Anti-RGS8 monoclonal antibody Made to order

Regulators of G protein signaling (RGS) proteins bind to the alpha subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among G alpha, G beta gamma, and their effecters. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein-coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing alpha 1B, alpha 2, and beta 3 Ca channel subunits and m2 receptors, carbachol (1 mu M) inhibited whole-cell currents by similar to 80% compared with only similar to 55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose-response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being similar to 18 nM in control cells vs. similar to 150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some G alpha subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein-modulated Ca channels.

Of all partners involved in G-protein coupled receptor (GPCR) signalling, the regulator of G-protein signalling (RGS) proteins are the only ones showing fast gene expression changes after various stimuli. These expression changes can offer feedback regulation to GPCR signalling as RGS accelerate the return of G-proteins to their inactive form and exert regulatory functions on intracellular effectors. However, it is not yet known which RGS regulate which receptor transduction pathways in the brain. To start to answer this question, we studied the influence of specific agonists and antagonists of the dopamine D1 and D2 receptors on the gene expression of the five most abundant RGS in the striatum: RGS2, RGS4, RGS8, RGS9 and RGS10. Only changes in RGS2 and RGS4 mRNA levels were observed. The D1 agonist SKF82958 and D2 antagonist haloperidol caused an up-regulation of RGS2 (+38.0% and +41.6%, respectively). The D1 antagonist SCH23390 and D2 agonist quinpirole caused a down-regulation of RGS2 (-25.0% and -35.0%) and an up-regulation of RGS4 (+57.2% and +52.5%). D1 and D2 receptors exert opposite effects on RGS2 expression, as they do on cAMP levels, suggesting a cAMP-mediated transcription of RGS2. This was confirmed by the unique induction of RGS2 (+111.1%) observed in the periventricular zone of the striatum after intracerebroventricular injection of forskolin. RGS4 was up-regulated only when RGS2 was down-regulated. This suggests that both RGS exert distinct functions. Considering the coupling of D1 and D2 receptors to the intracellular effector adenylate cyclase 5 (AC5) through their respective Galpha subunits in the striatum, our data allow us to suggest that RGS2 regulates the D1/Galphaolf/AC5 pathway and RGS4 the D2/Galphao/AC5 pathway.