inεerted coding εequence. The C100-R gene productε aε well aε host cells or cell lines transfected or tranεformed with recombinant C100-R expression vectors can be used for a variety of purposeε. Theεe include but are not limited to generating antibodieε (i.e.. monoclonal or polyclonal) that bind to the receptor, including thoεe that competitively inhibit binding and "neutralize" β- APP activity of BAPP or fragmentε of BAPP, and the screening and selection of β-APP analogs or drugs that act via the C100-R; etc. 5.2.1. EXPRESSION SYSTEMS Methods which are well known to those skilled in the art can be used to construct expreεsion vectors containing the C100-R coding sequence and appropriate transcriptional/tranεlational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniqueε and in vivo recombination/genetic recombination. See, for example, the techniqueε described in Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Asεociateε and Wiley Interεcience, N.Y. A variety of host-expresεion vector εyεtemε may be utilized to express the C100-R coding sequence. These include but are not limited to microorganiεmε εuch aε bacteria tranεformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expreε¬ εion vectors containing the C100-R coding sequence; yeast transformed with recombinant yeast expresεion vectorε containing the C100-R coding sequence; insect cell systems infected with recombinant virus expres¬ sion vectors (e.g.. baculovirus) containing the C100-R coding εequence; plant cell systems infected with recombinant virus expression vectors (e.g. , cauli¬ flower mosaic viruε, CaMV; tobacco moεaic viruε, TMV) or tranεformed with recombinant plasmid expression vectors (e.g. , Ti plasmid) containing the C100-R coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g.. adenovirus, vaccinia virus) including cell lines engineered to contain multiple copies of the C100-R DNA either stably amplified (e.g.. CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g.. murine cell lines) . The expresεion elementε of theεe systems vary in their strength and specificitieε. Depending on the host/vector system utilized, any of a number of suit¬ able transcription and translation elements, including constitutive and inducible promoters, may be used in the expresεion vector. For example, when cloning in bacterial εystems, inducible promoterε εuch aε pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be uεed; when cloning in inεect cell εyεtemε, promoterε εuch aε the baculoviruε polyhedrin promoter may be uεed; when cloning in plant cell εystems, promoters derived from the genome of plant cells (e.g.. heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruseε (e.g. , the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mamma- lian cell εyεtemε, promoterε derived from the genome of mammalian cells (e.g.. metallothionein promoter) or from mammalian viruses (e.g. , the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lineε that contain multiple copieε of the C100-R DNA, SV40-, BPV- and EBV-baεed vectorε may be uεed with an appropriate εelectable marker. In bacterial εyεtemε a number of expreεsion vectorε may be advantageouεly εelected depending upon the use intended for the C100-R expresεed. For example, when large quantitieε of C100-R are to be produced for the generation of antibodieε, vectorε which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited to the ______ coli expresεion vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the C100-R coding sequence may be ligated into the vector in frame with the lacZ coding region so that a hybrid ClOO-R/lacZ protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectorε may alεo be used to express foreign polypeptides as fuεion proteinε with glutathione S-transferase (GST) . In general, such fusion proteins are soluble and can eaεily be purified from lysed cells by affinity chromatography, e.g. , adsorption to glutathione-agaroεe beadε followed by elution in the preεence of free glutathione. The pGEX vectorε are deεigned to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety. See also Booth et al., 1988, Immunol. Lett. 19: 65-70; and Gardella et al., 1990, J. Biol. Chem. 265: 15854- 15859; Pritchett et al., 1989, Biotechniqueε 7:580. In yeast, a number of vectors containing consti- _ tutive or inducible promoterε may be uεed. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Auεubel et al. , Greene Publiεh. Aεεoc. & Wiley Interscience, Ch. 13; Grant et al., 1987, Expresεion and Secretion Vectorε for Yeaεt, m 0 Methods in Enzymology, Eds. Wu & Groεεman, 1987, Acad. Preεε, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Preεε, Waεh. , D.C., Ch. 3; and Bitter, 1987, Heterologouε Gene Expreεεion in Yeaεt, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyceε, 1982, Edε. Strathern et al.. Cold Spring Harbor Preεs, Vols. I and II. In caεeε where plant expreεsion vectors are used, the expression of the C100-R coding sequence may be driven by any of a number of promoters. For example, viral promoterε such as the 35S RNA and 19S RNA promoters of CaMV (Brisεon et al., 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatεu et al., 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small εubunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984, Science 224:838- 843); or heat shock promoters, e.g.. soybean hspl7.5-E or hεpl7.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be uεed. Theεe conεtructε can be introduced into plant cellε uεing Ti plaεmidε, Ri plaεmidε, plant viruε vectorε, direct DNA tranεforma- tion, microinjection, electroporation, etc. For reviews of such techniques see, for example, Weisεbach & Weiεεbach, 1988, Methods for Plant Molecular Biology, Academic Preεε, NY, Section VIII, pp. 421- 463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9. An alternative expression system which could be used to express C100-R is an inεect εyεtem. In one such system, Autograoha californica nuclear polyhidroεiε virus (AcNPV) is used as a vector to express foreign geneε. The viruε growε in Spodoptera frugiperda cellε. The C100-R coding εequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) . Successful insertion of the C100-R coding εequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e.. viruε lacking the proteinaceouε coat coded for by the polyhedrin gene) . Theεe recombinant viruεeε are then uεed to infect Spodoptera frugiperda cells in which the inεerted gene iε expressed. (E.g.. see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Patent No. 4,215,051). In mammalian host cellε, a number of viral based expression syεtems may be utilized. In cases where an adenovirus iε used as an expression vector, the C100-R coding εequence may be ligated to an adenoviruε trans¬ cription/translation control complex, e.g. , the late promoter and tripartite leader εequence. Thiε chimeric gene may then be inserted in the adenoviruε genome by in vitro or in vivo recombination. Inser¬ tion in a non-essential region of the viral genome (e.g.. region El or E3) will result in a recombinant virus that iε viable and capable of expreεεing C100-R in infected hosts. (E.g.. See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659) . Alterna¬ tively, the vaccinia 7.5K promoter may be used. (E.g.. εee Mackett et al., 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al. , 1984, J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931). Specific initiation εignalε may alεo be required for efficient tranεlation of inserted C100-R coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire C100-R gene, including its own initiation codon and adjacent sequences, is inserted into the appro¬ priate expression vector, no additional translational control signalε may be needed. However, in cases where only a portion of the C100-R coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the C100-R coding sequence to ensure translation of the entire insert. These exogenous translational control signalε and initiation codonε can be of a variety of origins, both natural and synthetic. The efficiency of expresεion may be enhanced by the incluεion of appropriate tranεcription enhancer elementε, transcription terminators, etc. (εee Bitter et al., 1987, Methodε in Enzymol. 