Garzón Javier, Rodríguez-Muñoz María, López-Fando Almudena, Sánchez-Blázquez Pilar
Department of Neuropharmacology, Cajal Institute, Consejo Superior de Investigaciones Científicas, E-28002 Madrid, Spain.
J Biol Chem. 2005 Mar 11;280(10):8951-60. doi: 10.1074/jbc.M407005200. Epub 2005 Jan 4.
In mouse periaqueductal gray matter (PAG) membranes, the mu-opioid receptor (MOR) coprecipitated the alpha-subunits of the Gi/o/z/q/11 proteins, the Gbeta1/2 subunits, and the regulator of G-protein signaling RGS9-2 and its partner protein Gbeta5. 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 Galpha subunits with MORs was reduced by up to 50%. Furthermore, the association between Galpha subunits and RGS9-2 proteins was increased. Twenty-four hours after receiving intracerebroventricular morphine, the Galpha 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 Galpha 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 Km 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 Galpha subunits by RGS9-2 proteins. This multistep process is initiated by the morphine-induced transfer of MOR-associated Galpha 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.
在小鼠中脑导水管周围灰质（PAG）膜中，μ-阿片受体（MOR）与Gi/o/z/q/11蛋白的α亚基、Gβ1/2亚基以及G蛋白信号调节因子RGS9-2及其伴侣蛋白Gβ5共沉淀。存在于该神经结构中的RGS7和RGS11与MOR无关联。体内脑室内注射吗啡并未改变MOR的免疫反应性，但给药后30分钟和3小时，Gα亚基与MOR的共沉淀减少了多达50%。此外，Gα亚基与RGS9-2蛋白之间的关联增加。脑室内注射吗啡24小时后，Gα亚基离开RGS9-2蛋白并重新与MOR结合。然而，能够诱导耐受性的阿片类药物剂量促进了Gα亚基向RGS9-2调控的稳定转移。这伴随着RGS9-2蛋白的丝氨酸磷酸化，增加了它们与14-3-3蛋白的共沉淀。在吗啡脱敏小鼠的PAG膜中，阿片类药物刺激G蛋白相关的鸟苷5'-O-(3-[35S]硫代三磷酸)结合以及低Km GTP酶活性的能力减弱。体内敲低RGS9-2的表达可防止吗啡改变MOR与G蛋白之间的关联，且不会产生耐受性。在RGS9-2敲低小鼠的PAG膜中，吗啡显示出完全激活G蛋白的能力。因此，适量吗啡给药后产生的耐受性是由RGS9-2蛋白对MOR激活的Gα亚基的稳定和保留所引起的。这个多步骤过程由吗啡诱导的与MOR相关的Gα亚基向RGS9-2蛋白的转移引发，随后是后者的丝氨酸磷酸化及其与14-3-3蛋白的结合。这种调节机制可能先于细胞膜上MOR的丢失，而其他阿片类激动剂已观察到这种情况。
