Abstract:
The present invention provides a human glutamate receptor and related DNA compounds useful not only in assays for potential pharmaceuticals but also in methods for molecular biology techniques.

Description:
This application is a division, of application Ser. No. 07/879,688 filed May 1, 1992 now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     In the mammalian central nervous system, L-glutamate serves as a major excitatory neurotransmitter. The interaction of glutamate with its membrane-bound receptors is believed to play a role in many important neuronal processes, including, for example, fast synaptic transmission, synaptic plasticity and long-term potentiation. These processes are fundamental to the maintenance of life and normal human abilities such as learning and memory. Monaghan D. T. et al., 8  Neuron  267 (1992). 
     Pharmacological characterization of receptors for L-glutamate has led to their classification into two families based on their biological function: the ionotropic receptors which are directly coupled to cation channels in the cell membrane, and the metabotropic receptors which function through coupling to G-proteins. A number of ionotropic receptors have been further characterized on the basis of the relatively specific agonists by which they can be activated. One major group comprises those receptors activated by N-methyl-D-aspartate (NMDA), which appears to have multiple allosteric modulatory sites. The other two groups consist of those receptors activated by kainate and/or amino-3-hydroxy-5-methyl-4-isoxozole propionate (AMPA). Collingridge G. L. et al., 40 Pharmacol. Rev. 143 (1989). 
     Molecular cloning studies of rodent ionotropic receptors have recently provided some information on the molecular structure of these proteins. The cDNAs for seven different subtypes of the kainate/AMPA group have been characterized. Heinemann S. et al., PCT publication, W091/06648 (1991), Keinanen K. et al., 249  Science  556 (1990), Sakimura K. et al., 272  FEBS Lett.  73 (1990), Werner P. et al., 341  Nature  742 (1991), Bettler B. et al., 8  Neuron  257 (1992). Splice variants, referred to as “flip” and “flop”, of some of these have been characterized as well. Sommer B. et al., 249  Science  1580 (1990). In addition, one member of the NMDA group has been cloned. Moriyoshi, K. et al., 354  Nature  31 (1991). An NNDA-related protein has also been reported. Kumar K. N. et al., 354  Nature  70 (1991). These proteins share varying degrees of homology with one another and are therefore believed to represent a gene superfamily. Based on analogy with other better characterized ion channel receptors, glutamate ionotropic receptors are expected to exist in vivo within the cell membrane as multisubunit assemblies of these subunits. Unwin N., 3  Neuron  665 (1989). 
     Moreover, at least two human glutamate receptors have been reported as cloned. The reported human receptors differ slightly from the present invention. Puckett C. et al., 88  Proc. Nat. Acad. Sci.  7557 (1991) and Sun W. et al., 89  Proc. Nat. Acad. Sci.  1443 (1992). The glutamate receptor cloned by Puckett et al. was named GluHI and was later identified to be the “flip” version of this particular receptor. The Sun W. et al. reference refers to the glutamate receptor they cloned as the HBGR1 receptor and explains that HBGR1 is presumed the “flop” version of GluHI. Sun et al. also discloses a partial clone of HBGR2, or GluH2. 
     In addition to its role in normal human physiology, interaction of L-glutamate with its receptors is believed to play a key role in many neurological disorders such as stroke, epilepsy and head trauma, as well as neurodegenerative processes such as Alzheimer&#39;s disease. Olney R. W., 17  Drug Dev. Resa.,  299 (1999). For this reason, understanding the molecular structure of human L-glutamate receptors will be important for understanding these disease processes as well as furthering the search for effective therapeutic agents. Up to the present, the search for therapeutic agents which will selectively bind and modulate the function of human glutamate receptors has been hampered by the unavailability of homogeneous sources of receptors to use for screens and tests of potential drug candidate compounds. The brain tissues commonly used by pharmacologists presently are derived from experimental animals (non-human) and furthermore contain mixtures of various types of glutamate receptors. 
     In searching for drugs for human therapy it is desirable to use receptors that are more analogous to those in the intact human brain than are the rodent receptors employed to date. The current invention provides a human receptor which can be used to search for drugs which modulate these receptors. 
     SUMMARY OF THE INVENTION 
     The present invention provides amino acid compounds which comprise the isolated amino acid sequence SEQ ID NO:1. In particular, the amino acid compound which is SEQ ID NO: 1 is preferred. 
     The invention also provides nucleic acid compounds which comprise an isolated nucleic acid sequence which encodes the amino acid compounds provided. Particularly, nucleic acid compounds which are DNA are preferred. Most preferred is the DNA compound SEQ ID NO:2. However, also preferred are those nucleic acid compounds which are sense mRNA. 
     Also provided by the present invention are recombinant nucleic acid vectors comprising the nucleic acids which encode SEQ ID NO:1. Preferred nucleic acid vectors are those which are DNA. Most preferred are recombinant DNA vectors which comprise SEQ ID NO:2. The recombinant DNA vector most preferred is plasmid pRS103. 
     Moreover, recombinant DNA vectors of the present invention preferably comprise a promoter positioned to drive expression of said isolated DNA sequence. A preferred recombinant DNA expression vector is one wherein the promoter functions in mammalian cells. A more preferred recombinant DNA expression vector is one wherein the promoter functions in COS-7 cells. Most preferred COS-7 cell DNA expression vectors further comprise SEQ ID NO:2. 
     Restriction fragments of the preferred vector are also provided. Particularly, the 4.2 kb (kilobase) EcoRI/Kpn1 and the 2.8 kb EcoRI/ClaI restriction fragment of pRS103 are provided. 
     The present invention also provides probes and primers useful for molecular biology techniques. Compounds which encode for SEQ ID NO:1 or a part thereof and which are at least 17 base pairs in length are provided. Preferably, the 17 base pair or more compound is DNA. Most preferred for this use are the DNA compounds which are SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. 
     Further, this invention provides cells in which the nucleic acid compounds of the invention may be harbored. Oocytes wherein nucleic acid compounds of the invention express functional HSGluR1 receptor are provided. Moreover, oocytes wherein nucleic acids of the present invention express functional HSGluR1 receptor and wherein functional GluR2 receptor is also expressed is provided. Oocytes wherein nucleic acids of the present invention express functional HSGluR1 receptor and wherein functional GluR2 receptor is co-expressed, and wherein functional GluR3 receptor is additionally expressed is also provided. An oocyte wherein DNA expresses functional HSGluRl receptor is preferred. Most preferred is an oocyte wherein sense mRNA expresses functional HSGluR1 receptor. 
     Other host cells provided by the present invention include those which are transfected with a nucleic acid compound which encodes SEQ ID NO:1. Preferred cells include host cells transfected with a recombinant DNA vector. Preferred transfected host cells which encodes SEQ ID NO:1 are  E. coli  cells. The most preferred  E. coli  host cell is  E. coli /pRS103. 
     Host cells which are transfected with a DNA vector which further comprise a promoter positioned to drive expression of functional HSGluR1 receptor are also provided. Preferably, the DNA vector comprises SEQ ID NO:2. Preferred host cells for expression of functional HSGluR1 are mammalian cells. Preferred mammalian cells for expression of functional HSGluR1 are COS-7 cells. Specifically, COS-7 cells which have been transfected with a DNA expression vector which expresses a functional HSGluR1 receptor and which further comprise a DNA vector which encodes a functional GluR2 receptor are provided. COS-7 cells which have been transfected with an DNA expression vector which expresses a functional HSGluR1 receptor, and which further comprise a DNA vector which encodes a functional GluR2 receptor, and which further comprise a DNA vector which encodes a functional GluR3 receptor are also provided. 
     Additionally, the invention provides a method for identifying DNA homologous to a probe of the present invention which comprises contacting test nucleic acid with the probe under hybridizing conditions and identifying DNA that is homologous to the probe. The preferred probes for use in this method are SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. 
     Assays utilizing the compounds provided by the present invention are also provided. The assays provided determine whether a substance evokes a glutaminergic response, said assays comprising introducing said substance and a functional compound of SEQ ID NO:1 into an acceptable medium, subsequently monitoring glutaminergic activity by physically detectable means, and thereby identifying those substances which effect a chosen response. Other assays further comprise a functional GluR2 receptor. A preferred assay further comprises both a functional GluR2 receptor and a functional GluR3 receptor. 
     Preferable, the physically detectable means are competition with radiolabeled glutamate, binding of glutaminergic ligand or generating a detectable ion flow. A preferred assay is the oocyte assay system. A most preferred oocyte assay system utilizes sense mRNA. 
     The invention also provides a method for constructing a recombinant host cell capable of expressing a nucleic acid compound which encodes a compound which comprises SEQ ID NO:1, said method comprising transfecting a host cell with a recombinant DNA vector which comprises said nucleic acid compound. A preferred method utilizes  E. coli  cells as the host cells. A more preferred method further comprises a DNA vector. A most preferred method further comprises a DNA vector which comprises SEQ ID NO:2. 
     Additionally, a method for expressing a nucleic acid sequence which encodes SEQ ID NO:1 in a recombinant host cell is provided. The method comprises transfecting host cells with nucleic acids of the present invention and culturing the transfected host cells under conditions suitable for gene expression. A preferred method utilizes COS-7 cells as the host cells. A more preferred method utilizes both COS-7 cells and a recombinant DNA vector comprising SEQ ID NO:2. 
     The following section provides a more detailed description of the present invention. For purposes of clarity and as an aid in understanding the invention, as disclosed and claimed herein, the following items are defined below. 
     All or part of SEQ ID NO:1—at least 6 consecutive amino acid residues or more of SEQ ID NO:1. 
     Functional compound of SEQ ID NO:1—A compound comprising SEQ ID NO:1 which is capable of generating ion flow, binding glutamate or binding glutaminergic ligand. 
     HSGluR1 receptor—the amino acid sequence SEQ ID NO:1. 
     GluR2 receptor—the amino acid sequence commonly associated with the rat ionotropic glutamate receptor 2. 
     GluR3 receptor—the amino acid sequence commonly associated with the rat ionotropic glutamate receptor 3. 
     SEQ ID NO:3—this segment is base 1 through 60 of SEQ ID NO:2, counting from the 5′ end: ATG CAG CAC ATT TTT GCC TTC TTC TGC ACC GGT TTC CTA GGC GCG GTA GTA GGT GCC AAT. 
     SEQ ID NO:4—this segment includes bases 130 through 189 of SEQ ID NO: 2, counting from the 5′ end: TTT GCT TTG TCG CAA CTC ACA GAG CCC CCG AAG CTG CTC CCC CAG ATT GAT ATT GTG AAC. 
     SEQ ID NO:5—this segment includes bases 2662 through 2718 of SEQ ID NO:2, with a TAA stop codon added at the 3′ end: CAA TCG ATT CCT TGC ATG AGC CAC AGT TCA GGG ATG CCC TTG GGA GCC ACG GGA TTG TAA. 
     Transfection—any transfer of nucleic acid into a host cell, with or without integration of said nucleic acid into genome of said host cell. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides an amino acid compound which comprises the isolated amino acid sequence SEQ ID NO:1. The preferred amino acid compound is SEQ ID NO:1, which is the following sequence of amino acids: 
     
