Prolonged activation of D2 dopamine receptors enhances subsequent drug-stimulated cyclic AMP accumulation.This heterologous sensitization of adenylate cyclase occurs following persistent activation of several Gai/o-coupled receptors, however, the precise mechanisms involved remain unknown. Previous studies support a hypothesis that persistent activation of a Gai/o-Coupled receptor promotes the dissociation of Ga and By'subunits in a pertussis toxinsensitive fashion that induces sensitization through a Gots-dependent mechanism. Recent evidence suggests that the pertussis toxin-dependent signaling events that modulate protein kinases may act directly or indirectly to regulate individual adenylate cyclases and play a role in heterologous sensitization. We propose to refine further this hypothetical model using experiments that will manipulate the expression of recombinant Got subunits and adenylate cyclase isoforms in characterized cell lines expressing recombinant D2 dopamine receptors. The first objective is to define the relative contributions of individual Gai/o and By subunits for D2 receptor-induced sensitization. This objective will be accomplished by expressing individual constitutively active Got subunits or pertussis toxin-resistant Ga subunits containing mutations that alter GTP hydrolysis. Additional studies will use recombinant proteins that modulate G protein signaling (GBy sequestering proteins and AGS proteins). These studies will use representative recombinant adenylate cyclases that show short-term sensitization. The second objective is to determine the role of protein kinases in sensitization using wild-type and mutant isoforms of adenylate cyclase. These studies will use selective adenylate cyclase activators in combination with site-directed mutagenesis to examine the kinase involvement and to identify potential cellular mechanisms for agonist-induced sensitization. The third objective will determine the absolute requirement of Gas and role of Gas-adenylate cyclase interactions in heterologous sensitization. These studies will use a newly discovered cellular model deficient in Gas signaling. The ability of wild-type and mutant Gas to rescue sensitization in that model will be explored. The isoform specificity and the role of Gas-adenylate cyclase interactions in heterologous sensitization will also be examined using recombinant adenylate cyclases that are deficient in Gas binding and using dominant negative mutants of Gas. The goals of the present studies are to examine and elucidate the biochemical pathways and mechanism(s) responsible for D2 receptorinduced heterologous sensitization. The data that we obtain are likely to increase our understanding of the ] pathophysiology of central nervous system disorders and may also lead to improved treatment strategies.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
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Molecular Neuropharmacology and Signaling Study Section (MNPS)
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Asanuma, Chiiko
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Purdue University
Schools of Pharmacy
West Lafayette
United States
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Brust, Tarsis F; Alongkronrusmee, Doungkamol; Soto-Velasquez, Monica et al. (2017) Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties. Sci Signal 10:
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