Mu opioid receptor activation enhances regulator of G protein signaling 4 association with the mu opioid receptor/G protein complex in a GTP‐dependent manner

The interaction of Regulator of G protein Signaling 4 (RGS4) with the rat mu opioid receptor (MOR)/G protein complex was investigated. Solubilized MOR from rat brain membranes was immunoprecipitated in the presence of RGS4 with antibodies against the N‐terminus of MOR (anti‐MOR(10–70)). Activation of MOR with [D‐Ala(2), N‐Me‐Phe(4), Gly(5)‐ol] enkephalin (DAMGO) during immunoprecipitation caused a 150% increase in Goα and a 50% increase in RGS4 in the pellet. When 10 μM GTP was included with DAMGO, there was an additional 72% increase in RGS4 co‐immunoprecipitating with MOR (p = 0.003). Guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) increased the amount of co‐precipitating RGS4 by 93% (compared to DAMGO alone, p = 0.008), and the inclusion of GTPγS caused the ratio of MOR to RGS4 to be 1 : 1 (31 fmoles : 28 fmoles, respectively). GTPγS also increased the association of endogenous RGS4 with MOR. In His(6)RGS4/Ni(2+)‐NTA agarose pull down experiments, 0.3 μM GTPγS tripled the binding of Goα to His(6)RGS4, whereas the addition of 100 μM GDP blocked this effect. Importantly, activation of solubilized MOR with DAMGO in the presence of 100 μM GDP and 0.3 μM GTPγS increased Goα binding to His(6)RGS4/Ni(2+)‐NTA agarose (p = 0.001). [Image: see text]Regulators of G protein Signaling (RGS) shorten the time that G proteins are active. Activation of the mu opioid receptor (MOR) causes GTP to bind to and to activate Go (αoβγ). RGS4 then binds to the activated αo‐GTP/MOR complex and accelerates the intrinsic GTPase of αo. After αo dissociates from MOR, RGS4 remains bound to the C‐terminal region of MOR.