       
         
               
               
             
           
               
                 Met Gln His Ile Phe Ala Phe Phe Cys Thr Gly Phe Leu Gly Ala Val 
                   
               
               
                   
               
               
                 Val Gly Ala Asn Phe Pro Asn Asn Ile Gln Ile Gly Gly Leu Phe Pro 
               
               
                   
               
               
                 Aen Gln Gln Ser Gln Glu His Ala Ala Phe Arg Phe Ala Leu Ser Gln 
               
               
                   
               
               
                 Leu Thr Glu Pro Pro Lye Leu Leu Pro Gln Ile Asp Ile Val Asn Ile 
               
               
                   
               
               
                 Ser Asp Ser Phe Glu Met Thr Tyr Arg Phe Cye Ser Gln Phe Ser Lys 
               
               
                   
               
               
                 Gly Val Tyr Ala Ile Phe Gly Phe Tyr Glu Arg Arg Thr Val Asn Met 
               
               
                   
               
               
                 Leu Thr Ser Phe Cys Gly Ala Leu His Val Cys Phe Ile Thr Pro Ser 
               
               
                   
               
               
                 Phe Pro Val Asp Thr Ser Asn Gln Phe Val Leu Gln Leu Arg Pro Glu 
               
               
                   
               
               
                 Leu Gln Asp Ala Leu Ile Ser Ile Ile Asp His Tyr Lye Trp Gln Lys 
               
               
                   
               
               
                 Phe Val Tyr Ile Tyr Asp Ala Asp Arg Gly Leu Ser Val Leu Gln Lys 
               
               
                   
               
               
                 Val Leu Asp Thr Ala Ala Glu Lys Asn Trp Gln Val Thr Ala Val Asn 
               
               
                   
               
               
                 Ile Leu Thr Thr Thr Glu Glu Gly Tyr Arg Met Leu Phe Gln Asp Leu 
               
               
                   
               
               
                 Glu Lys Lys Lys Glu Arg Leu Val Val Val Asp Cys Glu Ser Glu Arg 
               
               
                   
               
               
                 Leu Asn Ala Ile Leu Gly Gln Ile Ile Lys Leu Glu Lys Asn Gly Ile 
               
               
                   
               
               
                 Gly Tyr His Tyr Ile Leu Ala Asn Leu Gly Phe Met Asp Ile Asp Leu 
               
               
                   
               
               
                 Asn Lye Phe Lye Glu Ser Gly Ala Asn Val Thr Gly Phe Gln Leu Val 
               
               
                   
               
               
                 Asn Tyr Thr Asp Thr Ile Pro Ala Lys Ile Met Gln Gln Trp Lys Asn 
               
               
                   
               
               
                 Ser Asp Ala Arg Asp His Thr Arg Val Asp Trp Lys Arg Pro Lys Tyr 
               
               
                   
               
               
                 Thr Ser Ala Leu Thr Tyr Asp Gly Val Lys Val Met Ala Glu Ala Phe 
               
               
                   
               
               
                 Gln Ser Leu Arg Arg Gln Arg Ile Asp Ile Ser Arg Arg Gly Asn Ala 
               
               
                   
               
               
                 Gly Asp Cys Leu Ala Asn Pro Ala Val Pro Trp Gly Gln Gly Ile Asp 
               
               
                   
               
               
                 Ile Gln Arg Ala Leu Gln Gln Val Arg Phe Glu Gly Leu Thr Gly Asn 
               
               
                   
               
               
                 Val Gln Phe Asn Glu Lys Gly Arg Arg Thr Asn Tyr Thr Leu His Val 
               
               
                   
               
               
                 Ile Glu Met Lys His Asp Gly Ile Arg Lye Ile Gly Tyr Trp Asn Glu 
               
               
                   
               
               
                 Asp Asp Lys Phe Val Pro Ala Ala Thr Asp Ala Gln Ala Gly Gly Asp 
               
               
                   
               
               
                 Asn Ser Ser Val Gln Asn Arg Thr Tyr Ile Val Thr Thr Ile Leu Glu 
               
               
                   
               
               
                 Asp Pro Tyr Val Met Leu Lys Lys Asn Ala Asn Gln Phe Glu Gly Asn 
               
               
                   
               
               
                 Asp Arg Tyr Glu Gly Tyr Cys Val Glu Leu Ala Ala Glu Ile Ala Lys 
               
               
                   
               
               
                 His Val Gly Tyr Ser Tyr Arg Leu Glu Ile Val Ser Asp Gly Lys Tyr 
               
               
                   
               
               
                 Gly Ala Arg Asp Pro Asp Thr Lys Ala Trp Asn Gly Met Val Gly Glu 
               
               
                   
               
               
                 Leu Val Tyr Gly Arg Ala Asp Val Ala Val Ala Pro Leu Thr Ile Thr 
               
               
                   
               
               
                 Leu Val Arg Glu Glu Val Ile Asp Phe Ser Lys Pro Phe Met Ser Leu 
               
               
                   
               
               
                 Gly Ile Ser Ile Met Ile Lys Lys Pro Gln Lys Ser Lye Pro Gly Val 
               
               
                   
               
               
                 Phe Ser Phe Leu Aep Pro Leu Ala Tyr Glu Ile Trp Met Cye Ile Val 
               
               
                   
               
               
                 Phe Ala Tyr Ile Gly Val Ser Val Val Leu Phe Leu Val Ser Arg Phe 
               
               
                   
               
               
                 Ser Pro Tyr Glu Trp His Ser Glu Glu Phe Glu Glu Gly Arg Asp Gln 
               
               
                   
               
               
                 Thr Thr Ser Asp Gln Ser Asn Glu Phe Gly Ile Phe Asn Ser Leu Trp 
               
               
                   
               
               
                 Phe Ser Leu Gly Ala Phe Met Gln Gln Gly Cye Asp Ile Ser Pro Arg 
               
               
                   
               
               
                 Ser Leu Ser Gly Arg Ile Val Gly Gly Val Trp Trp Phe Phe Thr Leu 
               
               
                   
               
               
                 Ile Ile Ile Ser Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Thr Val 
               
               
                   
               
               
