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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH060397-08
Application #
7325721
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Asanuma, Chiiko
Project Start
2000-05-10
Project End
2009-11-30
Budget Start
2007-12-01
Budget End
2008-11-30
Support Year
8
Fiscal Year
2008
Total Cost
$250,084
Indirect Cost
Name
Purdue University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
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:
Brust, Tarsis F; Hayes, Michael P; Roman, David L et al. (2015) New functional activity of aripiprazole revealed: Robust antagonism of D2 dopamine receptor-stimulated G?? signaling. Biochem Pharmacol 93:85-91
Brust, Tarsis F; Conley, Jason M; Watts, Val J (2015) G?(i/o)-coupled receptor-mediated sensitization of adenylyl cyclase: 40 years later. Eur J Pharmacol 763:223-32
Brust, Tarsis F; Hayes, Michael P; Roman, David L et al. (2015) Bias analyses of preclinical and clinical D2 dopamine ligands: studies with immediate and complex signaling pathways. J Pharmacol Exp Ther 352:480-93
Conley, Jason M; Brust, Tarsis F; Xu, Ruqiang et al. (2014) Drug-induced sensitization of adenylyl cyclase: assay streamlining and miniaturization for small molecule and siRNA screening applications. J Vis Exp :e51218
Staffend, Nancy A; Hedges, Valerie L; Chemel, Benjamin R et al. (2014) Cell-type specific increases in female hamster nucleus accumbens spine density following female sexual experience. Brain Struct Funct 219:2071-81
Conley, Jason M; Watts, Val J (2013) Differential effects of AGS3 expression on D(2L) dopamine receptor-mediated adenylyl cyclase signaling. Cell Mol Neurobiol 33:551-8
Brand, Cameron S; Hocker, Harrison J; Gorfe, Alemayehu A et al. (2013) Isoform selectivity of adenylyl cyclase inhibitors: characterization of known and novel compounds. J Pharmacol Exp Ther 347:265-75
Conley, Jason M; Brand, Cameron S; Bogard, Amy S et al. (2013) Development of a high-throughput screening paradigm for the discovery of small-molecule modulators of adenylyl cyclase: identification of an adenylyl cyclase 2 inhibitor. J Pharmacol Exp Ther 347:276-87
Ejendal, Karin F K; Conley, Jason M; Hu, Chang-Deng et al. (2013) Bimolecular fluorescence complementation analysis of G protein-coupled receptor dimerization in living cells. Methods Enzymol 521:259-79

Showing the most recent 10 out of 50 publications