Multiple subtypes of dopamine receptor have been identified. They can be classified as D1-like or D2-like on the basis of their pharmacological properties, deduced amino acid sequences, and coupling to second- messenger systems. The existence of these receptors provides new therapeutic targets that will he exploited once their specific distribution and properties have been defined. On the other hand, because of the similarities between D1 and D5 receptors and among D2, D3, and D4 receptors, the results of conventional assays of receptors using available radioligands must be interpreted with caution. Alternative, more specific approaches for investigating the distribution, properties, and regulation of dopamine receptors using specific and selective molecular and immunological probes are likely to be required. The experiments described in the present proposal involve the use of nuclease protection assays to measure mRNA levels and to investigate the effects of physiological and pharmacological manipulations on transcription. Several types of anti-dopamine receptor antibodies will be used in the proposed study. These will include site-specific antibodies to peptides, antibodies against fusion proteins expressed in bacteria, and monoclonal antibodies against receptors expressed in Sf9 cells infected with recombinant baculovirus. Antibodies will be used to demonstrate expression and distribution of receptor protein and to define the regulation of receptor levels. Some experiments will measure the effects of lesions and of pharmacological interventions on receptors and receptor message expressed in rat brain. Most of the proposed studies will be carried out using cultured cells including a cell line derived from pituitary tumor 7315a, called SUP1 cells, and with cell lines stably transfected to express specific subtypes of dopamine receptor. Agonist- mediated changes in mRNA and receptor expression have been observed in SUP1 cells and/or transfected 293 cells. Mechanisms underlying these changes will be investigated. Studies of mRNA synthesis and receptor turnover will be carried out. The possibility that post-transcriptional regulation of mRNA stability occurs will also be investigated. Anti- receptor and anti-G protein antibodies will also be used to investigate the coupling of subtypes of dopamine receptor to specific subtypes of GTP-binding proteins. An understanding of the factors that regulate the density, properties, and expression of dopamine receptors may contribute to the design of more selective therapeutic agents and may help to elucidate mechanisms underlying the side effects that are seen following chronic administration of drugs acting through stimulation or inhibition of subtypes of dopamine receptors.
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