The long term objectives of this research are to understand the roles of brain dopamine neurotransmitter receptors in the etiology and treatment of severe psychiatric and neurological disorders. It is specifically proposed to clone and characterize the rat and human D2 receptor genes. As a first step in this process a mouse cell line has been transfected with rat genomic DNA (linked to 0X174 DNA fragments) and selected for stable expression of D2-like receptors. A genomic DNA library will be constructed from this cell line and recombinant clones screened for 0X174 sequences. Positive clones will be screened for the ability to express D2 receptors on retransfection and for homology with conserved regions of the G-protein coupled receptor family. Using detailed restriction enzyme mapping, DNA sequencing, and transfection/expression assays, the structure of the rat D2 gene will be determined. The human D2 gene will be isolated from a human genomic DNA library by homology with the rat gene. The chromosomal location of the human gene will be determined by in situ hybridization to metaphase chromosomes. The presence and frequency of restriction fragment length polymorphisms will be defined in a control population. These studies will provide significant information on the protein structure, genomic organization, and regulation of D2 receptors. In addition, these studies will provide the tools for a variety of future studies including 1) linkage analysis of D2 receptors in familial forms of disorders such as schizophrenia, 2) site-directed mutagenesis studies of D2 expression at the translational and transcriptional level, and 3) structure-function studies of D2 receptor interactions with drugs and coupling proteins.
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