We propose to study the molecular mechanism which regulates the interaction of psychoactive drugs (butyrophenones and phenothiazines) and neurotransmitters (dopamine) with components of the brain adenylate cyclase complex. We will investigate the role of sulfhydryl groups in the functional coupling of the components of the dopamine receptor with the guanine nucleotide binding protein (G) and the cyclase catalytic unit (C) in purified membranes and in a soluble extract from brain. We will establish and characterize the inactivation of the dopamine receptor and adenylate cyclase following pretreatment of membranes with alkylating agents such as DTNB and evaluate the role of guanine nucleotides and G in the reactivation process. Experiments are proposed that will identify the location of the reactive sulfhydryl group in the dopamine receptorcyclase complex and assess its chemical reactivity. We will develop and assay procedure for identifying soluble D1 and D2 receptor binding sites and establish a reconstitution protocol with soluble G-C and with S49 lymphoma cell membranes that are deficient in G protein (cyc-). This will enable us to assess the functional activity of the D1 and D2 dopamine receptor binding sites. In addition, studies are proposed to purify D1 and D2 sites from mammalian brain. Serum and monoclonal antibodies to each of these two sites will be prepared. Finally, radioimmunoassay procedures will be developed to study the cellular and subcellular location of these sites. Overall, the results from these studies should reveal important information about the molecular make-up and regulation of the dopamine receptor in mammalian brain and the mechanism of action of antipsychotic drugs. We believe this information will be useful for the design of novel psychotherapeutic drugs for the treatment of mental disease.
|Lingham, R B; Gottesfeld, Z (1986) Deafferentation elicits increased dopamine-sensitive adenylate cyclase and receptor binding in the olfactory tubercle. J Neurosci 6:2208-14|