                 Glu Arg Met Val Ser Pro Ile Glu Ser Ala Glu Asp Leu Ala Lys Gln 
               
               
                   
               
               
                 Thr Glu Ile Ala Tyr Gly Thr Leu Glu Ala Gly Ser Thr Lye Glu Phe 
               
               
                   
               
               
                 Phe Arg Arg Ser Lye Ile Ala Val Phe Glu Lye Met Trp Thr Tyr Met 
               
               
                   
               
               
                 Lye Ser Ala Glu Pro Ser Val Phe Val Arg Thr Thr Glu Glu Gly Met 
               
               
                   
               
               
                 Ile Arg Val Arg Lys Ser Lys Gly Lys Tyr Ala Tyr Leu Leu Glu Ser 
               
               
                   
               
               
                 Thr Met Asn Glu Tyr Ile Glu Gln Arg Lys Pro Cys Asp Thr Met Lys 
               
               
                   
               
               
                 Val Gly Gly Asn Leu Asp Ser Lys Gly Tyr Gly Ile Ala Thr Pro Lys 
               
               
                   
               
               
                 Gly Ser Ala Leu Arg Asn Pro Val Asn Leu Ala Val Leu Lys Leu Asn 
               
               
                   
               
               
                 Glu Gln Gly Leu Leu Asp Lys Leu Lys Asn Lys Trp Typ Tyr Asp Lys 
               
               
                   
               
               
                 Gly Glu Cys Gly Ser Gly Gly Gly Asp Ser Lys Asp Lys Thr Ser Ala 
               
               
                   
               
               
                 Leu Ser Leu Ser Asn Val Ala Gly Val Phe Tyr Ile Leu Ile Gly Gly 
               
               
                   
               
               
                 Leu Gly Leu Ala Met Leu Val Ala Leu Ile Glu Phe Cys Tyr Lys Ser 
               
               
                   
               
               
                 Arg Ser Glu Ser Lys Arg Met Lye Gly Phe Cys Leu Ile Pro Gln Gln 
               
               
                   
               
               
                 Ser Ile Asn Glu Ala Ile Arg Thr Ser Thr Leu Pro Arg Asn Ser Gly 
               
               
                   
               
               
                 Ala Gly Ala Ser Ser Gly Gly Ser Gly Glu Aen Gly Arg Val Val Ser 
               
               
                   
               
               
                 His Asp Phe Pro Lye Ser Met Gln Ser Ile Pro Cys Met Ser His Ser 
               
               
                   
               
               
                 Ser Gly Met Pro Leu Gly Ala Thr Gly Leu 
               
               
                   
               
             
          
         
       
     
     Skilled artisans will recognize that some alterations of SEQ ID NO:1 will fail to change the function of the amino acid compound. For instance, some hydrophobic amino acids may be exchanged for other hydrophobic amino acids. Those altered amino acid compounds which confer substantially the same function in substantially the same manner as the exemplified amino acid compound are also included in the present invention. 
     Skilled artisans will also recognize that these proteins can be synthesized by a number of different methods. All of the amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis or recombinant methods. Both methods are described in U.S. Pat. No. 4,617,149, herein incorporated by reference. Recombinant methods are preferred if a high yield is desired. A general method for the construction of any desired DNA sequence is provided in Brown et al., 68  Methods in Enzymology  109 (1979). 
     Other routes of production are well known to skilled artisans. Expression in eucaryotic cells can be achieved via SEQ ID NO:2. For example, the amino acid compounds can be produced in eucaryotic cells using SV40-derived expression vectors comprising DNA which encodes for SEQ ID NO:1. As is well known in the art, some viruses are also appropriate vectors. For example, the adenoviruses, the papovaviruses, the vaccinia viruses, the herpes viruses, and the baculoviruses as well as vectors derived from these viruses are useful. Such a method is described in U.S. Pat. No. 4,775,624, herein incorporated by reference. Several alternate methods of expression are described in J. Sambrook, E. F. Fritsch &amp; T. Maniatis,  Molecular Cloning: A Laboratory Manual  16.3-17.44 (1999) and  Methods in Enzymology , Vol. 185 (1990). 
     Other embodiments of the present invention are nucleic acid compounds which comprise isolated nucleic acid sequences which encode all or part of SEQ ID NO:1. As skilled artisans will recognize, the amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one nucleic acid triplet. Because these alternate nucleic acid sequences would encode substantially the same amino acid sequence, the present invention further comprises these alternate nucleic acid sequences. Preferably, the nucleic acid compound is DNA or sense mRNA. A most preferred embodiment of a DNA compound encoding the HSGluR1 compound has this sequence: 
     
       
         
               
               
             
           
               
                 ATG CAG CAC ATT TTT GCC TTC TTC TGC ACC GGT TTC CTA GGC GCG GTA 
                   
               
               
                   
               
               
                 GTA GGT GCC AAT TTC CCC AAC AAT ATC CAG ATC GGG GGA TTA TTT CCA 
               
               
                   
               
               
                 AAC CAG CAG TCA CAG GAA CAT GCT GCT TTT AGA TTT GCT TTG TCG CAA 
               
               
                   
               
               
                 CTC ACA GAG CCC CCG AAG CTG CTC CCC CAG ATT GAT ATT GTG AAC ATC 
               
               
                   
               
               
                 AGC GAC AGC TTT GAG ATG ACC TAT AGA TTC TGT TCC CAG TTC TCC AAA 
               
               
                   
               
               
                 GGA GTC TAT GCC ATC TTT GGG TTT TAT GAA CGT AGG ACT GTC AAC ATG 
               
               
                   
               
               
                 CTG ACC TCC TTT TGT GGG GCC CTC CAC GTC TGC TTC ATT ACG CCG AGC 
               
               
                   
               
               
                 TTT CCC GTT GAT ACA TCC AAT CAG TTT GTC CTT CAG CTG CGC CCT GAA 
               
               
                   
               
               
                 CTG ACC TCC TTT TGT GGG GCC CTC CAC GTC TGC TTC ATT ACG CCG AGC 
               
               
                   
               
               
                 TTT GTC TAC ATT TAT GAT GCC GAC CGG GGC TTA TCC GTC CTG CAG AAA 
               
               
                   
               
               
                 GTC CTG GAT ACA GCT GCT GAG AAG AAC TGG CAG GTG ACA GCA GTC AAC 
               
               
                   
               
               
                 ATT TTG ACA ACC ACA GAG GAG GGA TAC CGG ATG CTC TTT CAG GAC CTG 
               
               
                   
               
               
                 GAG AAG AAA AAG GAG CGG CTG GTG GTG GTG GAC TGT GAA TCA GAA CGC 
               
               
                   
               
               
                 CTC AAT GCT ATC TTG GGC CAG ATT ATA AAG CTA GAG AAG AAT GGC ATC 
               
               
                   
               
               
                 GGC TAC CAC TAC ATT CTT GCA AAT CTG GGC TTC ATG GAC ATT GAC TTA 
               
               
                   
               
               
                 AAC AAA TTC AAG GAG AGT GGC GCC AAT GTG ACA GGT TTC CAG CTG GTG 
               
               
                   
               
               
                 AAC TAC ACA GAC ACT ATT CCG GCC AAG ATC ATG CAG CAG TGG AAG AAT 
               
               
                   
               
               
                 AGT GAT GCT CGA GAC CAC ACA CGG GTG GAC TGG AAG AGA CCC AAG TAC 
               
               
                   
               
               
                 ACC TCT GCG CTC ACC TAC GAT GGG GTG AAG GTG ATG GCT GAG GCT TTC 
               
               
                   
               
               
                 CAG AGC CTG CGG AGG CAG AGA ATT GAT ATA TCT CGC CGG GGG AAT GCT 
               
               
                   
               
               
                 ATC CAG AGA GCT CTG CAG CAG GTG CGA TTT GAA GGT TTA ACA GGA AAC 
               
               
                   
               
               
                 GTG CAG TTT AAT GAG AAA GGA CGC CGG ACC AAC TAC ACG CTC CAC GTG 
               
               
                   
               
               
                 ATT GAA ATG AAA CAT GAC GGC ATC CGA AAG ATT GGT TAC TGG AAT GAA 
               
               
                   
               
               
                 GAT GAT AAG TTT GTC CCT GCA GCC ACC GAT GCC CAA GCT GGG GGC GAT 
               
               
                   
               
               
                 AAT TCA AGT GTT CAG AAC AGA ACA TAC ATC GTC ACA ACA ATC CTA GAA 
               
               
                   
               
               
                 GAT CCT TAT GTG ATG CTC AAG AAG AAC GCC AAT CAG TTT GAG GGC AAT 
               
               
                   
               
               
                 GAC CGT TAC GAG GGC TAC TGT GTA GAG CTG GCG GCA GAG ATT GCC AAG 
               
               
                   
