The goal of this proposal is to examine the molecular mechanism by which the Ca2+ -binding protein, calmodulin (CaM) and GTP interact and regulate dopamine (DA) -receptor coupled adenylate cyclase activity. We will continue to examine the contributions of these agents to the supersensitivity of DA receptors (mainly D1 subtype) after chronic treatment with psychoactive drugs. We have shown a potentiative interaction between CaM, DA and GTP in rat and bovine striatum. CaM was 3-10 times more potent in potentiating DA- and GTP-stimulated adenylate cyclase activities than in stimulating basal activity. After chronic treatment with haloperidol, rat striatal adenylate cyclase is more responsive to DA, CaM and guanine nucleotides. Thus CaM may be important in regulating DA-stimulated adenylate cyclase in the brain and its activities are altered by chronic treatment with psychoactive drugs. The first specific aim is to investigate the mechanism by which CaM, Da and GTP interact to activate adenylate cyclase activity in brain. Using bovine striatum, we will examine whether GTP and DA increase the binding of CaM to the catalytic subunit, C or other CaM-binding proteins using a photoaffinity labeled derivative of CaM. The ability of CaM to stabilize or promote coupling between the stimulatory GTP-binding protein, N-S and C will be examined using ultracentrifugation and protection against inactivation. Activation of C by CaM and N-S will be directly studied using purified soluble components and reconstitution into liposomes. The effect of CaM on D1 receptor binding and coupling will be examined.
The second aim i s to investigate whether there are selective alterations in DA receptor levels, coupling of DA receptors to GTP- and CaM-related components or their potentiative interactions in rat striatum and mesolimbic areas after chronic treatment with the psychoactive drugs: a) haloperidol which blocks D1 and D2 receptors; b) sulpiride, D2 selective; c) SCH23390, D1 selective, and d) amphetamine which releases DA and down-regulates receptors. We want to examine whether CaM-mediated interactions can be selectively regulated through chronic inhibition or activation of D1 or D2 receptors. These studies will further delineate the intramembrane role of GTP, Ca2+ and CaM in modulating normal and altered DA receptor activity which may be important in the etiology of Parkinsons disease, tardive dyskinesias, amphetamine psychosis, and schizophrenia.
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