153:516- 544) . In addition, a hoεt cell εtrain may be choεen which modulateε the expreεεion of the inεerted εequenceε, or modifieε and proceεεeε the gene product in the εpecific faεhion deεired. Such modificationε (e.g.. glycosylation) and processing (e.g. , cleavage) of protein products may be important for the function of the protein. Different host cells have charac¬ teristic and specific mechanismε for the poεt-tranεla- tional proceεεing and modification of proteinε. Appropriate cellε lineε or hoεt εyεtems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukary- otic host cells which possesε the cellular machinery for proper proceεsing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian hoεt cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc. For long-term, high-yield production of recombi¬ nant proteins, εtable expreεsion is preferred. For example, cell lines which stably expresε the C100-R may be engineered. Rather than uεing expreεsion vectorε which contain viral originε of replication, hoεt cellε can be tranεformed with the C100-R DNA controlled by appropriate expreεεion control elements (e.g.. promoter, enhancer, sequences, transcription terminators, polyadenylation εiteε, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The εelectable marker in the recombinant plaεmid conferε reεiεtance to the εelection and allows cells to stably integrate the plasmid into their chromosomeε and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which expresε the C100-R on the cell surface, and which respond to BAPP-CIOO mediated signal transduction. Such engineered cell lines are particularly useful in screening for BAPP-CIOO analogs. A number of selection εyεtems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltranεferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferaεe (Lowy, et al., 1980, Cell 22:817) geneε can be _ employed in tk " , hgprt " or aprt " cellε, respectively. Also, antimetabolite resistance can be used as the basiε of εelection for dhfr, which conferε resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. 0 Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resiεtance to the aminoglycoεide G-418 (Colberre- Garapin, et al., 1981, J. Mol. Biol. 150:1); and 5 hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147) geneε. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows Q cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylaεe) which conferε reεiεtance to the ornithine decarboxylaεe inhibitor, 2-(difluoromethyl) -DL-ornithine, DFMO 5 (McConlogue L. , 1987, In: Current Communicationε in Molecular Biology, Cold Spring Harbor Laboratory ed.). 5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS THAT EXPRESS THE C100-R The host cells which contain the coding sequence and which expreεε the biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or abεence of "marker" gene functionε; (c) aεεeεεing the level of tranεcription aε measured by the expres¬ sion of C100-R mRNA traήscriptε in the hoεt cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity. In the first approach, the presence of the C100-R _ coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequenceε that are homologouε to the C100-R coding εequence, reεpectively, or portionε or derivativeε thereof. Q In the εecond approach, the recombinant expreε¬ sion vector/hoεt system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g.. thymidine kinase activity, resistance to antibiotics, reεiεtance to methotrexate, tranεformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the C100-R coding sequence is inserted within a marker gene sequence of the vector, recombinants containing the C100-R coding sequence can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the C100-R sequence under the control of the same or different promoter used to control the expression of the C100-R coding sequence. Expresεion of the marker in response to induction or selection indicates expresεion of the C100-R coding εequence. In the third approach, tranεcriptional activity for the C100-R coding region can be aεsesεed by hybridization aεsays. For example, RNA can be iεolated and analyzed by Northern blot using a probe homologous to the C100-R coding sequence or particular portions thereof. Alternatively, total nucleic acids of the host cell may be extracted and asεayed for hybridization to εuch probeε. In the fourth approach, the expreεsion of the C100-R protein product can be assesεed immunologically, for example by Western blots, immunoassays such aε radioimmuno-precipitation, enzyme-linked immunoaεεayε and the like. The ultimate teεt of the success of the expresεion εyεtem, however, involves the detection of the biologically active C100-R gene product. A number of assays can be used to detect receptor activity including but not limited to B-APP binding aεεays; and β-APP biological asεayε uεing engineered cell lineε aε the teεt εubεtrate. 5.2.3. RECOVERY OF THE C100-R Once a clone that produceε high levelε of biolo¬ gically active C100-R iε identified, the clone may be expanded and used to produce large amounts of the receptor which may be purified using techniques well- known in the art including, but not limited to immuno- affinity purification, chromatographic methods including high performance liquid chromatography, affinity chromatography using immobilized ligand such as β-APP-ClOO or analogε thereof bound to beadε, immunoaffinity purification uεing antibodieε and the like. Where the C100-R coding εequence iε engineered to encode a cleavable fuεion protein, purification may be readily accompliεhed uεing affinity purification tech¬ niqueε. For example, a collagenase cleavage recog¬ nition consensus sequence may be engineered between the carboxy terminuε of C100-R and protein A. The resulting fusion protein may be readily purified using an IgG column that binds the protein A moiety. Unfused C100-R may be readily releaεed from the column by treatment with collagenaεe. Another example would be the use of pGEX vectors that expresε foreign polypeptides as fusion proteins with glutathionine S- tranεferaεe (GST) . The fuεion protein may be engi- neered with either thrombin or factor Xa cleavage sites between the cloned gene and the GST moiety. The fusion protein may be easily purified from cell extracts by adsorption to glutathione agarose beadε followed by elution in the preεence of glutathione. In thiε aεpect of the invention, any cleavage εite or enzyme cleavage substrate may be engineered between the C100-R sequence and a second peptide or protein that has a binding partner which could be uεed for purification, e.g. _ any antigen for which an immuno- affinity column can be prepared. 5.3. GENERATION OF ANTIBODIES THAT DEFINE THE C100-R Various procedures known in the art may be used for the production of antibodieε to epitopeε of the recombinantly produced C100-R. Neutralizing antibodieε i.e.. thoεe which compete for the BAPP-CIOO binding site of the receptor are especially preferred for diagnostics and therapeutics. Such antibodieε include but are not limited to polyclonal, monoclonal, chimeric, εingle chain, Fab fragmentε and fragments produced by an Fab expreεεion library. For the production of antibodies, various host animals may be immunized by injection with the C100-R including but not limited to rabbitε, mice, ratε, etc. Various adjuvants may be used to increase the immuno- logical response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gelε such as aluminum hydroxide, surface active subεtanceε εuch aε lyεolecithin, pluronic polyols, polyanions, peptides, oil emulsionε, keyhole limpet hemocyanin, dinitrophenol, and poten¬ tially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corvnebacterium parvum. by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the EBV-hybridoma tech¬ 10 nique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Lisε, Inc., pp. 77-96). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 15 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the geneε from a mouεe antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be 2 _0_ used. Antibody fragments which contain specific binding sites of C100-R may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragmentε which can be produced • > - by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments. Alterna¬ tively, Fab expreεsion libraries may be constructed (Huεe et al., 1989, Science, 246:1275-1281) to allow 30 rapid and eaεy identification of monoclonal Fab fragmentε with the deεired εpecificity to C100-R. 5.4. ANTI-SENSE RNA AND RIBOZYMES Alεo within the scope of the invention are oligo- 5 ribonucleotide sequenceε, that include anti-εenεe RNA and DNA molecules and ribozymes that function to inhibit the translation of C100-R mRNA. Anti-senεe RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. In regard to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g.. between -10 and +10 regions of the C100-R nucleotide sequence, are preferred. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mecha¬ nism of ribozyme action involveε εequence εpecific hybridization of the ribozyme molecule to complemen¬ tary target RNA, followed by a endonucleolytic cleav- age. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of C100-R RNA sequences. Specific ribozyme cleavage siteε within any potential RNA target are initially identified by εcanning the target molecule for ribozyme cleavage εiteε which include the following εequences, GUA, GUU and GUC. Once identified, short RNA εequenceε of between 15 and 20 ribonucleotideε corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligo¬ nucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complemen¬ tary oligonucleotides, uεing ribonucleaεe protection aεεayε. Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniqueε for chemically synthesizing oligodeoxyribonucleotideε well known in the art such as for example solid phase phosphoramidite chemical synthesiε. Alternatively, RNA moleculeε may be generated by in vitro and in vivo tranεcription of DNA sequences encoding the antisense RNA molecule. Such DNA εequenceε may be incorporated into a wide variety of vectorε which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antiεense cDNA constructε that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lineε. Variouε modifications to the DNA molecules may be introduced as a means of increaεing intracellular stability and half-life. Posεible modificationε include but are not limited to the addition of flanking εequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the uεe of phosphorothioate or 2' O-methyl rather than phospho- diesterase linkageε within the oligodeoxyribonucleo- tide backbone. 5.5. USES OF THE C100-R, DNA AND ENGINEERED CELL LINES The C100-R DNA, antisense oligonucleotides and ribozymes, APP-R expreεεion products, antibodies and engineered cell lines described above have a number of uses for the diagnosiε and treatment of Alzheimer'ε Diseaεe. For example, the C100-R DNA εequence may be uεed in hybridization assayε of biopsies or autopεieε to diagnoεe abnormalitieε of C100-R expreεεion; e.g.. Southern or Northern analyεiε, including i-n εitu hybridization aεsays. In therapeutic applicationε, antiεenεe or ribozyme moleculeε designed on the basis of the C100-R DNA sequence may be utilized to block transcription and expression of the C100-R gene product. Alternatively, the C100-R DNA could be used in gene therapy approach to introduce the normal recombinant gene into the defective cells of an individual or to correct an endogenouε mutation in order to reconstitute the C100-R and its function. In another embodiment of the invention, anti¬ bodies specific for the C100-R may be used to determine the pattern of receptor expreεεion in biopsy tisεue, or for diagnoεtic imaging in vivo; in εuch applicationε, "neutralizing" antibodieε may be preferred. For example, an antibody conjugated to an imaging compound could be adminiεtered to a patient to "map" the locationε and diεtribution of the C100-R in vivo. In another embodiment of the invention, the APP-R itself, or a fragment containing its BAPP-CIOO binding site, could be administered in vivo. The free C100-R or the peptide fragment could competitively bind to β- APP and inhibit its interaction with the native receptor in vivo . In yet another embodiment of the invention, the engineered cell lines which express the entire C100-R or its ligand binding domain may be utilized to screen and identify active /3APP-C100 agonists or antagonists. Alternatively, cell lines that endogenously express the C100-R may be used for this purpose; e.g. , neuroblastoma cell lineε εuch aε SK-N-MC aε exemplified herein may be used. /SAPP-derived peptides, other peptides, synthetic compounds, natural products, and other sourceε of potentially biologically active materialε can be εcreened in the following manners. The ability of a teεt compound to inhibit the binding of / SAPP-CIOO (which can be labelled e.g.. with 35 Met or a "flag" sequence or detected with antibodies to the peptide) to the C100- R, and thus its potential to act as either agonistε or antagonists may be meaεured. Aε a εource of the receptor either whole ClOO-R-expreεεing cellε homogenateε, or subcellular fractions may be used. The binding may be measured using standard receptor binding techniques, such as those deεcribed in Section 0 6.1.3. The ability of agentε to prevent, or mimic, the effectε of 3APP-C100 on signal transduction responseε in the ClOO-R-expreεεing cellε may be measured. For example, responseε εuch aε changes in cytosolic concentrationε of calcium, activation of 5 specific protein kinaseε, altered εecretion of hormoneε or neurotranεmitterε, modulation of second mesεenger production, or changeε in cellular metaboliεm may be monitored. These assays may be performed in whole cells or membrane preparations Q using conventional techniqueε developed for theεe purpoεeε. In engineered cellε or cell lineε that reεpond to the toxic effectε / SAPP-CIOO, the ability of εubεtanceε to induce or prevent the toxicity may be meaεured. Toxicity may be monitored aε deεcribed in 5 the literature (Yankner et al., 1989, Science 245:417- 420) , or by other eεtabliεhed techniqueε. Tranεgenic animals that contain the C100-R DNA as the transgene may be engineered to determine the in vivo effectε of the / SAPP-CIOO agoniεts or antagonistε found in the above or other εcreenε, or to profile other agentε that are potentially therapeutic for Alzheimer'ε Diεeaεe. Recently, computer generated modelε for ligand- receptor interactions have been developed, and in a εpecific embodiment of the invention, information derived from computer modeling of C100-R may be used for design of receptor agonist or antagoniεt. Changeε made to C100-R sequences, using for example techniques for site directed mutagenesis, and expression of mutant receptors in cell lines may be used to further define the functional role of particular receptor regions and residues. 6. EXAMPLES The subεection below deεcribeε the initial characterization of an APP binding protein expressed on neuronally derived cells and the cloning and sequencing of a complementary DNA representing part of the rat C100-R. The deduced amino acid sequence of C100-R reveals several motifs shared in common between "B" type calcium channels, Ryanodine calcium channels, calreticulin and protein Serine/Threonine Kinases. 6.1. MATERIAL AND METHODS 6.1.1. IN VITRO TRANSLATION OF FLAG-βAPP-ClOO The following "flag" εequence-containing oligonucleotide waε εyntheεized and inserted into pGEM7, ACC ATG GAC TAC AAA GAC GAT GAC GAT AAA TCG AT MET ASP TYR LYS ASP ASP ASP ASP LYS SER. (Immunex; Prickett et al. 1989 BioTechniques 7:580). In addition, prior to the MET codon, the ACC triplet gives a consensus sequence for eucaryotic translation start and the Clal site after the flag εequence waε added for cloning aε waε Hindlll linker at the five prime end of the oligio. The pGEM7 flag conεtruct waε cut with Smal and Clal and the Clal εite waε filled in. The Bgll/Smal fragment from jSAPP-695 was cloned into this construct. Cloneε with the proper orientation were selected. This recombinant was utilized to make 35 S- / SAPP-C100 peptide in two ways. Firεt, in the coupled transcription/translation system, TNT Coupled Reticulocyte Lysate Syεtem (Promega Corp.). Cloεed circular plaεmid DNA waε 5 added to the lysate along with 35 S-methionine (6 μC,) following the recommended procedure. Second, in uncoupled reactions, the recombinant was linearized with Spe I, and 5 μg DNA was utilized to produce RNA in vitro («20-30μg) following the manufacturer•ε 0 protocol (Promega Corp.). 