Defective dopamine (DA) neurotransmission has been implicated in neurodegenerative diseases such as Parkinson's disease (PD) and in other neuropathological conditions such as hyperprolactemia, hypertension, schizophrenia and drug addiction. The long term objective of this laboratory is to identify specific functional roles of DA and its receptors in neurotransmission, in the normal and diseased human brain. The applicants hypothesize that different cellular systems contain DA receptors which interact differentially with different effectors of different signal transducing systems, resulting in activation of diverse signalling responses. To this end, they have examined the stimulatory D-1 DA receptors at the pharmacological, physiological and molecular levels, in different cellular systems: GH4C1 transfected with human D-1 cDNA, human SK-N-MC neuroblastoma cells and rat striata. Progress to date has verified the validity of this hypothesis. In this grant proposal, the applicants will extend their studies to include the novel and genetically distinct DA receptor, D-5, which is similar to and yet different form D-1 receptors. Using transfected GH4C1 cells, they will analyze and compare the molecular properties of D-1 and D-5 receptors through biochemical and physiological studies. D-5 receptors will be solubilized and studied using methods developed for D-1 receptors. The importance of the redox state of D-5 receptors and its sensitivity to oxidizing/reducing agents will be analyzed, as the applicants have described for D-1 receptors. Using specific anti-G protein antibodies, purified G proteins and by developing """"""""knock-out"""""""" cells in which G protein expression is selectively suppressed with the use of antisense oligonucleotides, they will identify the different G proteins which couple to D-1 (G0?) and D-5 (G2?) receptors. The novel mechanism by which D-5 receptors inhibit phosphoinositide metabolism will be characterized in detail. They will identify the G proteins and the protein kinases which may be involved in this inhibition, using specific antibodies and inhibitors of possible candidate components. The ability of D-1 receptors to down-regulate Gs-alpha at both the protein and mRNA levels will be further investigated in detail, since this has direct therapeutic significance in the agonist-based therapies of PD. From molecular studies, they will assess the effect of DA in altering the stability and rate of synthesis of Gs-alpha mRNA in SK-N-MC cells. Studies will also be conducted to analyze the processes which can reverse the down-regulation of Gs-alpha by DA. These systematic studies may enable an elucidation of the mechanisms of D-1 and D-5 DA receptor activation and function in the brain.
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