               
               
                 CAC GTG GGC TAC TCC TAC CGT CTG GAG ATT GTC AGT GAT GGA AAA TAC 
               
               
                   
               
               
                 GGA GCC CGA GAC CCT GAC ACG AAG GCC TGG AAT GGC ATC GTG GGA GAG 
               
               
                   
               
               
                 CTG GTC TAT GGA AGA GCA GAT GTG GCT GTG GCT CCC TTA ACT ATC ACT 
               
               
                   
               
               
                 TTG GTC CGG GAA GAA GTT ATA GAT TTC TCC AAA CCA TTT ATG AGT TTG 
               
               
                   
               
               
                 GGG ATC TCC ATC ATG ATT AAA AAA CCA CAG AAA TCC AAG CCG GGT GTC 
               
               
                   
               
               
                 TTC TCC TTC CTT GAT CCT TTG GCT TAT GAG ATT TGG ATG TGC ATT GTT 
               
               
                   
               
               
                 TTT GCC TAC ATT GGA GTG AGT GTT GTC CTC TTC CTG GTC AGC CGC TTC 
               
               
                   
               
               
                 AGT CCC TAT GAA TGG CAC AGT GAA GAG TTT GAG GAA GGA CGG GAC CAG 
               
               
                   
               
               
                 ACA ACC AGT GAC CAG TCC AAT GAG TTT GGG ATA TTC AAC AGT TTG TGG 
               
               
                   
               
               
                 TTC TCC CTG GGA GCC TTC ATG CAG CAA GGA TGT GAC ATT TCT CCC AGG 
               
               
                   
               
               
                 TCC CTG TCT GGT CGC ATC GTT GGT GGC GTC TGG TGG TTC TTC ACC TTA 
               
               
                   
               
               
                 ATC ATC ATC TCC TCA TAT ACA GCC AAT CTG GCC GCC TTC CTG ACC GTG 
               
               
                   
               
               
                 GAG AGG ATG GTG TCT CCC ATT GAG AGT GCA GAG GAC CTA GCG AAG CAG 
               
               
                   
               
               
                 ACA GAA ATT GCC TAC GGG ACG CTG GAA GCA GGA TCT ACT AAG GAG TTC 
               
               
                   
               
               
                 TTC AGG AGG TCT AAA ATT GCT GTG TTT GAG AAG ATG TGG ACA TAC ATG 
               
               
                   
               
               
                 ATC ATC ATC TCC TCA TAT ACA GCC AAT CTG GCC GCC TTC CTG ACC GTG 
               
               
                   
               
               
                 GAG AGG ATG GTG TCT CCC ATT GAG AGT GCA GAG GAC CTA GCG AAG CAG 
               
               
                   
               
               
                 ACC ATG AAT GAG TAC ATT GAG CAG CGG AAA CCC TGT GAC ACC ATG AAG 
               
               
                   
               
               
                 GTG GGA GGT AAC TTG GAT TCC AAA GGC TAT GGC ATT GCA ACA CCC AAG 
               
               
                   
               
               
                 GGG TCT GCC CTG AGA AAT CCA GTA AAC CTG GCA GTG TTA AAA CTG AAC 
               
               
                   
               
               
                 CTG AGC CTC AGC AAT GTG GCA GGC GTG TTC TAC ATC CTG ATC GGA GGA 
               
               
                   
               
               
                 GGC GAG TGC GGC AGC GGG GGA GGT GAT TCC AAG GAC AAG ACA AGC GCT 
               
               
                   
               
               
                 CTG AGC CTC AGC AAT GTG GCA GGC GTG TTC TAC ATC CTG ATC GGA GGA 
               
               
                   
               
               
                 CTT GGA CTA GCC ATG CTG GTT GCC TTA ATC GAG TTC TGC TAC AAA TCC 
               
               
                   
               
               
                 CGT AGT GAA TCC AAG CGG ATG AAG GGT TTT TGT TTG ATC CCA CAG CAA 
               
               
                   
               
               
                 TCC ATC AAC GAA GCC ATA CGG ACA TCG ACC CTC CCC CGC AAC AGC GGG 
               
               
                   
               
               
                 GCA GGA GCC AGC AGC GGC GGC AGT GGA GAG AAT GGT CGG GTG GTC AGC 
               
               
                   
               
               
                 CAT GAC TTC CCC AAG TCC ATG CAA TCG ATT CCT TGC ATG AGC CAC AGT 
               
               
                   
               
               
                 TCA GGG ATG CCC TTG GGA GCC ACG GGA TTG 
               
               
                   
               
             
          
         
       
     
