A variety of neuropsychiatric diseases, including Parkinson's disease and schizophrenia, appear to involve dopamine or dopamine-sensitive neurons. Therapy of these and other diseases is based on the use of drugs that stimulate or inhibit dopamine receptors, and changes in the number or properties of dopamine receptors may complicate the use of these drugs. The overall aim of the experiments described in this proposal is to investigate factors involved in the regulation of dopamine receptors at a cellular and molecular level. There appear to be two major subtypes of dopamine receptors that can be distinguished both pharmacologically and in terms of the biochemical effector mechanisms that mediate their effects. The so-called D-1 receptor has a high affinity for antagonists such as SCH-23390. Dopamine, acting at D-1 receptors, stimulates the activity of adenylate cyclase. D-2 receptors selectively binding drugs such as domperidone and sulpiride. These receptors appear to function through inhibition of the activity of adenylate cyclase. D-1 and D-2 receptors are independently distributed and regulated in the mammalian central nervous system. Progress in understanding at a molecular level the regulation of dopamine receptors has been hindered by the need to use homogenates of heterogeneous tissues such as the basal ganglia. The present experiments will focus on the use of clonal lines of cultured cells that express the subtypes of dopamine receptors. The pituitary tumor 7315a contains D-2 receptors while some gliomas and neuroblastomas express D-1 receptors. Cells from these tumors are being or have been adapated to grow as maintained lines in tissue culture. Ligands with high specific activity labeled with 125I are particularly useful in studies of receptors of cultured cells in that tissue availability may be a limiting factor. 125I-SCH-23982 and an iodinated analog of raclopride (125I-IBZM) will be used to study D-1 and D-2 receptors, respectively. Studies of desensitization and receptor sequestration will be carried out using cultured cells that express D-1 and D-2 receptors. An additional major avenue of investigation will involve preparation of anti-receptor antibodies and anti-idiotypic antibodies that cross-react with dopamine receptors. Purified receptor proteins will be used to immunize rabbits and BALB/c mice. Monoclonal antibodies with a high affinity for spiroperidol have been produced. These antibodies will be purified and used to elicit monoclonal anti- idiotypic antibodies that will be tested for their ability to cross- react with D-2 receptors. Similar protocols will be developed that are designed to elicit anti-D-1-receptor antibodies. These antibodies will be used to determine the role of phosphorylation in desensitization of D-1 and D-2 receptors. An immunologic approach will also be used to determine the rates of synthesis of dopamine receptors in naive and desensitized cells.
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