Kainate-binding human CNS receptors of the EAA4 family

Abstract

Neurotransmission by excitatory amino acids (EAAs) such as glutamate is mediated via membrane-bound surface receptors. DNA coding for one family of these receptors of the kainate-binding type of EAA receptors, has now been isolated and the receptor protein characterized. Herein described are recombinant cell lines which produce the EAA receptor as a heterologous membrane-bound product. Also described are related aspects of the invention, which are of commercial significance. Included is use of the cell lines as a tool for discovery of compounds which modulate EAA receptor stimulation.

What is claimed is:  
     
         1 . An isolated polynucleotide comprising nucleic acids arranged in a sequence that codes for a human EAA4 receptor, or for a kainate-binding fragment of a human EAA4 receptor.  
     
     
         2 . An isolated polynucleotide according to  claim 1 , consisting of DNA.  
     
     
         3 . An isolated polynucleotide according to  claim 2 , wherein said nucleic acids are arranged in a sequence that codes for the human EAA4a receptor, or for a kainate-binding fragment thereof.  
     
     
         4 . An isolated polynucleotide according to  claim 2 , wherein said nucleic acids are arranged in a sequence that codes for the human EAA4a receptor.  
     
     
         5 . An isolated polynucleotide according to  claim 2 , wherein said nucleic acids are arranged in a sequence that codes for the human EAA4b receptor, or for a kainate-binding variant thereof.  
     
     
         6 . An isolated polynucleotide according to  claim 2 , wherein said nucleic acids are arranged in a sequence that codes for the human EAA4b receptor.  
     
     
         7 . A recombinant DNA construct having incorporated therein a polynucleotide as defined in  claim 1 .  
     
     
         8 . A recombinant DNA construct according to  claim 7 , wherein said polynucleotide comprises nucleic acids arranged in a sequence that codes for the human EAA4a receptor.  
     
     
         9 . A recombinant DNA construct according to  claim 7 , wherein said construct is plasmid pBS/humEAA4a (ATCC 75245).  
     
     
         10 . A 2.9 kilobase EcoRI/EcoRI fragment of the recombinant DNA construct according to  claim 9 .  
     
     
         11 . A cell that has been engineered genetically to produce a kainate-binding human EAA receptor, said cell having incorporated expressibly therein a heterologous DNA molecule comprising nucleic acids arranged in a sequence that codes for a human EAA4 receptor or for a kainate-binding fragment thereof.  
     
     
         12 . A cell as defined in  claim 11 , which is a mammalian cell.  
     
     
         13 . A cell as defined in  claim 11 , which is an oocyte.  
     
     
         14 . A cell according to  claim 11 , wherein said heterologous DNA molecule codes for the human EAA4a receptor.  
     
     
         15 . A cell as defined in  claim 14 , wherein said cell is a mammalian cell.  
     
     
         16 . A cell as defined in  claim 14 , wherein said cell is an oocyte.  
     
     
         17 . A membrane preparation derived from a cell as defined in  claim 11 .  
     
     
         18 . A membrane preparation derived from a cell as defined in  claim 14 .  
     
     
         19 . A membrane preparation derived from a cell as defined in  claim 15 .  
     
     
         20 . A process for obtaining a substantially homogeneous source of a human EAA receptor, which comprises the steps of culturing cells having incorporated expressibly therein a polynucleotide comprising nucleic acids arranged in a sequence that encodes a human EAA4 receptor, and then recovering the cultured cells.  
     
     
         21 . A process for obtaining a substantially homogeneous source of a human EAA receptor according to  claim 20 , comprising the subsequent step of obtaining a membrane preparation from the cultured cells.  
     
     
         22 . A method of assaying a test ligand for binding to a human CNS receptor, which comprises the steps of incubating the test ligand under appropriate conditions with a human EAA4 receptor-producing cell as defined in  claim 11  or with membrane preparation derived therefrom, and then determining the extent of binding between the human EAA4 receptor and the test ligand.  
     
     
         23 . A method according to  claim 22 , wherein the human EAA4 receptor producing cell is a human EAA4a receptor-producing cell.  
     
     
         24 . A method according to  claim 23 , wherein the cell is a mammalian cell.  
     
     
         25 . A method according to  claim 23 , wherein the test ligand is incubated with a membrane preparation derived from said human EAA4a-producing cell.  
     
     
         26 . A method of detecting interaction between a test ligand and a human CNS receptor, which comprises the steps of incubating the test ligand with a human EAA4 receptor-producing cell as defined in  claim 11 , or with a membrane preparation derived therefrom, and then measuring ligand-induced electrical current across said cell or membrane.  
     
     
         27 . A method according to  claim 26 , wherein the receptor-producing cell is an EAA4a receptor-producing cell.  
     
     
         28 . A method according to  claim 27 , wherein the cell is a mammalian cell.  
     
     
         29 . A method according to  claim 28 , wherein the cell is an oocyte.  
     
     
         30 . A human EAA4 receptor, in a form essentially free from other proteins of human origin.  
     
     
         31 . A human EAA4 receptor as defined in  claim 30 , which is the human EAA4a receptor.  
     
     
         32 . A kainate-binding fragment of a human EAA4 receptor.  
     
     
         33 . A kainate-binding fragment according to  claim 32 , which is a kainate-binding fragment of a human EAA4 receptor.  
     
     
         34 . An antibody which binds the human EAA4a receptor.  
     
     
         35 . An antibody according to  claim 34 , having a reporter molecule coupled thereto.  
     
     
         36 . An immunogenic fragment of the human EAA4a receptor.  
     
     
         37 . An oligonucleotide which comprises at least about 17 nucleic acids which hybridizes with a polynucleotide defined in  claim 2 .  
     
     
         38 . An oligonucleotide according to  claim 37 , having a reporter molecule conjugated thereto.