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J. Biol. Chem., Vol. 280, Issue 10, 8951-8960, March 11, 2005

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

ROLE IN RECEPTOR DESENSITIZATION^*

Javier Garzón, María Rodríguez-Muñoz, Almudena López-Fando, and Pilar Sánchez-Blázquez

From the Department of Neuropharmacology, Cajal Institute, Consejo Superior de Investigaciones Científicas, E-28002 Madrid, Spain

In mouse periaqueductal gray matter (PAG) membranes, the µ-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 G₅. 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 co-precipitation 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-[³⁵S]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.

Received for publication, June 23, 2004 , and in revised form, January 3, 2005.

^* This work was supported by Ministerio de Ciencia y Tecnología Grants SAF2003-01121 and BMC2002-03228 and Comunidad Autonoma de Madrid Grant 08.8/0003/2003-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Neurofarmacología, Inst. Cajal, CSIC, Avd. Dr. Arce 37, E-28002 Madrid, Spain. Tel.: 34-91-585-4733; Fax: 34-91-585-4754; E-mail: jgarzon{at}cajal.csic.es.

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