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The complex of G protein regulator RGS9-2 and G?(5) controls sensitization and signaling kinetics of type 5 adenylyl cyclase in the striatum.

ABSTRACT: Multiple neurotransmitter systems in the striatum converge to regulate the excitability of striatal neurons by activating several heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that signal to the type 5 adenylyl cyclase (AC5), the key effector enzyme that produces the intracellular second messenger cyclic adenosine monophosphate (cAMP). Plasticity of cAMP signaling in the striatum is thought to play an essential role in the development of drug addiction. We showed that the complex of the ninth regulator of G protein signaling (RGS9-2) with the G protein ? subunit (G?(5)) critically controlled signaling from dopamine and opioid GPCRs to AC5 in the striatum. RGS9-2/G?(5) directly interacted with and suppressed the basal activity of AC5. In addition, the RGS9-2/G?(5) complex attenuated the stimulatory action of G?? on AC5 by facilitating the GTPase (guanosine triphosphatase) activity of G?(o), thus promoting the formation of the inactive heterotrimer and inhibiting G??. Furthermore, by increasing the deactivation rate of G?(i), RGS9-2/G?(5) facilitated the recovery of AC5 from inhibition. Mice lacking RGS9 showed increased cAMP production and, upon withdrawal from opioid administration, enhanced sensitization of AC5. Our findings establish RGS9-2/G?(5) complexes as regulators of three key aspects of cAMP signaling: basal activity, sensitization, and temporal kinetics of AC5, thus highlighting the role of this complex in regulating both inhibitory and stimulatory GPCRs that shape cAMP signaling in the striatum.

SUBMITTER: Xie K

PROVIDER: S-EPMC3580943 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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The complex of G protein regulator RGS9-2 and Gβ(5) controls sensitization and signaling kinetics of type 5 adenylyl cyclase in the striatum.

Xie Keqiang K Masuho Ikuo I Brand Cameron C Dessauer Carmen W CW Martemyanov Kirill A KA

Science signaling 20120828 239

Multiple neurotransmitter systems in the striatum converge to regulate the excitability of striatal neurons by activating several heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that signal to the type 5 adenylyl cyclase (AC5), the key effector enzyme that produces the intracellular second messenger cyclic adenosine monophosphate (cAMP). Plasticity of cAMP signaling in the striatum is thought to play an essential role in the development of drug addiction. ...[more]

PMID: 22932702

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Project description:Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of Gαi/o-linked receptors including D2 dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e. CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Dataset Information

The complex of G protein regulator RGS9-2 and G?(5) controls sensitization and signaling kinetics of type 5 adenylyl cyclase in the striatum.

Publications

The complex of G protein regulator RGS9-2 and Gβ(5) controls sensitization and signaling kinetics of type 5 adenylyl cyclase in the striatum.

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