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.

ABSTRACT: 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-MOR10-70 ). Activation of MOR with [D-Ala(2) , N-Me-Phe(4) , Gly(5) -ol] enkephalin (DAMGO) during immunoprecipitation caused a 150% increase in Go? and a 50% increase in RGS4 in the pellet. When 10 ?M 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) (GTP?S) increased the amount of co-precipitating RGS4 by 93% (compared to DAMGO alone, p = 0.008), and the inclusion of GTP?S caused the ratio of MOR to RGS4 to be 1 : 1 (31 fmoles : 28 fmoles, respectively). GTP?S also increased the association of endogenous RGS4 with MOR. In His6 RGS4/Ni(2+) -NTA agarose pull down experiments, 0.3 ?M GTP?S tripled the binding of Go? to His6 RGS4, whereas the addition of 100 ?M GDP blocked this effect. Importantly, activation of solubilized MOR with DAMGO in the presence of 100 ?M GDP and 0.3 ?M GTP?S increased Go? binding to His6 RGS4/Ni(2+) -NTA agarose (p = 0.001). 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 (?o??). RGS4 then binds to the activated ?o-GTP/MOR complex and accelerates the intrinsic GTPase of ?o. After ?o dissociates from MOR, RGS4 remains bound to the C-terminal region of MOR.

SUBMITTER: Santhappan R 

PROVIDER: S-EPMC5034817 | biostudies-other | 2015 Oct

REPOSITORIES: biostudies-other