1-5 μg of thiε RNA waε tranεlated in either a wheat germ or rabbit reticulocyte tranεlation system according to manufac¬ turer's protocol (Promega Corp.). The in vitro tranεlationε either had one-tenth volume of 100 mM N- 5 acetyl glucoεamine (Sigma) added, and were aliquotted and frozen at -80°C, and dialyzed prior to uεe; were aliquotted and frozen directly, then dialyzed and εpin column purified prior to uεe (Boehringer-Mannheim) ; or were affinity purified using the commercially avail- Q able Flag Anti-body (CA ++ -dependent binding Ml Ab, IBI) coupled to Affigel 10 (Biorad) . Affinity chroma¬ tography was accomplished by pasεing the in vitro translations over the Affigel 10-coupled Ml antibody column (1 ml bed volume) equilibrated in PBS (120 mM 5 NaCl, 2.7 mM KCl, 10 mM phoεphate buffer, pH 7.4) with 0.5 mM CaCl 2 . After thorough waεhing (5-10 column volumeε) , the peptide was eluted from the column with PBS with Na 2 EDTA (2 mM) . The peptide eluted as a sharp 0.7 ml peak. EDTA waε removed by dialyεiε for use in binding or autoradiography. The flag- / SAPP-C100 construct was sequenced and the product peptide from the in vitro translation analyzed for amino acid composition by masε spectroscopic techniqueε to confirm itε identity. 6.1-2. CELL CULTURE PC12 cells (ATCC) for the binding asεayε were grown in RPMI1640 supplemented with 10% heat-inacti- _ vated horse serum and 5% fetal bovine serum. T-175 flaεks were seeded with 5 x 10 s cells/ml in 60 ml of growth medium containing 10 ng/ml NGF. An additional 60 ml of this medium were added every 2 days until the cells were harvested (usually at 6 days) . Where 0 indicated, PC12 cellε were cultured aε above except in the abεence of NGF. Cells were collected by centrifu- gation at 138 X g for 8 minutes. The supernatant was discarded and the pellet resuspended in 5 ml of the growth medium. The cells were triturated 3 times through a 23g needle attached to a 10 cc εyringe to provide a εingle cell suspenεion or, at moεt, εmall cell aggregateε. Trypan blue dye excluεion indicated that over 99% of the cellε were viable. SK-N-MC cellε (ATCC) were grown in DME εupplemented with 10% calf serum. The SK-N-MC cells were cultured aε deεcribed for PC-12 cellε except NGF is not included. 6.1.3. BINDING TO CELLS For / 8APP-C100 binding experiments, PC-12 or SK-N- MC cells grown and prepared as described above, were counted, collected by low speed centrifugation (138xg) , and resuspended in phosphate-buffered saline (PBS, pH 7.4) with 1% BSA and 1% glucose (PBG) . An 0.08 ml volume of the cell suεpension, containing 2 x 10 6 cells, was mixed with 0.01 ml of 35 S- / 8APP-C100 and 0.01 ml of PBG, or PBG containing either unlabeled / SAPP-CIOO or its vehicle. For kinetic and inhibition experiments, the concentration of 35 S- / SAPP-C100 was either 25 pM or 50 pM. For saturation experiments, the ligand concentration ranged from 15 pM to 6 nM. The mixture was incubated at 4°C for 3 hourε, unleεε otherwise indicated. At the end of the incubation period, the cells were pelleted by centrifugation at 138 X g for 90 sec, the supernatant (containing the unbound ligand) was removed, and 0.2 ml of freεh, ice- cold PBG waε added. The cells and fresh buffer were briefly agitated, and again centrifuged and εuεpended in fresh, ice-cold buffer as above. Finally, the 0 cells were pelleted by centrifugation, filtered through BSA (0.1%) treated filters and washed with 4 1-ml rinseε of ice-cold PBS with 0.1% BSA. The duration of the period between resuspenεion of the cellε in freεh, ice-cold buffer and filtration of the 5 cells was less than 10 min. In the dissociation experiments, and to collect a sample of bound ligand, the cellε were incubated in the buffer added aε the εecond waεh for up to 3 hourε to allow the bound ligand to dissociate. The cells were then pelleted by 0 centrifugation and the supernatant (containing the bound ligand that had disεociated) waε removed. The cells were then rewashed and harvested as described above. Radioactivity retained by filters was meaεured by εcintillation counting. Nonεpecific binding was _ taken as that occurring in the presence of excesε (3.2 nM to 18 nM) 8APP-C100. Saturation data were analyzed by computer uεing commercially available εoftware. The binding model that best fitted the experimental data was determined by F-test (p<.05). 0 6.1.4. BINDING AUTORADIOGRAPHY Sprague-Dawley ratε were εacrificed by hyper- capnia and placed on ice until their brainε could be removed (less than an hour) . The brains were rapidly frozen in isopentane chilled to -20°C with dry ice and either used immediately or stored at -80°C until uεe. The brains were affixed to the specimen holder of a cryostat and 25 μM coronal sections were collected onto gelatin-coated microscope slides. The sections were either frozen overnight at -80°C or used imme¬ diately. The sections were air-dried at 4°C and then preincubated with the binding buffer (PBS, 1% BSA) for 30 minutes at 4°C. This buffer waε replaced by freεh buffer containing either the ligand alone ( 35 S- / 8APP- C100, O.OlnM) or the ligand with up to a 10-fold excess of unlabeled /SAPP-CIOO or its vehicle. The incubation was continued for 3 h at 4°C. The ligand- containing buffer was then removed and the sections were washed twice with freεh buffer (15 min. and 1 min., 4°C) . The εectionε were then dipped into ice cold distilled water 10 times and dried under a stream of air at room temperature. The sectionε were then apposed to a sheet of X-ray film for a period of 1 to 14 days and the film developed. Some sections were stained with Cresyl violet for identification of brain region. The optical densities of regions of the autoradiographs were determined quantitatively uεing an image analyεiε system. 6.1.5. SCREENING FOR THE C100-R CDNA BY EXPRESSION CLONING cDNA waε conεtructed from embryonic day 18 rat brain mRNA according to the protocol of Neve et al. (1986; delineated in detail by Klickεtein and Neve in Current Protocols in Molecular Biology) . λgtll libraries (rat brain cDNA library, RL Neve; human brain cDNA library, Clontech) were plated on Y1090 E. coli cells at 150,000 pfu/150mm plate. Plates were incubated at 42°C for 3 h, at which time plaques were just viεible. Plateε were overlaid with Schleicher and Schuell 132 mm nitrocelluloεe filterε which had been saturated with 10 mM IPTG (isopropyl-/?.D- thiogalactoside) and slightly air dried. Plateε were set at 37°C for 3 hours, and the first filter removed _ and placed in TBS (50 mM Tris, pH 7.7, 150 mM NaCl). A second, duplicate IPTG-soaked nitrocelluloεe filter waε placed on the plate surface and left for two hours. The second filter was removed and placed in TBS. The filters were allowed to εit in TBS without 0 blocking agent for at leaεt 12 hours and up to four days to allow refolding. Filters were blocked in TBS with 5% nonfat skimmed milk for periods ranging from 2 h to overnight. Filters were waεhed in binding buffer (50 m Tris-HCl, Ph 7.7, 50 mM NaCl, 2 mM MgCl 2 , ImM 5 Dithiothreitol) . Filters were incubated with in vitro translated 35 S-/8APP-C100 at 3 nM for two hourε at 4°C. Three 3 minuteε waεheε (4°C) were uεed to remove non- bound labeled peptide: (i) binding buffer alone, (ii) binding buffer + 0.1% NP40, and (iii) binding buffer. Q Filters were immediately removed from the wash and air dried. The processed filters were exposed to X-OMAT RP film with a screen at -70°C for 6 days to two weeks. It was found that wet plates were necessary to get sufficient protein/plaque to get significant 5 signal. Alεo, throughout the procedure it waε neceεsary to plate many more phage than routinely required at the comparable stage of purification for nucleic acid probes. Even when a "plaque"-pure phage population containing the / SAPP-CIOO binding protein site was plated as described above, only a low percentage of the plaques bound to the βAPP-ClOO, posεibly due to the requirement of proper folding for binding. Duplicate filterε were compared to identify plaques expressing protein which bound to the 35 S-βAPP- C100. Positive plaques were picked and taken through further roundε of binding and purification. Six roundε of isolation were required to obtain a single poεitive clone from a rat brain cDNA library. Thiε rat clone is designated λABlR. Five rounds of isola¬ tion were required to isolate five positive clones from a human brain cDNA library. The inεert from λABlR was subcloned by PCR amplification using λgtll sequencing primers which include restriction enzyme sites (Sal I and Not I) . The amplified fragment was purified, cut with the restriction enzymes as