The process by which dopamine (DA) receptors are desensitized is important not only for understanding the regulatory events of DA action on its receptors, but also for determining the mechanisms underlying the progressively diminished response to agonist-based therapies seen in neurological diseases, such as Parkinson's disease. Such desensitization studies may also be useful in alleviation of harmful side- effects of current therapeutic procedures. The desensitization of human D-1 DA receptors in SK-N-MC neuroblastoma cells is homologous, yet the D-1 receptor is phosphorylated by both protein kinase A (PKA) and receptor-specific kinase. Desensitization of D-1 DA receptors results in rapid loss of adenylyl cyclase activity, with a slower loss in receptor binding sites. At the molecular level, D-1 receptor mRNA transcription is bimodal: a 60 percent increase in mRNA with 2 hours is followed by a 50 percent decrease, relative to control receptor mRNA levels. The mechanisms regulating the homologous, yet PKA- dependent desensitization, of these receptors will be studied in this grant at both the protein and mRNA level and their overall impact on neuronal function will be ascertained using SK-N-MC cells and GH4C1 cells expressing the cloned human D-1 and D-5 DA receptor. The function of putative D-1 receptor-specific kinase will also be analyzed in detail. The effects of DA on D-1 mRNA transcription will be studied, by analyzing both the 5' promoter regions and by measurements of mRNA stability, during the stages of up- and down-regulation of receptor message. Using nuclear run-on studies, the rate of transcription of receptor mRNA will be analyzed, during the up-and down-regualtion cycles. The requirements for receptor occupancy by agonist and functional Gs-receptor couplings will be examined in detail. The effects of inhibitory D-4 DA receptors in modulating the actions of D-1 receptors in SK-N-MC cells will be studied. Regulatory and transcription factors such as CREB and immediate early genes, will be analyzed and their role in promoting receptor activation and increases in total cellular mRNA, by DA, will be determined. These studies will help elucidate the role of DA on neuronal plasticity. In addition to biochemical analyses, electrophysiological studies will be conducted to analyze the role of DA and D-1/D-5 receptors on long term potentiation and neurite growth, especially after desensitization of receptors. The information obtained after tetanic stimulation of cells will be correlated with the results obtained from biochemical studies, and will provide unique insight to the DA-mediated processes underlying neuronal cell firing.
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