     This is the sequence identified as SEQ ID NO:2. 
       E. coli /pRS103, which contains a cloning vector comprising SEQ ID NO:2, was deposited and made part of the stock culture collection of the Northern Regional Research Laboratories (NRRL), Agricultural Research Service, U.S. Department of Agriculture, Peoria, Ill., 61604 on Apr. 22, 1992, under the accession number NRRL B-18967. SEQ ID NO:2 can be isolated from the plasmid, for example, as a 4.2 kb EcoR1/Kpn1 restriction fragment. Other fragments are also useful in obtaining SEQ ID NO:2. 
     Additionally, the DNA sequences can be synthesized using automated DNA synthesizers, such as the ABS (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, Calif. 94404) 380B DNA synthesizer. The DNA sequences can also be generated by the polymerase chain reaction (PCR) as described in U.S. Pat. No. 4,189,818, herein incorporated by reference. 
     Because skilled artisans will recognize that many vectors are available for expression and cloning, those expression and cloning vectors which comprise nucleic acids which encode SEQ ID NO:1 are included in the present invention. The preferred nucleic acid vectors are those which are DNA. Most preferred are recombinant DNA vectors which comprise the isolated DNA sequence which is SEQ ID NO:2. The recombinant DNA vector most preferred is plasmid pRS103. 
     DNA vectors which further comprise a promoter positioned to drive expression of functional HSGluR1 receptor are also provided. Preferred recombinant DNA expression vectors are those wherein the promoter functions in mammalian cells. More preferred recombinant DNA expression vectors are those wherein the promoter functions in COS-7 cells. Most preferred COS-7 DNA expression vectors further comprise SEQ ID NO:2. 
     Restriction fragments of these vectors are also provided. The preferred fragments are the 4.2 kb EcoR1/Kpn1 restriction fragment and the 2.8 kb EcoRI/ClaI restriction fragment of pRS103. 
     Plasmid pRS103 may be isolated from the deposited  E. coli| /pRS103, using an ordinary cesium chloride DNA isolation procedure. Plasmid pRS103 is readily utilized to construct expression vectors that produce HSGluR1 receptors in a variety of organisms and cell lines, including, for example, CV1 cells, COS cells, CHO cells,  E. coli , Sf9 (as host for baculovirus), Pichia and Saccharomyceyes. The current literature contains techniques for constructing expression vectors and for transfecting host cells. For example, Sambrook et al.,  Molecular Cloning: A Laboratory Manual  Chapters 16 and 17 (1989), explains these techniques. 
     The construction protocols discussed in Sambrook et al. can be followed to construct analogous vectors for other organisms merely by substituting, if necessary, the appropriate regulatory elements using techniques well known to skilled artisans. Promoters which may be used, for example, are the thymidine kinase promoter, the metallothionin promoter or various viral and immunoglobulin promoters. 
     The DNA compounds of the present invention also include primers or probes. Nucleic acid compounds of at least 17 base pairs which encode all or a part of SEQ ID NO: 1 are included in the present invention. DNA is the preferred nucleic acid used as a probe or primer. Most preferred DNA compounds useful as probes or primers are: SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. A skilled artisan would recognize the techniques associated with probes and primers as well known in the art. Any sequence of at least 17 base pairs in length of the nucleic acids of the present invention may be used to screen any other nucleic acid. For example, all or part of SEQ ID NO:3 and all or part of the reverse complement of SEQ ID NO:5 may be used to hybridize to the terminal ends of the coding sequence. Then, through PCR amplification, the full length sequence may be generated. The full length sequence can be subsequently subcloned into any vector of choice. 
     Alternatively, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 may be radioactively labeled at the 5′ end in order to screen cDNA libraries by conventional means. Furthermore, any piece of HSGluR1 DNA which has been bound to a filter may be flooded with total mRNA transcripts, in order to then reverse-transcribe the mRNA transcripts which bind. 
     Primers and probes may be obtained by means well known in the art. For example, once pRS103 is isolated, restriction enzymes and subsequent gel separation may be used to isolate the fragment of choice. 
     Host cells which harbor the nucleic acids provided by the present invention are also provided. Oocytes, mammalian cells and  E. coli  cells are specifically preferred. COS-7 cells are the most preferred mammalian cells provided. 
     Oocytes which, in addition to harboring nucleic acids capable of expressing functional HSGluR1 receptor, further harbors nucleic acids capable of expressing functional GluR2 receptor are provided. Oocytes which, in addition to harboring nucleic acids capable of expressing functional HSGluR1 receptor, also harbors nucleic acids capable of expressing functional GluR2 receptor and also harbors nucleic acids capable of expressing functional GluR3 receptor are also provided. Most preferred oocytes of the present invention are those which harbor sense mRNA. 
     Host cells which are transfected with a DNA vector having a promoter positioned to drive expression of functional HSGluR1 receptor are also provided. Preferably, the DNA vector comprises SEQ ID NO:2. Preferred host cells for expression of functional HSGluR1 are mammalian cells. Preferred mammalian cells for expression of functional HSGluR1 are COS-7 cells. Specifically, COS-7 cells which have been transfected with a DNA expression vector which expresses a functional HSGluR1 receptor and which further comprise a DNA vector which encodes a functional GluR2 receptor are provided. COS-7 cells which (a) have been transfected with an DNA expression vector which expresses a functional HSGluR1 receptor, and (b) further comprise a DNA vector which encodes a functional GluR2 receptor, and (c) further comprise a DNA vector which encodes a functional GluR3 receptor are also provided. Wigler M. et al., 16 Cell 777 (1979), describe such a cotransfection procedure. 
     Preferred host cells also include  E. coli  cells. The more preferred  E.coli  cells are those which have been transfected with a DNA vector. Most preferred  E.coli  host cells are those which have been transfected with a DNA expression vector which comprises SEQ ID NO:2. The most preferred  E.coli  cell is one transfected with plasmid pRS/103. 
     Oocytes harboring foreign nucleic acids can be constructed according to the procedures described in Lübbert, et al. 84  Proc. Mat. Acad. Sci.  4332 (1987) and Berger, Methods in Enzymology, Vol. 152 (1987). Other host cell transfection procedures are well known in the art. Nucleic acids which encode GluR2 and GluR3 can be obtained according to Heinemann S. et al., PCT publication WO91/06648 (1992). 
     The present invention also provides a method for constructing a recombinant host cell capable of expressing SEQ ID NO:1, said method comprising transfecting a host cell with a recombinant DNA vector that comprises an isolated DNA sequence which encodes SEQ ID NO:1. 
     A preferred host cell for this method is COS-7. An especially preferred expression vector in COS-7 is one which is DNA. An especially preferred method comprises a DNA expression vector which comprises SEQ ID NO:2. Transformed host cells may be cultured under conditions well known to skilled artisans such that SEQ ID NO:1 is expressed, thus producing HSGluR1 activity in the recombinant host cell. 
     Therefore, also provided by the present invention is a method for expressing a gene which encodes SEQ ID NO:1 in a recombinant host cell, said method comprising culturing said transfected host cell under conditions suitable for gene expression. A preferred method utilizes mammalian cells. A most preferred method utilizes COS-7 cells. A more preferred method utilizes COS-7 cells as host cells for a recombinant DNA vector. A most preferred method utilizes COS-7 cells as host cells for a recombinant DNA vector comprising SEQ ID NO:2. Expression in host cells may be accomplished according to the procedures outlined in Sambrook et al.,  Molecular Cloning: A Laboratory Manual  16-17 (1989). 
     Additionally, the invention provides a method for identifying DNA homologous to a probe of the present invention, which comprises contacting the test nucleic acid with the probe under hybridizing conditions and identified as being homologous to the probe. The preferred probes for use in this method are SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. Hybridization techniques are well known in the art. Sambrook et al.,  Molecular Cloning: A Laboratory Manual  11 (1989) describe such procedures. 
     Assays utilizing the compounds provided by the present invention are also provided. Assays provided include a method for determining whether a substance evokes a glutaminergic response, said method comprising introducing said substance and a functional compound of SEQ ID NO:1 into an acceptable medium, and subsequently monitoring glutaminergic activity by physically detectable means, and thereby identifying those substances which effect a chosen response. Other assays further comprise a functional GluR2 receptor. A preferred assay further comprises both the GluR2 and the GluR3 receptor. 
     Preferably, the physically detectable means is competition with radiolabeled glutamate, binding of glutaminergic ligand or generating a detectable ion flow. A preferred assay is an oocyte assay system. A most preferred oocyte assay system utilizes sense mRNA. Most preferred is an assay wherein the oocyte expression system utilizes sense mRNA. 
     The oocyte expression system can be constructed according to the procedure described in Lübbert, et al. 84  Proc. Nat. Acad. Sci.  4332 (1987) and Berger, Methods in Enzymology, Vol.152 (1987). The radiolabeled HSGluR1 competition assay may be accomplished according to Nelson, et al., 41  Life Sciences  1567 (1987). The assay which measures ion flow in mammalian cells may be accomplished according to Hamill O.P. et al., 391 (No. 2) Pflugers Archiv:European J. of Physiology, 85 (1981). 
     Skilled artisans will recognize that competition assays results are described in terms of K i  values. Moreover, skilled artisans realize that desirable K i  values are dependent on the selectivity of the compound tested. For example, a compound with a K i  which is less than 10 nM is generally considered an excellent candidate for drug therapy. However, a compound which has a lower affinity, but is selective for the particular receptor, may be an even better candidate. 
     The present invention provides assays, which indicate whether a substance has either a high affinity or low affinity to HSGluR1 receptor, because skilled artisans will recognize that any information regarding binding or selectivity of a particular compound is beneficial in the pharmaceutical development of drugs. 
     The following are examples of the present invention: 
    
    
     EXAMPLE 1 
     Growth of  E. coli /pRS103 
     A lyophilized culture of  E. coli  containing plasmid pRS103 can be obtained from the American Type Culture Collection, Rockville, Md. 20852, and inoculated into a suitable broth for the growth of  E. coli  using standard microbiological procedures. 
     The contents of a lyophil vial containing  E. coli /pRS103 were transferred into 100 ml of sterile YT (tryptone-yeast extract) broth containing 100 μg/ml ampicillin in a one liter fermentation flask and shaken at 37° C. on an orbital shaker at 250-300 rpm. After the optical density (OD, measured at 600 millimicrons) had reached approximately 1-2 OD, the bacterial cells were recovered and used for the isolation of plasmid pRS103 according to the procedures detailed in J. Sambrook et al.,  Molecular Cloning , Chapter 1, (1999). 
     Once isolated from the bacterial cells, the plasmid DNA served as a source for the DNA encoding the human HSGluR1 receptor protein. One convenient method to remove the receptor-encoding DNA from plasmid pRS103 was to digest the plasmid with restriction enzymes EcoRI and Kpn I. These enzymes cut the plasmid at unique sites to produce a DNA fragment of approximately 4.2 kb containing the entire coding sequence of the human HSGluR1 receptor. 
     EXAMPLE 2 
     In Vitro Transcription of RNA using pRS103 as a DNA Template 
     RNA transcripts encoding the HSGluR1 receptor were produced by enzymatic transcription from pRS103 using an RNA polymerase which recognizes the transcription promoter contained in the plasmid adjacent to the amino terminal coding end of the receptor subunit cDNA. Plasmid pRS103 was treated with the restriction enzyme SalI which made a single cut distal to the 3′ end of the cDNA insert in the circular DNA and converted the plasmid DNA into a linear form. This DNA was then incubated with T7 RNA polymerase in the presence of GpppG cap nucleotide, rATP, rCTP, rUTP and rGTP. The synthetic RNA transcript obtained was purified by passage over a Sephadex G-50 column. For a detailed description of in vitro RNA synthesis using bacteriophage RNA polymerase such as T7, see P. A. Krieg and D. A. Melton, Vol 155,  Methods in Enzymology , Ch. 25, 1987. 
     EXAMPLE 3 
     Functional Expression of Human HSGluR1 Receptor in Xenopus Oocytes. 
     Oocytes suitable for injection were obtained from the adult female  Xenopus laevis  using procedures described in C. J. Marcus-Sekura and M. J. M. Hitchcock,  Methods in Enzymology , Vol. 152 (1997). After treatment with collagenase type la (Sigma) at a concentration of 2 mg/ml, the defolliculated oocytes were injected essentially as described by M. J. M. Hitchcock et al.,  Methods in Enzymology , Vol. 152 Chapter 22, (1997). Subsequently, 5 ng of RNA transcript in a total volume of 50 nl, prepared as described in Example 2, were injected into each oocyte and they were then incubated in Barth&#39;s saline solution at 18° C. until needed for electrophysiological measurements. 
     In order to detect the presence of HSGluR1 receptor, the ability of the receptor to assemble into functional ion channels was determined by voltage recording of electrical current flowing across the oocyte membrane in response to glutamate agonists. Individual oocytes were placed in a diffusion chamber (0.5 ml vol.) through which solutions were perfused rapidly. Drugs (agonists and antagonists) were applied to the oocytes by adding them to the perfusing solutions and subsequently washing them out with control solution. The control solution contained 96 nM NaCl, 2 mM KCl, 1.8 nM CaCl2, 1 MgCl2, and 5 mM HEPES buffer, pH 7.6. After insertion of electrodes into the oocytes, voltage recordings were made using the bridge circuit of an Axoclamp 1A voltage-clamp unit. Microelectrodes were filled with 3 M CsCl. Electrophysiological recordings of the oocytes clamped at −70 mV were made at room temperature (20-25° C.), 3 days or more after injection of RNA into the oocytes. In response to perfusion of the cells with 100 μM kainic acid, an inward current across the oocyte membrane of 10-30 nano-amperes was observed. For a detailed discussion of the electrophysiology of Xenopus oocytes see N. Dascal, 22 CRC  Critical Reviews in Biochemistry,  317 (1987). As those skilled in the art appreciate these results are indicative of a glutamate receptor. 
     