per manufacturer'ε specifica¬ tions and subcloned into Sall/Notl-cut Blueεcript (SK+, Stratagene) . The rat εubclone waε deεignated pABlR-rat. A second cloning strategy was developed utilizing the commercially available antibody to the "flag" tag. Libraries were plated, filter lifts produced, procesεed, probed and waεhed aε deεcribed above with the exception that the /3APP-C100 was not labeled with 35 S-methiόnine. Filters were cross-linked in a Stratalinker cross-linker (Stratagene) and blocked by 2 h incubation in TBS with 5% nonfat milk. The filters were then probed with a commercial anti-flag antibody at 10 μg/ml (M2, IBI) in TBS. Excess anti¬ body was removed by three 10 minute washeε in TBS (4°C), with the middle wash solution εupplemented with 0.1% NP40. Filterε were incubated with a second antibody, goat anti-mouse IgG (heavy and light chain) biotin conjugate (NEN) in the same manner as with the first antibody. After washing, the filters were incubated with [ 125 I]-Avidin (NEN) at 0.5 μC,/ml in binding buffer as above. The last three washeε were in TBS with the middle wash including 0.1% NP40. Filters were air dried, and exposed to XOMAT-RP film with screen at -70°C for 6 d to three weeks. Autora- diographs of the filters were compared for duplicates. Positive plaques were picked and carried through further rounds of εcreening. Phage containing the human cognate of the / SAPP- CIOO binding protein were cloned by both the direct binding method and the flag-antibody method. These cDNAs have been subcloned and are being sequenced. 6.1.6. CDNA CHARACTERIZATION cDNAε were either subcloned into high copy plasmid vectors and tranεformed into bacteria or were amplified directly from the phage reco binants by the polymerase chain reaction. In the former caεe, double-εtranded miniprep DNA waε sequenced uεing Sequenaεe (U.S. Biochemicalε) ; in the latter caεe, the PCR products were purified and sequenced with the femtomole sequencing εyεtem (Promega) . Sequencing of the cloneε was done by standard dideoxy methodology (Sequenase; USBiochem) . Computer analysiε was done primarily with GenePro (Riverside Scientific Enterpriseε) and PCGene (Intelligenicε) . Southern tranεfers of human DNA or rat DNA (PC12) were probed with a 32 P-labeled 260 bp fragment (EcoRI/Scal) repreεenting the 5' end of the AB1R cDNA. 8 μg of DNA was cut with various reεtriction enzymes, run on a 0.8% agarose gel, and transferred to nylon or nitrocellulose. Northern analyεiε waε carried out on total cellular RNA iεolated with the guanidinium thiocyanate method (Chirgwin et al. 1979 Biochemiεtry 18 5294). Glyoxal gels, formaldehyde-agarose gels and dot blots were utilized to quantitate and evaluate the RNA under different conditions and from different sources (Tanzi et al., 1987, Science 235:880-884; Tanzi et al., 1988, Nature 331:528-530) . RNA from PC12 cells treated with nerve growth factor for various periods of time, RNA from brains of patients with Alzheimer's Diseaεe and Down's Syndrome, RNA from brain subregionε, RNA from variouε developmental stages, etc. was run. Samples from AD or Downs brains were attained from the Children' Hospital (Boston) Pathology Dept. and from the McLean Hoεpital Brain Tissue Resource. A lysogen of the 1985 bp ABIR cDNA in λgtll waε conεtructed in Y1089 by infecting Y1089 bacteria with the recombinant phage at a moi of 10,000:1, and identifying temperature sensitive clones from the unlysed bacteria. The lysogen was induced with heat and IPTG, and the resultant cell lysate reεuspended in Lae mil gel sample buffer. A portion of the εample waε electrophoresed on 10% PAGE, transferred to nitrocellulose, and incubated in succesεively more dilute concentrationε of urea in PBS-Triton-X at 4°C over two dayε after the method of Kageyama and Pastan (1989, Cell 59:815). Binding to 35 S-/3APP-C100 was then carried out according to the protocol used for isolating the ABIR clone from the cDNA libraries, with the change that PBS instead of TBS waε uεed. The cDNA inεert from λABlR waε εubcloned into the mammalian expreεεion vector pCDNAl in the antiεenεe orientation, and PC12 cellε were tranεfected by electroporation. Theεe tranεient cellε were grown in DME media for 3 d, treated with NGF and teεted for their ability to bind 35 S-labeled / SAPP-CIOO. Stable tranεfectant cellε were εelected for by the incluεion of G418 in the media and neomycin reεistant cells were subεequently teεted in the binding aεεay for their ability to bind the labeled / SAPP-CIOO after treatment with NGF. 6.1.7. IN SITU HYBRIDIZATION USING θAPP-ClOO BINDING PROTEIN PROBE Hybridization probes were prepared by label¬ ing the pABlR-rat construct linearized at variouε restriction sites. Anti-εense RNA probes were made from the plasmid template linearized at the Ncol site utilizing the T3 promoter of Bluescript, and sense- control RNA probes were made from the plasmid linearized at the BamHI site utilizing the T7 promoter of Bluescript. Probes were made in various ways. In vitro tranεcription waε uεed to produce RNA probeε. RNA waε labeled with either 35 S-UTP (Promega reagents) or Digoxigenin-UTP (Boehringer-Mannheim reagents) . Digoxigenin-labeled DNA probes were produced by PCR (Lanzillo, 1991; M. McKenna and J. Carlεon, perεonal communication) ; PCR primerε were made to produce the appropriate fragmentε (Genoεys Biotechnologies, Inc.). Sections for in situ hybridization were prepared in the same manner as for binding autoradiography, only uεing RNAεe free conditionε. Prior to hybrid¬ ization, the sections were given two 3m washes with phosphate buffer (0.1 M) containing glycine (0.75 g/ml) followed by two 15 m washeε with phoεphate buffer alone. They were then treated with proteinaεe K (1 μg/ml in 15 mM Tris-HCl, pH 7.5 and 15 mM EDTA) for 30 m at 37°C and washed with acetic acid anhydride (0.25% in 0.1 M triethanolamine, pH 7.5) for 10 m. This was followed by two 15 m waεheε with SSC (0.15 M NaCl, 0.015 M εodiu citrate, pH 7.0) at twice itε normal concentration (2X) , defatting with EtOH and chloroform, and air drying. The hybridization probe, prepared aε deεcribed above, waε then placed onto the sections in a solution containing 50% formamide, 2OX Denhardt's reagent, 300 μg/ml single εtranded DNA, 150 μg/ml tRNA, 20 mM /3-mercaptoethanol (BME) , and 2X SSC. Coverεlipε were placed over the εectionε and hybrid¬ ization solution and sealed to the slides with rubber cement. The hybridization was carried out overnight at 60°C. The coverslipε were then removed and the εectionε were washed twice for 30 m at 60°C with 4X SSC supplemented with 300 mM BME. Following this, they were treated with RNAse (20μg/ml in 2X SSC, 20 m BME) for 30 m at 45°C. The sections were then washed four times with 2X SSC (60 m, 30 m, 30 m, and 30 m at 60°C) , washed one time with IX SSC (30 m, 60°C) , and left overnight in 2X SSC. The following day the εectionε were quickly rinsed in 0.05 M phosphate buffer and then diεtilled water. They were then air dried and placed in contact with X-ray film. The filmε were developed Id to 2 weekε later and uεed to eεtabliεh groεε diεtribution of hybridized probe. Finer localization of the probe waε achieved by coating the εectionε with a photoemulεion then developing the emulεion and measuring the grain densitieε over cell bodies in various brain regions. 