Modulatory proteins of human CNS receptors

Abstract

Neurotransmission by excitatory amino acids (EAAs) such as glutamate is mediated via membrane-bound surface receptors. This neurotransmission has been found to be modulated by certain modulatory proteins. DNA coding for a family of such modulatory proteins has now been isolated and the modulatory proteins have been characterized. Herein described are recombinant cell lines which produce these modulatory proteins as heterologous membrane-bound products. Also described are related aspects of the invention, which are of commercial significance, including the use of cell lines which express the modulatory proteins either homomerically, or heteromerically in a complex with an NMDA receptor, as a tool for discovery of compounds which affect the function of the modulatory proteins.

We claim:  
     
         1 . An isolated polynucleotide comprising a region that encodes a human NR3-1 modulatory protein, or a functional fragment thereof which retains the modulatory activity of the NR3-1 protein.  
     
     
         2 . An isolated polynucleotide comprising a region encoding a functional variant of a human NR3-1 receptor, wherein said variant shares greater than 98.5% amino acid identity with said NR3-1 protein and retains the modulatory activity of said NR3-1 protein.  
     
     
         3 . An isolated polynucleotide as defined in  claim 2 , comprising a region that encodes a human NR3-2 protein.  
     
     
         4 . A recombinant DNA construct having incorporated therein a polynucleotide as defined in  claim 1 .  
     
     
         5 . A recombinant DNA construct having incorporated therein a polynucleotide as defined in  claim 2 .  
     
     
         6 . A cell that has been engineered genetically to produce a human NR3 protein or a fragent thereof, said cell having incorporated expressibly therein a heterologous polynuclotide as defined in  claim 1 .  
     
     
         7 . A cell that has been engineered genetically to produce a human NR3 protein or a fragment thereof, said cell having incorporated expressibly therein a heterologous polynucleotide as defined in  claim 2 .  
     
     
         8 . A membrane preparation derived from a cell as defined in  claim 6 .  
     
     
         9 . A membrane preparation derived from a cell as defined in  claim 7 .  
     
     
         10 . A cell that has been engineered genetically to produce a heteromeric human receptor complex comprising an NR3 protein and an NMDA receptor, said cell having incorporated expressibly therein a heterologous polynucleotide encoding a human NR3 protein and a heterologous polynucleotide encoding a human NMDA receptor.  
     
     
         11 . A cell as defined in  claim 10  wherein said human NR3 protein is selected from the group consisting of the NR3-1 and the NR3-2 proteins, and the human NMDA receptor is selected from the group consisting of the NMDAR1-1, NMDAR1-2, NMDAR1-3, NMDAR1-4, NMDAR1-5, NMDAR1-6, NMDAR1-7 and NMDAR1-8 receptors.  
     
     
         12 . A process for obtaining a substantially homogeneous source of a human NR3 protein selected from the group consisting of the NR3-1 and NR3-2 proteins, said process comprising the steps of culturing cells having incorporated expressibly therein a heterologous polynucleotide encoding NR3-1 or NR3-2, and then recovering the cultured cells.  
     
     
         13 . A process for obtaining a substantially homogeneous source of a human NR3 protein according to  claim 12 , comprising the subsequent step of obtaining a membrane preparation from the cultured cells.  
     
     
         14 . A method of assaying a candidate ligand for interaction with a human NR3 protein selected from the group consisting of the NR3-1 and NR3-2 proteins, which comprises the steps of incubating the candidate ligand under appropriate conditions with a cell having incorporated expressibly therein a heterologous polynucleotide encoding NR3-1 or NR3-2, or with a membrane preparation derived therefrom, and then determining the extent of binding between the human NR3 protein and the candidate ligand.  
     
     
         15 . A method of assaying a candidate ligand for interaction with a human heteromeric receptor complex comprising an NR3 protein and an NMDA receptor, which comprises the steps of incubating the candidate ligand under appropriate conditions with a cell as defined in  claim 10 , or with membrane preparation derived therefrom, and then determining the extent of binding between the complex and the candidate ligand, or determining ligand-induced electrical current across said cell or membrane.  
     
     
         16 . A human NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins, in a form essentially free from other proteins of human origin.  
     
     
         17 . A functional fragment of an NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins.  
     
     
         18 . An antibody which binds a human NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins.  
     
     
         19 . An immunogenic fragment of a human NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins.  
     
     
         20 . An oligonucleotide comprising at least about 17 nucleic acids which hybridizes with a polynucleotide encoding an NR3 protein selected from the group consisting of the NR3-1 and NRe-2 proteins.