The Gs and Gi pathways interact to control the levels of intracellular cAMP. Although coincident signaling through Gs and Gi-coupled receptors can attenuate Gs-stimulated cAMP levels, it is not known if prior activation of the Gi pathway can affect signaling by Gs-coupled receptors. We have found that activated Gαo/i interact with RGS20, a GTPase activating protein for members of the Gαο/i family. Interaction between Gαo/i and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Gαo and Gαi2 but not by Gαq, Gαi1 or Gαi3. The Gαo/i-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Gαo stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to Go/i reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that Go/i promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of β-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that Go/i-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the Gs and Gi pathways controls cellular cAMP levels, thus allowing for signal integration. © 2008.