6.1.8. CLONING OF THE FULL LENGTH RAT APP-4 In order to obtain the full-length C100-R the rat libraries are being screened with various probes made from pABlR-rat. These probes are specific for the most 5' sequence of the rat clone. After more rat sequence is available, subεequent roundε of εcreening will utilize probeε εpecific to the moεt 5' εequence available at each round of εcreening. In addition, a εecond rat-brain cDNA library was constructed in λZAP (Stratagene) with a primer εpecific for the 5* region of the cDNA inεert. The library was constructed using the RACE (rapid amplification of cDNA ends) protocol (Frohmann et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998-9002) with the following modifications: (1) The cDNA syntheεiε waε primed with AB1RT3-1 or with AB1RT3-3, specific for the 3' UTR of the ABIR mRNA, instead of with oligo(dT) . (2) The mRNA waε denatured with methyl- mercury hydroxide prior to cDNA synthesiε. (3) MMLV reverεe transcriptase ("Superεcript, " Bethesda Research Labs) was used inεtead of AMV reverse trans- criptaεe. (4) Notl adapters instead of EcoRI linkers were attached to the ends of the cDNAs so that no subsequent digestion of the cDNA waε necessary before cloning. The cDNAs were cloned into Notl digested, and partially filled in λ ZAP II, to make two libraries of approximately 10 5 cDNA cloneε each.Thiε library will be screened with probes specific for the most 5' sequence available at each round of cloning. Another approach to cloning the full-length gene will be to utilize PCR technology. Using hippocampal meεsenger RNA we will use a primer specific for the 5* end of the cDNA insert and perform the reverse transcriptase reaction. The RNA/DNA hybrid will then be "tailed" with guanines by terminal transferase, the RNA digested with RNAεe H and the second strand primed with a poly-C primer. By using primers with restric¬ tion siteε incorporated into them, theεe "clone- specific" cDNAs will be cloned and εequenced. The proceεε will be repeated through multiple roundε until the entire 11 kb sequence has been attained (Frohmann et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998- 9002) . 6.2. RESULTS 6.2.1. RADIOLIGAND BINDING Because 3APP-C100 is toxic to NGF-differentiated _ PC12 cells, we tested the binding of the in vitro synthesized /3APP-C100 fragment to the surface of PC12 cells that had been treated with NGF (Kozlowski et al. 1992 J. Neuroscience 12:1679). 35 S-/8APP-C100 binding to the differentiated PC12 cells was inhibitable by 0 unlabeled / SAPP-CIOO (FIG. IA) . Similar resultε were obtained with the neuroblaεtoma cell line, SK-N-MC (see FIG. IB) . In the NGF-treated PC-12 cells, the inhibitable fraction of the binding accounted for 40%-60% of the total binding. The IC 50 value for the inhibitable binding was 1.7 + 0.7 nM (n=5) . Inhibitable binding reached a maximum after 3 hours of incubation and was completely dissociable (FIG. 2) . Saturation experiments indicated the presence of a single class of binding sites with a K d value of 0.81 + 0.37 nM, in approximate agreement with the IC 50 value determined above, and a B ^ value of 0.37 + 0.05 fmole/10 6 cells. Binding of 35 S-/3APP-C100 to NGF-treated PC12 cells was not significantly inhibited by other peptides, including a number of tachykinins. In rat brain εectionε, the higheεt levelε of εpecific binding were found in regionε of the hippocampuε and olfactory tubercle. The amount of inhibitable 35 S- / 8APP-C100 binding waε dependent upon the duration of expoεure of the PC12 cellε to NGF. PC12 cellε that were not exposed to NGF showed little inhibitable binding, whereas those cultured in the presence of NGF for 4 or 6 days showed progreεεively greater amounts of binding. Another variable affecting binding was pH. The maximum amount of binding occurred at pH 7. Binding waε only 25% of maximum at pH 6 and only 52% of maximum at pH 8.5. To aεεess the stability of 35 S-/3APP-C100 in the aεsay conditions, samples of both free ligand at the end of the incubation period (unbound) and ligand which had bound to the cells and been released were examined by SDS-polyacrylamide gel electrophoresis (PAGE) . Before addition to the assay, most of the radioactivity was contained in a single band with an apparent molecular weight of 15 kDa. This represents monomer / SAPP-CIOO. A small amount of material waε alεo present in two additional diffuse bands. One migrated at approximately twice the molecular weight of the major band and possibly represents a dimer of / SAPP-CIOO. The other band had a much higher molecular weight and may be an aggregate. The gel profile of the unbound ligand at the end of the incubation period was identical to that of the ligand before incubation, indicating that the ligand had not been degraded. Similarly, the ligand released from binding had the same profile aε the ligand originally added, with the poεεible exception of a εlight reduction in the amount of high molecular weight material. These data show that the binding of /3APP-C100 ligand does not cause itε modification. A mutation of the tyrosine in /3APP-C100, made to examine the importance of a consenεuε εequence for phoεphotyroεine (Tyr 687 to Phe) , produced a peptide that waε not toxic to differentiated PC-12 cellε. Phe 6g7 - / SAPP-C100 doeε not εignificantly inhibit 35 S- / SAPP-C100 binding at concentrationε up to 14nM (n=3) , making it at leaεt 20 timeε leεε potent a ligand at the /3APP- C100 binding εite than the native form. 6.2.2. CLONING AND CHARACTERIZATION OF THE C100-R The autoradiographic signals from the 35 S- / 8APP- C100 binding to λgtll plaques induced to express protein in the late lytic cycle were atypical and variable. We used the XOMAT-RP high resolution film and long exposures in order to establish clear duplicates on the autoradiographs (FIG. 3) . The size of the cDNA insert of the rat clone, λABlR, was 1970 bp. The complete sequence is given in Figure 4 (SEQ. ID. NOS: 1-2) . The sequence given includes a portion of the / 8-gal gene derived from the vectors, λgtll and Bluescript. The clone's open reading frame is not in register with the /8-Gal gene, so the clone is being expreεεed as a nonfusion protein. The ATG at position 175 haε an adjacent sequence eight base pairs away that could serve as an E. coli ribosomal binding site, - AGAAA (the canonical site is AGGA) . Translation of the open reading frame initiating at this Methionine would yield a protein of 407 amino acids with a molecular weight of 45,253D. Such expreεεion would probably be a product of re¬ initiation and haε been previouεly noted for expreεεion of a 100-kD nonfusion protein (Yang CH. Lambie, E.J., and Snyder, M. J. of Cell Biology 116: 1303-1317) . Sequence analysiε haε revealed that the clone containε 1349 baεe pairε of the carboxyl- terminal εection of the gene (1395 minus /3-Gal sequence) and 575 base pairs of 3 ' untranslated region. Nucleic acid sequence analysis shows homology with "B" type calcium channels, the Ryanodine calcium channel, and a high affinity calcium binding protein, calreticulin (Figure 5) . Analysiε of the tranεlated protein εequence revealε several potential phosphorylation siteε (variouε kinaεe target εiteε. including cAMP- and cGMP-dependent protein kinases, protein kinaεe C, caεein kinase II) , potential N- glycoεylation εiteε, potential N-myriεtoylation sites, tyrosine sulfatation sites, and a serine/threonine protein kinase specific signature. This ser/thr kinase "signature" or conserved catalytic core sequence is found in the ABIR clone from 246-258 in the tranεlated sequence (FIG. 4) . The signature fits the -[LIVMFYC]-X- [HY]-D[LIVMFY]-K-X(2)-N-[LIVMFC] consensus pattern found by Hunter and his colleagues (Hanks, S.K., Quinn, A.M., and Hunter, T. (1988) Science 241 42-52) . In the rat ABIR clone the sequence is V-I-H-R-D-I-K-S-D-N-I-L-L. (FIG. 4) . A number of additional rat cDNA clones have been isolated and sequenced (Fig. 9) . The new sequence information extends, in the 5' direction, the coding region of C100-R represented in Figure 4. The * above nucleotide 686 in Figure 9 representε the junction with the rat sequence depicted in Figure 4 at nucleotide 40. Northern analyεiε demonεtrates that the messenger RNA corresponding to this cDNA is highly expresεed throughout the brain. The cDNA hybridizes to a prominent band at 11 kb and more weakly to bands at about 3kb. There is an NGF-inducible hybridizing band in PC12 cells at 9.5 kb. There iε alεo a 4.4 kb hybridizing band in PC12 cellε that appears with prolonged culturing, aε well aε with NGF treatment. Theεe more weakly hybridizing bands of lower molecular weightε may represent crosε-hybridization to messenger RNAs coding for moleculeε like calreticulin which demonεtrate homology at the nucleic acid level. Norther analysis of RNA from Alzheimer's Disease brains indicate that the /3APP-C100 binding protein is not expressed at a higher or lower level than in controls. Southern analysiε of human and rat genomic DNA demonεtrateε that ABIR cDNA hybridizes to a small number of bands, which is consistent with the ABIR representing a single copy gene (Figure 6) . A strongly hybridizing 3.0 kb band was observed as well as two more weakly hybridizing bands, one at 8.5 kb and a high molecular weight band between 25 and 30 kb. depending on species. 