       
         
           
             5 
           
           
             
               906 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               not provided 
             
             1
Met Gln His Ile Phe Ala Phe Phe Cys Thr Gly Phe Leu Gly Ala Val
  1               5                  10                  15
Val Gly Ala Asn Phe Pro Asn Asn Ile Gln Ile Gly Gly Leu Phe Pro
             20                  25                  30
Asn Gln Gln Ser Gln Glu His Ala Ala Phe Arg Phe Ala Leu Ser Gln
         35                  40                  45
Leu Thr Glu Pro Pro Lys Leu Leu Pro Gln Ile Asp Ile Val Asn Ile
     50                  55                  60
Ser Asp Ser Phe Glu Met Thr Tyr Arg Phe Cys Ser Gln Phe Ser Lys
 65                  70                  75                  80
Gly Val Tyr Ala Ile Phe Gly Phe Tyr Glu Arg Arg Thr Val Asn Met
                 85                  90                  95
Leu Thr Ser Phe Cys Gly Ala Leu His Val Cys Phe Ile Thr Pro Ser
            100                 105                 110
Phe Pro Val Asp Thr Ser Asn Gln Phe Val Leu Gln Leu Arg Pro Glu
        115                 120                 125
Leu Gln Asp Ala Leu Ile Ser Ile Ile Asp His Tyr Lys Trp Gln Lys
    130                 135                 140
Phe Val Tyr Ile Tyr Asp Ala Asp Arg Gly Leu Ser Val Leu Gln Lys
145                 150                 155                 160
Val Leu Asp Thr Ala Ala Glu Lys Asn Trp Gln Val Thr Ala Val Asn
                165                 170                 175
Ile Leu Thr Thr Thr Glu Glu Gly Tyr Arg Met Leu Phe Gln Asp Leu
            180                 185                 190
Glu Lys Lys Lys Glu Arg Leu Val Val Val Asp Cys Glu Ser Glu Arg
        195                 200                 205
Leu Asn Ala Ile Leu Gly Gln Ile Ile Lys Leu Glu Lys Asn Gly Ile
    210                 215                 220
Gly Tyr His Tyr Ile Leu Ala Asn Leu Gly Phe Met Asp Ile Asp Leu
225                 230                 235                 240
Asn Lys Phe Lys Glu Ser Gly Ala Asn Val Thr Gly Phe Gln Leu Val
                245                 250                 255
Asn Tyr Thr Asp Thr Ile Pro Ala Lys Ile Met Gln Gln Trp Lys Asn
            260                 265                 270
Ser Asp Ala Arg Asp His Thr Arg Val Asp Trp Lys Arg Pro Lys Tyr
        275                 280                 285
Thr Ser Ala Leu Thr Tyr Asp Gly Val Lys Val Met Ala Glu Ala Phe
    290                 295                 300
Gln Ser Leu Arg Arg Gln Arg Ile Asp Ile Ser Arg Arg Gly Asn Ala
305                 310                 315                 320
Gly Asp Cys Leu Ala Asn Pro Ala Val Pro Trp Gly Gln Gly Ile Asp
                325                 330                 335
Ile Gln Arg Ala Leu Gln Gln Val Arg Phe Glu Gly Leu Thr Gly Asn
            340                 345                 350
Val Gln Phe Asn Glu Lys Gly Arg Arg Thr Asn Tyr Thr Leu His Val
        355                 360                 365
Ile Glu Met Lys His Asp Gly Ile Arg Lys Ile Gly Tyr Trp Asn Glu
    370                 375                 380
Asp Asp Lys Phe Val Pro Ala Ala Thr Asp Ala Gln Ala Gly Gly Asp
385                 390                 395                 400
Asn Ser Ser Val Gln Asn Arg Thr Tyr Ile Val Thr Thr Ile Leu Glu
                405                 410                 415
Asp Pro Tyr Val Met Leu Lys Lys Asn Ala Asn Gln Phe Glu Gly Asn
            420                 425                 430
Asp Arg Tyr Glu Gly Tyr Cys Val Glu Leu Ala Ala Glu Ile Ala Lys
        435                 440                 445
His Val Gly Tyr Ser Tyr Arg Leu Glu Ile Val Ser Asp Gly Lys Tyr
    450                 455                 460
Gly Ala Arg Asp Pro Asp Thr Lys Ala Trp Asn Gly Met Val Gly Glu
465                 470                 475                 480
Leu Val Tyr Gly Arg Ala Asp Val Ala Val Ala Pro Leu Thr Ile Thr
                485                 490                 495
Leu Val Arg Glu Glu Val Ile Asp Phe Ser Lys Pro Phe Met Ser Leu
            500                 505                 510
Gly Ile Ser Ile Met Ile Lys Lys Pro Gln Lys Ser Lys Pro Gly Val
        515                 520                 525
Phe Ser Phe Leu Asp Pro Leu Ala Tyr Glu Ile Trp Met Cys Ile Val
    530                 535                 540
Phe Ala Tyr Ile Gly Val Ser Val Val Leu Phe Leu Val Ser Arg Phe
545                 550                 555                 560
Ser Pro Tyr Glu Trp His Ser Glu Glu Phe Glu Glu Gly Arg Asp Gln
                565                 570                 575
Thr Thr Ser Asp Gln Ser Asn Glu Phe Gly Ile Phe Asn Ser Leu Trp
            580                 585                 590
Phe Ser Leu Gly Ala Phe Met Gln Gln Gly Cys Asp Ile Ser Pro Arg
        595                 600                 605
Ser Leu Ser Gly Arg Ile Val Gly Gly Val Trp Trp Phe Phe Thr Leu
    610                 615                 620
Ile Ile Ile Ser Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Thr Val
625                 630                 635                 640
Glu Arg Met Val Ser Pro Ile Glu Ser Ala Glu Asp Leu Ala Lys Gln
                645                 650                 655
Thr Glu Ile Ala Tyr Gly Thr Leu Glu Ala Gly Ser Thr Lys Glu Phe
            660                 665                 670
Phe Arg Arg Ser Lys Ile Ala Val Phe Glu Lys Met Trp Thr Tyr Met
        675                 680                 685
Lys Ser Ala Glu Pro Ser Val Phe Val Arg Thr Thr Glu Glu Gly Met
    690                 695                 700
Ile Arg Val Arg Lys Ser Lys Gly Lys Tyr Ala Tyr Leu Leu Glu Ser
705                 710                 715                 720
Thr Met Asn Glu Tyr Ile Glu Gln Arg Lys Pro Cys Asp Thr Met Lys
                725                 730                 735
Val Gly Gly Asn Leu Asp Ser Lys Gly Tyr Gly Ile Ala Thr Pro Lys
            740                 745                 750
Gly Ser Ala Leu Arg Asn Pro Val Asn Leu Ala Val Leu Lys Leu Asn
        755                 760                 765
Glu Gln Gly Leu Leu Asp Lys Leu Lys Asn Lys Trp Trp Tyr Asp Lys
    770                 775                 780
Gly Glu Cys Gly Ser Gly Gly Gly Asp Ser Lys Asp Lys Thr Ser Ala
785                 790                 795                 800
Leu Ser Leu Ser Asn Val Ala Gly Val Phe Tyr Ile Leu Ile Gly Gly
                805                 810                 815
Leu Gly Leu Ala Met Leu Val Ala Leu Ile Glu Phe Cys Tyr Lys Ser
            820                 825                 830
Arg Ser Glu Ser Lys Arg Met Lys Gly Phe Cys Leu Ile Pro Gln Gln
        835                 840                 845
Ser Ile Asn Glu Ala Ile Arg Thr Ser Thr Leu Pro Arg Asn Ser Gly
    850                 855                 860
Ala Gly Ala Ser Ser Gly Gly Ser Gly Glu Asn Gly Arg Val Val Ser
865                 870                 875                 880
His Asp Phe Pro Lys Ser Met Gln Ser Ile Pro Cys Met Ser His Ser
                885                 890                 895
Ser Gly Met Pro Leu Gly Ala Thr Gly Leu
            900                 905 
           
