Activation of μ-Opioid Receptors Transfers Control of Gα Subunits to the Regulator of G-protein Signaling RGS9-2

Abstract

In mouse periaqueductal gray matter (PAG) membranes, the -opioid receptor (MOR) coprecipitated the ␣-subunits of the G i/o/z/q/11 proteins, the G␤ 1/2 subunits, and the regulator of G-protein signaling RGS9-2 and its partner protein G␤ 5 . RGS7 and RGS11 present in this neural structure showed no association with MOR. In vivo intracerebroventricular injection of morphine did not alter MOR immunoreactivity, but 30 min and 3 h after administration, the coprecipitation of G␣ subunits with MORs was reduced by up to 50%. Furthermore, the association between G␣ subunits and RGS9-2 proteins was increased. Twenty-four hours after receiving intracerebroventricular morphine, the G␣ subunits left the RGS9-2 proteins and re-associated with the MORs. However, doses of the opioid able to induce tolerance promoted the stable transfer of G␣ subunits to the RGS9-2 control. This was accompanied by Ser phosphorylation of RGS9-2 proteins, which increased their coprecipitation with 14-3-3 proteins. In the PAG membranes of morphine-desensitized mice, the capacity of the opioid to stimulate G-protein-related guanosine 5- O-(3-[35 S]thiotriphosphate) binding as well as low K m GTPase activity was attenuated. The in vivo knockdown of RGS9-2 expression prevented morphine from altering the association between MORs and G-proteins, and tolerance did not develop. In PAG membranes from RGS9-2 knockdown mice, morphine showed full capacity to activate G-proteins. Thus, the tolerance that develops following an adequate dose of morphine is caused by the stabilization and retention of MOR-activated G␣ subunits by RGS9-2 proteins. This multistep process is initiated by the morphine-induced transfer of MOR-associated G␣ subunits to the RGS9-2 proteins, followed by Ser phosphorylation of the latter and their binding to 14-3-3 proteins. This regulatory mechanism probably precedes the loss of MORs from the cell membrane, which has been observed with other opioid agonists.

Sumoylated RGS-Rz Proteins Act as Scaffolds for Mu-Opioid Receptors and G-Protein Complexes in Mouse Brain

“…Male albino CD-1 mice (Charles River, Barcelona, Spain) weighing 22-25 g were used, from which PAG membranes enriched in synaptosomes were prepared as described previously (Garzón et al, 2005b). Briefly, 2 mm-thick coronal sections of the PAG from six mice were pooled in ice-cold 25 mM Tris HCl (pH 7.7), 1 mM EGTA, 0.32 M sucrose, protease inhibitor cocktail (Sigma, Madrid, Spain, #P8340), phosphatase inhibitor mixture (Sigma, #P2850) and H89 (Sigma, #B1427).…”

“…The procedure used to immunoprecipitate membrane proteins from the solubilized P2 fraction of mouse PAG has been described in detail (Garzón et al, 2005b). Briefly, for nondenaturing conditions about 1 mg protein from PAG membranes was sonicated in 50 mM Tris HCl (pH 7.7), 50 mM NaCl, 1% Nonidet P-40, in the presence of protease and phosphatase inhibitors, and H89.…”

Opioid Regulation of Mu Receptor Internalisation: Relevance to the Development of Tolerance and Dependence

“…RGS9-2 is particularly highly enriched in striatum and, although expressed at substantially lower levels in PAG and spinal cord, is present [45]. In vitro experiments performed with mouse PAG membranes showed that mu opioid receptor could be co-immunoprecipitated with α-subunits of G i/o/z/q/11 proteins, Gβ 1/2 subunits as well as both RGS9-2 and its partner protein Gβ 5 [46]. These interactions were modulated by morphine since 30 minutes and 3 hours after drug administration the coimmunoprecipitation of mu receptor-Gα subunit was reduced up to 50% whilst the interaction Gα-RGS9-2 was conversely increased.…”

Involvement of NOX1/NADPH Oxidase in Morphine-Induced Analgesia and Tolerance

“…The signaling pathways involved in the recruitment of PKC␥ to RGS proteins and MOR have been intensively studied , 2011. The physical interaction with activated PKC may easily phosphorylate RGS proteins and increase their binding to 14-3-3 proteins (Garzó n et al, 2005).…”

“…One of the mechanisms documented for uncoupling is the formation of a G␣, RGS9-2, and 14-3-3 proteins complex (Garzó n et al, 2005). The formation of this complex is accompanied by serine phosphorylation of RGS9-2.…”