6-2.3. IN SITU HYBRIDIZATION In rat brain section, levels of hybridization greater than background were obviouε only in the hippocampuε and olfactory tubercle (FIG. 8) . Thiε agreeε with the diεtribution of binding εiteε aε determined autoradioagraphically (FIG. 7) . 7. EXAMPLE: CLONING OF HUMAN C100-R An adult human hippocampal cDNA library in lambda ZAP 11 (Stratagene) waε screened using a labeled fragment from the rat C100-R cDNA insert. The labeled fragment is represented as the EcoRI fragment, from nucleotide 31 through 409, in Figure 9. The rat C100-R EcoRI fragment waε labeled using a random priming reaction and the library was screened under stringent conditions using methods routinely employed by one skilled in the art. For a review of screening strategies see e.g. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Preεε, N.Y. Several cloneε were obtained, ranging from 600 to 1500 baεe pairε in length. Reεultε from partial εequencing of the 1500 baεe pair clone iε εhown in Figure 11. Regionε of εequence homology between the human C100-R sequence and the rat C100-R sequence are depicted in Figure 12. 8. DEPOSIT OF MICROORGANISMS The following microorganisms have been deposited with the American Type Culture Collection, (ATCC) , Rockville, Maryland and have been asεigned the following accession numbers: Microorganism Date of Deposit Accession No. pABIR-rat August 7, 1992 69047 The present invention iε not to be limited in scope by the microorganismε deposited since the deposited embodiments are intended as illustrationε of single aspectε of the invention and any microorganiεmε which are functionally equivalent are within the scope of the invention. The present invention is not to be limited in εcope by the exemplified embodimentε which are intended aε illustrations of single aspectε of the invention, and any cloneε, DNA or amino acid sequences which are functionally equivalent are within the scope of the invention. Indeed, various modificationε of the invention in addition to thoεe skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It is also to be understood that all base pair sizes given for nucleotides are approximate and are uεed for purposes of description. International Application No: PCT/ MICROORGANISMS Optional Sheet in connection with the microorganism referred to on page _V__, lines -16 of the description ' A. IDENTIFICATION OF DEPOSIT ' Further deposits are identified on an additional sheet ' Name of depositary institution ' American Type Culture Collection Address of depositary institution (including postal code and country) ' 12301 Parklawn Drive Rockville, MD 10582 US Date of deposit ' August 7. 1992 Accession Number * 69047 B. ADDITIONAL INDICATIONS ' (leave blank if not applicable). This information is continued on a separate attached aheet C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ■ «__, D. SEPARATE FURNISHING OF INDICATIONS * O a∞ Han if not applk-_bie) The indications listed below will be submitted to the International Bureau later' (Specify the general nature of the indications e.g., "Accession Number of Deposit") E. D This sheet was received with the International application when filed (to be checked by the receiving Office) ELNORA Y. RIVERA INTERNATIONAL DIVISION (Authorized Officer) D The date of receipt (from the applicant) by the International Bureau " (Authorized Officer) Form PCT/RO/134 (January 1981 ) SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Manly, S. et al. (ii) TITLE OF INVENTION: Cloning And Expression Of BAPP-CIOO Receptor (ClOO-R) (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Pennie £ Edmonds (B) STREET: 1155 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: U.S.A. (F) ZIP: 10036-2711 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy,disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNE /AGENT INFORMATION: (A) NAME: Misroc , S. Leslie (B) REGISTRATION NUMBER: 18,072 (C) REFERENCE/DOCKET NUMBER: 6013-09-1 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212 790-9090 (B) TELEFAX: 212 869-9711 (C) TELEX: 66141 PENNIE (2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1971 base pairs (B) TYPE: nucleic acid (C) STRΛNDEI.NESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1395 (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: CCC CCT CGA GGT CGΛ CTC CTG GΛG CCC GTC ΛGT ΛTC GGC GGΛ ΛTT CCT -18 Pro Pro Λrg Gly Λrg Leu Leu Glu Pro Val Ser lie Gly Gly lie Pro 1 5 10 15 GAA CAΛ TGG GCT CGA CTG CTC CΛΛ ΛCC TCC ΛΛC ΛTT ΛCΛ ΛΛΛ CTG GΛΛ 96 Glu Gin Trp Λla Arg Leu Leu Gin Thr Ser Λsn lie T r Lys Leu Glu 20 25 30 CΛG ΛΛG ΛΛG ΛΛC CCΛ CΛG GCT GTT CTG GAT GTT CTC GΛG TTT TΛC GΛC 14<1 Gin Lys Lys Λsn Pro Gin Λla Val Leu Asp Val Leu Glu Pho Tyr Λsp 35 40 45 TCC AAA GAA ACA GTC AAC AAC CAG AAA TAC ATG AGC TTT ACA TCA GGΛ 192 Ser LyB Glu Thr Val ABΠ Asn Gin Lye Tyr Met Ser Phe Thr Ser Gly 50 55 60 GAT AAA AGT GCC CAT GGA TAT ATA GCA GCA CAT CAG TCG AAT ACC AAA 240 Aβp Lys Ser Ala His Gly Tyr lie Ala Ala His Gin Ser Asn Thr Lys 65 70 75 80 ACA GCT TCA GAA CCT CCT TTG GCT CCT CCT GTA TCT GAA GAA GAG GAT 288 Thr Ala Ser Glu Pro Pro Leu Ala Pro Pro Val Ser Glu Glu Glu Asp 85 90 95 GAA GAA GAG GAA GAG GAA GAA GAT GAT AAT GAG CCC CCG CCT GTC ATT 336 Glu Glu Glu Glu Glu Glu Glu Asp Asp Asn Glu Pro Pro Pro Val lie 100 105 110 GCA CCA AGA CCA GΛG CAT ACA AAA TCA ΛTC TAT ACT CGT TCT GTG GTT 384 Ala Pro Arg Pro Glu His Thr Lys Ser lie Tyr Thr Arg Ser Val Val 115 120 125 GAG TCA ATT GCT TCA CCA GCA GCA CCA AAT AAA GAA GCC ACC CCA CCT 432 Glu Ser lie Λla Ser Pro Ala Ala Pro Asn Lys Glu Ala Thr Pro Pro 130 135 140 TCT GCT GAG AAT GCC AAT TCC AGT ACT TTG TAC AGG AAT ACA GAT CGG 480 Ser Ala Glu Asn Ala Asn Ser Ser Thr Leu Tyr Arg Asn Thr Asp Arg 145 150 155 160 CAA AGA AAA AAA TCC AAG ATG ACA GAT GAG GAG ATC CTA GAG AAG CTA 528 Gin Arg Lys Lys Ser Lys Met Thr Asp Glu Glu lie Leu Glu Lys Leu 165 170 175 AGA AGC ΛTT GTG ΛGT GTT GGG GAC CCA ΛΛG ΛΛG AAA TAT ACA AGΛ TTT 576 Arg Ser lie Val Ser Val Gly Asp Pro Lys LyB Lys Tyr Thr Arg Phe 180 185 190 GAA AAA ATT GGC CAA GGG GCA TCA GGA ACT GTT TAC ACΛ GCA CTA GAC 624 Glu Lys lie Gly Gin Gly Ala Ser Gly Thr Val Tyr Thr Ala Leu Asp 195 200 205 ATT GCG ACA GGA CAA GAG GTG GCT ATA AAG CAA ATG AAC CTT CAA CAG 672 lie Ala Thr Gly Gin Glu Val Ala He Lys Gin Met ABΠ Leu Gin Gin 210 215 220 CΛG CCC AAA AAG GΛΛ TTΛ ATT ΛTT AAT GΛΛ ΛTT CTT GTC ΛTG AGG GAA 720 Gin Pro LyB Lys Glu Leu He He Asn Glu He Leu Val Met Λrg Glu 225 230 235 240 AAT AAG AAC CCC AAT ATT GTC AAT TAT TTA GAT AGC TAC TTA GTG GGT 768 Asn Lys Asn Pro Asn He Val Asn Tyr Leu Asp Ser Tyr Leu Val Gly 245 250 255 GAT GAA CTG TGG GTA GTC ATG GAA TAC TTG GCT GGT GGC TCT TTG ACT 816 Asp Glu Leu Trp Val Val Met Glu Tyr Leu Ala Gly Gly Ser Leu Thr 260 265 270 GAC GTG GTC ACA GAA ΛCC TGT ATG GAT GAA GGA CAG ATA GCA GCC GTC 864 Asp Val Val Thr Glu Thr Cys Met Asp Glu Gly Gin He Λla Ala Val 275 280 285 TGT AGA GAG TGC CTC CAA GCT TTG GAT TTC TTG CAC TCA AAA CAA GTG 912 Cys Arg Glu Cys Leu Gin Ala Leu Asp Phe Leu His Ser Lys Gin Val 290 295 300 ATC CAC AGA GAT ATA AAG AGT GAC AAT ATT CTC CTC GGG ATG GAT GGT 960 He His Arg Asp He Lys Ser Asp Asn He Leu Leu Gly Met Asp Gly 305 310 315 320 TCT GTT AAA CTG ACT GAT TTT GGA TTC TGT GCC CAA ATC ACT CCT GΛG 1008 Ser Val Lys Leu Thr Asp Phe Gly Phe CyB Ala Gin He Thr Pro Glu 325 330 335 CAA AGT AAA CGA AGC ACT ATG GTG GGA ACT CCC TAT TGG ATG GCA CCT 1056 Gin Ser Lys Arg Ser Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro 340 345 350 GAG GTG GTA ACT CGΛ AAA GCT TAT GGC CCG AAA GTT GAT ATC TGG TCT 1104 Glu Val Val Thr Λrg LyB Ala Tyr Gly Pro Lys Val Asp He Trp Ser 355 360 365 CTG GGA ATC ATG GCC ATT GAA ATG GTG GAA GGT GAA CCC CCT TAC CTT 1152 Leu Gly He Met Ala He Glu Met Val Glu Gly Glu Pro Pro Tyr Leu 370 375 380 AAT GAA AAT CCA CTC AGG GCC TTA TAT CTG ATA GCC ACT AAT GGΛ ACC 1200 Asn Glu Asn Pro Leu Arg Ala Leu Tyr Leu He Ala Thr Asn Gly Thr 385 390 395 400 CCA GAG CTC CAG AAT CCC GAG AGA CTG TCA GCT GTA TTC CGT GΛC TTC 1248 Pro Glu Leu Gin Λsn Pro Glu Arg Leu Ser Ala Val Phe Λrg Asp Phe 405 410 415 TTA AAT CGC TGT CTT GAG ATG GAT GTG GAT AGA CGA GGG TCT GCC AAG 1296 Leu Asn Arg Cys Leu Glu Met Asp Val Asp Arg Arg Gly Ser Ala Lys 420 425 430 GAG CTT TTG CAG CAT CCA TTT TTA AAA TTA GCC AAG CCC CTG TCC AGC 1344 Glu Leu Leu Gin His Pro Phe Leu Lys Leu Ala Lys Pro Leu Ser Ser 435 440 445 CTC ACT CCT CTG ATT CTT GCT GCA AAG GAA GCC ATT AAG AAC AGT AGC ]392 Leu Thr Pro Leu He Leu Ala Ala Lys Glu Ala He Lys Asn Ser Ser 450 455 460 CGT TAGAΛGTGCΛ ΛGCCTTΛCCC CTCΛCCGTCT CCCGGΛTGAG TΛΛGACTGAΛ 1445 Λrg 465 CTAAAACTCT GCTGCAGGAT CCACAGAAGA AAAGACAGTC AAATGGAGTG GGGGTTCTTT 1505 AACTTTCAAG TGAATAGAAA CTTCTTATAA ACCTTTTTCC TACTCCCTCA GATTΛTGTΛA 1565 TTTATTTGTA AGCCTGAACC GCAGCCCACA CAGGGCAGCA ATGTCGAAGT AGCCATTΛAG 1625 TGGCCACTTC CACCGTGAAG CGAGAGAGCC AGTAGTGAAT CCCCTCATTC GTGCATTTAC 1685 TTTGAAGAAA AAGAGATTTC TCAΛAGATGC ΛCACTCCCTC TTCATAGTGC TGTGTGTTTT 1745 TΛΛGTTAGAG AGTAGTCCCC CTTCCΛTTCΛ AΛCCTCTTTC AAAATCCCTT ΛCCCΛΛCGTG 1805 ATGTTTTTTC ACTTGCATTG TCATTAGATG TCCAGAAAAA AAGATGTCAA AATGTTTTTT 1865 TTAAAAAAAA GAAAGCAAAΛ ΛAGCAAAGAA AAAAGGAATT CCAGCTGΛGC GCCGGTCGCT 1925 ACCΛTTΛCCA GTTGGTCTGG TGTCAAGCGG CCGCCΛCCGC GGTGGA 1971 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 465 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2: Pro Pro Arg Gly Arg Leu Leu Glu Pro Val Ser He Gly Gly He Pro 1 5 10 15 Glu Gin Trp Ala Arg Leu Leu Gin Thr Ser Asn He Thr Lys Leu Glu 20 25 30 Gin Lys LyB Asn Pro Gin Ala Val Leu Asp Val Leu Glu Phe Tyr Asp 35 40 45 Ser LyB Glu Thr Val Asn ABΠ Gin Lys Tyr Met Ser Phe Thr Ser Gly 50 55 60 Asp LyB Ser Ala His Gly Tyr He Ala Ala His Gin Ser Asn Thr Lys 65 70 75 80 Thr Ala Ser Glu Pro Pro Leu Ala Pro Pro Val Ser Glu Glu Glu Asp 85 90 95 Glu Glu Glu Glu Glu Glu Glu Asp Asp Asn Glu Pro Pro Pro Val He 100 105 110 Ala Pro Arg Pro Glu His Thr Lys Ser He Tyr Thr Arg Ser Val Val 115 120 125 Glu Ser He Ala Ser Pro Ala Ala Pro Λsn Lye Glu Ala Thr Pro Pro 130 135 140 Ser Ala Glu Asn Ala Asn Ser Ser Thr Leu Tyr Arg Asn Thr Asp Arg 145 150 155 160 Gin Arg Lys Lys Ser Lys Met Thr Asp Glu Glu He Leu Glu Lys Leu 165 170 175 Arg Ser He Val Ser Val Gly Asp Pro Lys Lys Lys Tyr Thr Arg Phe 180 185 190 Glu Lys He Gly Gin Gly Ala Ser Gly Thr Val Tyr Thr Ala Leu Asp 195 200 205 He Ala Thr Gly Gin Glu Val Ala He Lys Gin Met ΛBΠ Leu Gin Gin 210 215 220 Gin Pro Lys Lys Glu Leu He He Asn Glu He Leu Val Met Λrg Glu 225 230 235 240 Asn Lys Asn Pro Asn He Val Asn Tyr Leu Asp Ser Tyr Leu Val Gly 245 250 255 Asp Glu Leu Trp Val Val Met Glu Tyr Leu Ala Gly Gly Ser Leu Thr 260 265 270 Asp Val Val Thr Glu Thr Cys Met Asp Glu Gly Gin He Λla Ala Val 275 280 285 Cys Arg Glu Cys Leu Gin Ala Leu Asp Phe Leu HiB Ser Lys Gin Val 290 295 300 He His Arg Asp He Lys Ser Asp Asn He Leu Leu Gly Met Asp Gly 305 310 315 320 Ser Val Lys Leu Thr Asp Phe Gly Phe Cys Ala Gin He Thr Pro Glu 325 330 335 Gin Ser Lys Arg Ser Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro 340 345 350 Glu Val Val Thr Arg Lys Λla Tyr Gly Pro Lys Val Asp He Trp Ser 355 360 365 Leu Gly He Met Λla He Glu Met Val Glu Gly Glu Pro Pro Tyr Leu 370 375 380 ABΠ Glu Asn Pro Leu Arg Λla Leu Tyr Leu He Ala Thr Asn Gly Thr 385 390 395 400 Pro Glu Leu Gin Asn Pro Glu Arg Leu Ser Ala Val Phe Arg Asp Phe 405 410 415 Leu Asn Arg Cys Leu Glu Met ABp Val Asp Arg Arg Gly Ser Ala Lys 420 425 430 Glu Leu Leu Gin His Pro Phe Leu Lye Leu Ala Lye Pro Leu Ser Ser 435 440 445 Leu Thr Pro Leu He Leu Ala Ala Lys Glu Ala He Lys ABΠ Ser Ser 450 455 460 Arg 465

Claims: WHAT IS CLAIMED IS; 1. A cDNA encoding an C100-R. 5 2. A recombinant DNA vector containing a nucleotide sequence that encodes an C100-R. 3. A recombinant DNA vector containing a 10 nucleotide sequence that encodes a C100-R fusion protein. 4. A recombinant DNA vector of Claim 2 in which the C100-R nucleotide sequence is operatively 15 associated with a regulatory sequence that controls gene expression in a host. 5. A recombinant DNA vector of Claim 3 in which the C100-R fusion protein nucleotide sequence iε _ operatively associated with a regulatory sequence that controls gene expression in a host. 6. The DNA of Claim 1, 2, 3, 4 or 5 in which the C100-R εequence encodeε the rat C100-R. 25 7. The DNA of Claim 1, 2, 3, 4 or 5 in which the C100-R εequence encodeε the human C100-R. 8. The DNA of Claim 1, 2, 3, 4 or 5 which iε 0 capable of hybridizing under stringent conditions, or which would be capable of hybridizing under stringent conditions but for the degeneracy of the genetic code to the C100-R DNA sequence of FIG. 4 (SEQ. ID. NO.: 1). 5 9. An engineered hoεt cell that containε the recombinant DNA vector of Claim 1, 2, 3, 4 or 5. 10. An engineered cell line that containε the recombinant DNA expreεεion vector of Claim 4 and expreεεes C100-R. 11. The engineered cell line of Claim 10 which expresses the C100-R on the surface of the cell. 12. The engineered cell line of Claim 10 or 11 which expresses human C100-R. 13. An engineered cell line that contains the recombinant DNA expresεion vector of Claim 5 and expreεεeε the C100-R fuεion protein. 14. The engineered cell line of Claim 13 that expreεεeε the C100-R fuεion protein on the surface of the cell. 15. The engineered cell line of Claim 13 or 14 which expresses a human C100-R fusion protein. 16. A method for producing recombinant C100-R, compriεing: (a) culturing a hoεt cell transformed with the recombinant DNA expression vector of Claim 4 and which expresεeε the C100-R; and (b) recovering the C100-R gene product from the cell culture. 17. The method of Claim 16 in which the human C100-R iε produced. 18. A method for producing recombinant ClOO-R fusion protein, comprising: (a) culturing a host cell transformed with the recombinant DNA expreεεion vector of Claim 5 and which expresseε the C100-R fusion protein; and (b) recovering the C100-R fusion protein from the cell culture. 19. The method of Claim 18 in which a human CIOO-R fusion protein iε produced. 20. An isolated recombinant C100-R. 21. The isolated C100-R of Claim 20 having a rat C100-R amino acid εequence. 22. The isolated C100-R of Claim 20 having a human C100-R amino acid sequence. 23. A fusion protein comprising a CIOO-R linked to a heterologous protein or peptide sequence. 24. The fusion protein of Claim 23 in which the C100-R moiety has a rat C100-R amino acid sequence. 25. The fusion protein of Claim 23 in which the C100-R moiety has a human C100-R amino acid sequence. 26. An oligonucleotide which encodes an antisense sequence complementary to a portion of the C100-R nucleotide sequence, and which inhibits translation of the C100-R gene in a cell. 27. The oligonucleotide of Claim 26 which is complementary to a nucleotide sequence encoding the amino terminal region of the ClOO-R. 28. A monoclonal antibody which immunospecifically bindε to an epitope of the C100-R. 29. The monoclonal antibody of Claim 28 which competitively inhibitε the binding of / SAPP-CIOO to the C100-R. 30. The monoclonal antibody of Claim 28 or 29 which bindε to the human C100-R. 31. A method for screening and identifying antagonists of 0APP-C1OO ligand, comprising: (a) contacting a cell line that expresses C100-R with a test compound in the presence of the |3APP-C100 ligand; and (b) determining whether the test compound inhibitε the binding and cytotoxic effects of the /3APP-C100 ligand on the cell line, in which antagonistε are identified as those compounds that inhibit both the binding and cytotoxic effects of the /3APP-C100 ligand on the cell line. 32. A method for screening and identifying agonists of the /8APP-C100 ligand, comprising: (a) contacting a cell line that expresεes the C100-R with a test compound in the presence and in the absence of the βAPP-ClOO ligand; (b) determining whether, in the presence of the /8APP-C100 ligand, the test compound inhibits the binding of the / 8APP-C100 ligand to the cell line; and (c) determining whether, in the absence of the /SAPP-CIOO ligand, the test compound mimics the cytotoxic effects of the 3APP-C100 ligand on the cell line, in which agonists are identified as those test compounds that inhibit the binding but mimic the cytotoxic effects of the / SAPP-CIOO ligand on the cell line. 33. The method according to Claim 31 or 32 in which the cell line is a genetically engineered cell line. 34. The method according to Claim 31 or 32 in which the cell line endogenously expreεεeε the C100-R. 35. The method according to Claim 34 in which the cell line is a neuroblastoma cell line. 36. The method according to Claim 35 in which the neuroblastoma cell line is SK-N-MC.

Inventors: KOZLOWSKI MICHAEL R, MANLY SUSAN P, NEVE RACHAEL L, KOZLOWSKI, MICHAEL, R., MANLY, SUSAN, P., NEVE, RACHAEL, L.
Applicants: MCLEAN HOSPITAL CORP, SQUIBB BRISTOL MYERS CO, BRISTOL-MYERS SQUIBB COMPANY, MCLEAN HOSPITAL CORPORATION
Application year: 1993
Main classification: C12Q1
IPC codes: C07K14, C12P21, G01N33