           
             
               2718 base pairs 
               nucleic acid 
               both 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             
               CDS 
                1..2718 
             
             2
ATG CAG CAC ATT TTT GCC TTC TTC TGC ACC GGT TTC CTA GGC GCG GTA       48
Met Gln His Ile Phe Ala Phe Phe Cys Thr Gly Phe Leu Gly Ala Val
  1               5                  10                  15
GTA GGT GCC AAT TTC CCC AAC AAT ATC CAG ATC GGG GGA TTA TTT CCA       96
Val Gly Ala Asn Phe Pro Asn Asn Ile Gln Ile Gly Gly Leu Phe Pro
             20                  25                  30
AAC CAG CAG TCA CAG GAA CAT GCT GCT TTT AGA TTT GCT TTG TCG CAA      144
Asn Gln Gln Ser Gln Glu His Ala Ala Phe Arg Phe Ala Leu Ser Gln
         35                  40                  45
CTC ACA GAG CCC CCG AAG CTG CTC CCC CAG ATT GAT ATT GTG AAC ATC      192
Leu Thr Glu Pro Pro Lys Leu Leu Pro Gln Ile Asp Ile Val Asn Ile
     50                  55                  60
AGC GAC AGC TTT GAG ATG ACC TAT AGA TTC TGT TCC CAG TTC TCC AAA      240
Ser Asp Ser Phe Glu Met Thr Tyr Arg Phe Cys Ser Gln Phe Ser Lys
 65                  70                  75                  80
GGA GTC TAT GCC ATC TTT GGG TTT TAT GAA CGT AGG ACT GTC AAC ATG      288
Gly Val Tyr Ala Ile Phe Gly Phe Tyr Glu Arg Arg Thr Val Asn Met
                 85                  90                  95
CTG ACC TCC TTT TGT GGG GCC CTC CAC GTC TGC TTC ATT ACG CCG AGC      336
Leu Thr Ser Phe Cys Gly Ala Leu His Val Cys Phe Ile Thr Pro Ser
            100                 105                 110
TTT CCC GTT GAT ACA TCC AAT CAG TTT GTC CTT CAG CTG CGC CCT GAA      384
Phe Pro Val Asp Thr Ser Asn Gln Phe Val Leu Gln Leu Arg Pro Glu
        115                 120                 125
CTG CAG GAT GCC CTC ATC AGC ATC ATT GAC CAT TAC AAG TGG CAG AAA      432
Leu Gln Asp Ala Leu Ile Ser Ile Ile Asp His Tyr Lys Trp Gln Lys
    130                 135                 140
TTT GTC TAC ATT TAT GAT GCC GAC CGG GGC TTA TCC GTC CTG CAG AAA      480
Phe Val Tyr Ile Tyr Asp Ala Asp Arg Gly Leu Ser Val Leu Gln Lys
145                 150                 155                 160
GTC CTG GAT ACA GCT GCT GAG AAG AAC TGG CAG GTG ACA GCA GTC AAC      528
Val Leu Asp Thr Ala Ala Glu Lys Asn Trp Gln Val Thr Ala Val Asn
                165                 170                 175
ATT TTG ACA ACC ACA GAG GAG GGA TAC CGG ATG CTC TTT CAG GAC CTG      576
Ile Leu Thr Thr Thr Glu Glu Gly Tyr Arg Met Leu Phe Gln Asp Leu
            180                 185                 190
GAG AAG AAA AAG GAG CGG CTG GTG GTG GTG GAC TGT GAA TCA GAA CGC      624
Glu Lys Lys Lys Glu Arg Leu Val Val Val Asp Cys Glu Ser Glu Arg
        195                 200                 205
CTC AAT GCT ATC TTG GGC CAG ATT ATA AAG CTA GAG AAG AAT GGC ATC      672
Leu Asn Ala Ile Leu Gly Gln Ile Ile Lys Leu Glu Lys Asn Gly Ile
    210                 215                 220
GGC TAC CAC TAC ATT CTT GCA AAT CTG GGC TTC ATG GAC ATT GAC TTA      720
Gly Tyr His Tyr Ile Leu Ala Asn Leu Gly Phe Met Asp Ile Asp Leu
225                 230                 235                 240
AAC AAA TTC AAG GAG AGT GGC GCC AAT GTG ACA GGT TTC CAG CTG GTG      768
Asn Lys Phe Lys Glu Ser Gly Ala Asn Val Thr Gly Phe Gln Leu Val
                245                 250                 255
AAC TAC ACA GAC ACT ATT CCG GCC AAG ATC ATG CAG CAG TGG AAG AAT      816
Asn Tyr Thr Asp Thr Ile Pro Ala Lys Ile Met Gln Gln Trp Lys Asn
            260                 265                 270
AGT GAT GCT CGA GAC CAC ACA CGG GTG GAC TGG AAG AGA CCC AAG TAC      864
Ser Asp Ala Arg Asp His Thr Arg Val Asp Trp Lys Arg Pro Lys Tyr
        275                 280                 285
ACC TCT GCG CTC ACC TAC GAT GGG GTG AAG GTG ATG GCT GAG GCT TTC      912
Thr Ser Ala Leu Thr Tyr Asp Gly Val Lys Val Met Ala Glu Ala Phe
    290                 295                 300
CAG AGC CTG CGG AGG CAG AGA ATT GAT ATA TCT CGC CGG GGG AAT GCT      960
Gln Ser Leu Arg Arg Gln Arg Ile Asp Ile Ser Arg Arg Gly Asn Ala
305                 310                 315                 320
GGG GAT TGT CTG GCT AAC CCA GCT GTT CCC TGG GGC CAA GGG ATC GAC     1008
Gly Asp Cys Leu Ala Asn Pro Ala Val Pro Trp Gly Gln Gly Ile Asp
                325                 330                 335
ATC CAG AGA GCT CTG CAG CAG GTG CGA TTT GAA GGT TTA ACA GGA AAC     1056
Ile Gln Arg Ala Leu Gln Gln Val Arg Phe Glu Gly Leu Thr Gly Asn
            340                 345                 350
GTG CAG TTT AAT GAG AAA GGA CGC CGG ACC AAC TAC ACG CTC CAC GTG     1104
Val Gln Phe Asn Glu Lys Gly Arg Arg Thr Asn Tyr Thr Leu His Val
        355                 360                 365
ATT GAA ATG AAA CAT GAC GGC ATC CGA AAG ATT GGT TAC TGG AAT GAA     1152
Ile Glu Met Lys His Asp Gly Ile Arg Lys Ile Gly Tyr Trp Asn Glu
    370                 375                 380
GAT GAT AAG TTT GTC CCT GCA GCC ACC GAT GCC CAA GCT GGG GGC GAT     1200
Asp Asp Lys Phe Val Pro Ala Ala Thr Asp Ala Gln Ala Gly Gly Asp
385                 390                 395                 400
AAT TCA AGT GTT CAG AAC AGA ACA TAC ATC GTC ACA ACA ATC CTA GAA     1248
Asn Ser Ser Val Gln Asn Arg Thr Tyr Ile Val Thr Thr Ile Leu Glu
                405                 410                 415
GAT CCT TAT GTG ATG CTC AAG AAG AAC GCC AAT CAG TTT GAG GGC AAT     1296
Asp Pro Tyr Val Met Leu Lys Lys Asn Ala Asn Gln Phe Glu Gly Asn
            420                 425                 430
GAC CGT TAC GAG GGC TAC TGT GTA GAG CTG GCG GCA GAG ATT GCC AAG     1344
Asp Arg Tyr Glu Gly Tyr Cys Val Glu Leu Ala Ala Glu Ile Ala Lys
        435                 440                 445
CAC GTG GGC TAC TCC TAC CGT CTG GAG ATT GTC AGT GAT GGA AAA TAC     1392
His Val Gly Tyr Ser Tyr Arg Leu Glu Ile Val Ser Asp Gly Lys Tyr
    450                 455                 460
GGA GCC CGA GAC CCT GAC ACG AAG GCC TGG AAT GGC ATG GTG GGA GAG     1440
Gly Ala Arg Asp Pro Asp Thr Lys Ala Trp Asn Gly Met Val Gly Glu
465                 470                 475                 480
CTG GTC TAT GGA AGA GCA GAT GTG GCT GTG GCT CCC TTA ACT ATC ACT     1488
Leu Val Tyr Gly Arg Ala Asp Val Ala Val Ala Pro Leu Thr Ile Thr
                485                 490                 495
TTG GTC CGG GAA GAA GTT ATA GAT TTC TCC AAA CCA TTT ATG AGT TTG     1536
Leu Val Arg Glu Glu Val Ile Asp Phe Ser Lys Pro Phe Met Ser Leu
            500                 505                 510
GGG ATC TCC ATC ATG ATT AAA AAA CCA CAG AAA TCC AAG CCG GGT GTC     1584
Gly Ile Ser Ile Met Ile Lys Lys Pro Gln Lys Ser Lys Pro Gly Val
        515                 520                 525
TTC TCC TTC CTT GAT CCT TTG GCT TAT GAG ATT TGG ATG TGC ATT GTT     1632
Phe Ser Phe Leu Asp Pro Leu Ala Tyr Glu Ile Trp Met Cys Ile Val
    530                 535                 540
TTT GCC TAC ATT GGA GTG AGT GTT GTC CTC TTC CTG GTC AGC CGC TTC     1680
Phe Ala Tyr Ile Gly Val Ser Val Val Leu Phe Leu Val Ser Arg Phe
545                 550                 555                 560
AGT CCC TAT GAA TGG CAC AGT GAA GAG TTT GAG GAA GGA CGG GAC CAG     1728
Ser Pro Tyr Glu Trp His Ser Glu Glu Phe Glu Glu Gly Arg Asp Gln
                565                 570                 575
ACA ACC AGT GAC CAG TCC AAT GAG TTT GGG ATA TTC AAC AGT TTG TGG     1776
Thr Thr Ser Asp Gln Ser Asn Glu Phe Gly Ile Phe Asn Ser Leu Trp
            580                 585                 590
TTC TCC CTG GGA GCC TTC ATG CAG CAA GGA TGT GAC ATT TCT CCC AGG     1824
Phe Ser Leu Gly Ala Phe Met Gln Gln Gly Cys Asp Ile Ser Pro Arg
        595                 600                 605
TCC CTG TCT GGT CGC ATC GTT GGT GGC GTC TGG TGG TTC TTC ACC TTA     1872
Ser Leu Ser Gly Arg Ile Val Gly Gly Val Trp Trp Phe Phe Thr Leu
    610                 615                 620
ATC ATC ATC TCC TCA TAT ACA GCC AAT CTG GCC GCC TTC CTG ACC GTG     1920
Ile Ile Ile Ser Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Thr Val
625                 630                 635                 640
GAG AGG ATG GTG TCT CCC ATT GAG AGT GCA GAG GAC CTA GCG AAG CAG     1968
Glu Arg Met Val Ser Pro Ile Glu Ser Ala Glu Asp Leu Ala Lys Gln
                645                 650                 655
ACA GAA ATT GCC TAC GGG ACG CTG GAA GCA GGA TCT ACT AAG GAG TTC     2016
Thr Glu Ile Ala Tyr Gly Thr Leu Glu Ala Gly Ser Thr Lys Glu Phe
            660                 665                 670
TTC AGG AGG TCT AAA ATT GCT GTG TTT GAG AAG ATG TGG ACA TAC ATG     2064
Phe Arg Arg Ser Lys Ile Ala Val Phe Glu Lys Met Trp Thr Tyr Met
        675                 680                 685
AAG TCA GCA GAG CCA TCA GTT TTT GTG CGG ACC ACA GAG GAG GGG ATG     2112
Lys Ser Ala Glu Pro Ser Val Phe Val Arg Thr Thr Glu Glu Gly Met
    690                 695                 700
ATT CGA GTG AGG AAA TCC AAA GGC AAA TAT GCC TAC CTC CTG GAG TCC     2160
Ile Arg Val Arg Lys Ser Lys Gly Lys Tyr Ala Tyr Leu Leu Glu Ser
705                 710                 715                 720
ACC ATG AAT GAG TAC ATT GAG CAG CGG AAA CCC TGT GAC ACC ATG AAG     2208
Thr Met Asn Glu Tyr Ile Glu Gln Arg Lys Pro Cys Asp Thr Met Lys
                725                 730                 735
GTG GGA GGT AAC TTG GAT TCC AAA GGC TAT GGC ATT GCA ACA CCC AAG     2256
Val Gly Gly Asn Leu Asp Ser Lys Gly Tyr Gly Ile Ala Thr Pro Lys
            740                 745                 750
GGG TCT GCC CTG AGA AAT CCA GTA AAC CTG GCA GTG TTA AAA CTG AAC     2304
Gly Ser Ala Leu Arg Asn Pro Val Asn Leu Ala Val Leu Lys Leu Asn
        755                 760                 765
GAG CAG GGG CTT TTG GAC AAA TTG AAA AAC AAA TGG TGG TAC GAC AAG     2352
Glu Gln Gly Leu Leu Asp Lys Leu Lys Asn Lys Trp Trp Tyr Asp Lys
    770                 775                 780
GGC GAG TGC GGC AGC GGG GGA GGT GAT TCC AAG GAC AAG ACA AGC GCT     2400
Gly Glu Cys Gly Ser Gly Gly Gly Asp Ser Lys Asp Lys Thr Ser Ala
785                 790                 795                 800
CTG AGC CTC AGC AAT GTG GCA GGC GTG TTC TAC ATC CTG ATC GGA GGA     2448
Leu Ser Leu Ser Asn Val Ala Gly Val Phe Tyr Ile Leu Ile Gly Gly
                805                 810                 815
CTT GGA CTA GCC ATG CTG GTT GCC TTA ATC GAG TTC TGC TAC AAA TCC     2496
Leu Gly Leu Ala Met Leu Val Ala Leu Ile Glu Phe Cys Tyr Lys Ser
            820                 825                 830
CGT AGT GAA TCC AAG CGG ATG AAG GGT TTT TGT TTG ATC CCA CAG CAA     2544
Arg Ser Glu Ser Lys Arg Met Lys Gly Phe Cys Leu Ile Pro Gln Gln
        835                 840                 845
TCC ATC AAC GAA GCC ATA CGG ACA TCG ACC CTC CCC CGC AAC AGC GGG     2592
Ser Ile Asn Glu Ala Ile Arg Thr Ser Thr Leu Pro Arg Asn Ser Gly
    850                 855                 860
GCA GGA GCC AGC AGC GGC GGC AGT GGA GAG AAT GGT CGG GTG GTC AGC     2640
Ala Gly Ala Ser Ser Gly Gly Ser Gly Glu Asn Gly Arg Val Val Ser
865                 870                 875                 880
CAT GAC TTC CCC AAG TCC ATG CAA TCG ATT CCT TGC ATG AGC CAC AGT     2688
His Asp Phe Pro Lys Ser Met Gln Ser Ile Pro Cys Met Ser His Ser
                885                 890                 895
TCA GGG ATG CCC TTG GGA GCC ACG GGA TTG                             2718
Ser Gly Met Pro Leu Gly Ala Thr Gly Leu
            900                 905 
           
           
             
               60 base pairs 
               nucleic acid 
               both 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             
               CDS 
                1..60 
             
             3
ATG CAG CAC ATT TTT GCC TTC TTC TGC ACC GGT TTC CTA GGC GCG GTA       48
Met Gln His Ile Phe Ala Phe Phe Cys Thr Gly Phe Leu Gly Ala Val
  1               5                  10                  15
GTA GGT GCC AAT                                                       60
Val Gly Ala Asn
             20 
           
           
             
               60 base pairs 
               nucleic acid 
               both 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             
               CDS 
                1-60 
             
             4
                                        TTT GCT TTG TCG CAA           15
                                        Phe Ala Leu Ser Gln
                                          1               5
CTC ACA GAG CCC CCG AAG CTG CTC CCC CAG ATT GAT ATT GTG AAC           60
Leu Thr Glu Pro Pro Lys Leu Leu Pro Gln Ile Asp Ile Val Asn
                 10                  15                  20 
           
           
             
               60 base pairs 
               nucleic acid 
               both 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             
               CDS 
                1-60 
             
             5
                        CAA TCG ATT CCT TGC ATG AGC CAC AGT           27
                        Gln Ser Ile Pro Cys Met Ser His Ser
                          1               5
TCA GGG ATG CCC TTG GGA GCC ACG GGA TTG TAA                           60
Ser Gly Met Pro Leu Gly Ala Thr Gly Leu
 10                  15