A Structural/Behavioral Analysis of the Regulation of Dopamine Signaling by Striatal RGS Proteins
Waugh, Jeffrey Lynn
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The regulators of G-protein signaling (RGS) proteins negatively modulate heterotrimeric G protein signaling by acting as GTPase activating proteins for Galpha subunits. In the striatum and nucleus accumbens, brain regions critical for control of movement, motivation and reward, overlapping RGS expression profiles suggested that functional specificity could not be explained by anatomical localization alone. We set out to assess striatal specificity within two distinct RGS pools, the R7 RGS subfamily and RGS10. The highly striatal-specific splice form RGS9-2 is a negative modulator of dopamine D2 receptor signaling, and has been shown to inhibit drug stimulated (cocaine or direct dopamine receptor agonists) locomotor activity. RGS9-2 is a member of the R7 subfamily, comprised of RGS6, -7, -9, and -11, which share highly similar subdomain structure. We analyzed the specificity of R7 modulation of dopamine receptor signaling using a novel behavioral assay. R7 RGS proteins were virally-overexpressed in rat or mouse accumbens via a stereotaxic injection of an engineered HSV virus. Following this surgery, drug-stimulated locomotor responses were assayed. We found that in rats and RGS9 KO mice, overexpression of R7 RGS proteins produces distinct locomotor and drug sensitization phenotypes, each of which occurs only during the period of RGS overexpression. Moreover, studies using truncation and chimeric RGS mutants demonstrated that while all tested subdomains were necessary for activity, only the C-terminus of RGS9-2 was sufficient to convey activity to RGS7. Lastly, RGS overexpression leads to distinct acute changes in weight: loss (RGS9-2) or gain (RGS7, RGS11). To elucidate RGS10 function in the brain, we mapped RGS10 protein in rodent brain using light microscopic and electron microscopic immunohistochemical techniques. Light microscopic analyses showed that RGS10 immunoreactivity labels all subcompartments of neurons and microglia, including their nuclei. Electron microscopy confirmed the presence of dense RGS10 immunoreactivity in euchromatin and resolved dense staining on terminals at symmetric synapses onto pyramidal cell somata. Dual-labeling histochemistry showed that RGS10 is expressed in specific neuronal cell types and circuits. Taken together, these data support a role for RGS10 in diverse processes including modulation of pre- and postsynaptic G-protein signaling and a potential role in modulating gene expression.