In the CNS, the regulators of G-protein signaling (RGS) proteins belonging to the Rz subfamily, RGS19 (Ga interacting protein (GAIP)) and RGS20 (Z1), control the activity of opioid agonists at m but not at d receptors. Rz proteins show high selectivity in deactivating Gaz-GTP subunits. After reducing the expression of RGSZ1 with antisense oligodeoxynucleotides ( Knockdown of GAIP and of the GAIP interacting protein C-terminus (GIPC) led to changes in agonist effects at m but not at d receptors. The impairment of RGSZ1 extended the duration of morphine analgesia by at least 1 h beyond that observed in control animals. CTOP (Cys 2 , Tyr 3 , Orn 5 , Pen 7 -amide) antagonized morphine analgesia when given during the period in which the effect of morphine was enhanced by RGSZ1 knockdown. Thus, in naive mice, morphine tachyphylaxis originated in the presence of the opioid agonist and during the analgesia time course. The knockdown of RGSZ1 facilitated the development of tolerance to a single dose of morphine and accelerated tolerance to continuous delivery of the opioid. These results indicate that m but not d receptors are linked to Rz regulation. The m receptor-mediated activation of Gz proteins is effective at recruiting the adaptive mechanisms leading to the development of opioid desensitization.
The regulator of G-protein signaling RGS17(Z2) is a member of the RGS-Rz subfamily of GTPase-activating proteins (GAP) that efficiently deactivate GazGTP subunits. We have found that in the central nervous system (CNS), the levels of RGSZ2 mRNA and protein are elevated in the hypothalamus, midbrain, and pons-medulla, and that RGSZ2 is glycosylated in synaptosomal membranes isolated from CNS tissue. In analyzing the function of RGSZ2 in the CNS, we found that when the expression of RGSZ2 was impaired, the antinociceptive response to morphine and [D-Ala 2 , N-MePhe 4 , Gly-ol 5 ]-enkephalin (DAMGO) augmented. This potentiation involved m-opioid receptors and increased tolerance to further doses of these agonists administered 24 h later. High doses of morphine promoted agonist desensitization even within the analgesia time-course, a phenomenon that appears to be related to the great capacity of morphine to activate Gz proteins. In contrast, the knockdown of RGSZ2 proteins did not affect the activity of d receptor agonists, [D-Pen 2,5 ]-enkephalin (DPDPE), and [D-Ala 2 ] deltorphin II. In membranes from periaqueductal gray matter (PAG), both RGSZ2 and the related RGS20(Z1) co-precipitated with m-opioid receptors. While a morphine challenge reduced the association of Gi/o/z with m receptors, it increased their association with the RGSZ2 and RGSZ1 proteins. However, only Gaz subunits co-precipitated with RGSZ2. Doses of morphine that produced acute tolerance maintained the association of Ga subunits with RGSZ proteins even after the analgesic effects had ceased. These results indicate that both RGSZ1 and RGSZ2 proteins influence m receptor signaling by sequestering Ga subunits, therefore behaving as effector antagonists.
This paper reports that regulators of G‐protein signalling (RGS) proteins modulate the timing and amplitude of opioid signals by a push–pull mechanism. This is achieved without noticeable changes in the binding properties of opioids, e.g. β‐endorphin to mu‐opioid receptors. The expression of RGS proteins was reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Knock down of RGS2 or RGS3 diminished morphine and β‐endorphin analgesia, whereas that of RGS9 or RGS12 enhanced this activity. In mice with impaired RGS9, but not impaired RGS2, the potency and, in particular, the duration of opioid antinociception increased. Further, the animals did not exhibit acute tolerance generated by a single and efficacious dose of morphine, nor did they develop tolerance after a daily i.c.v. injection of the opioid for 4 days. In a model of sustained morphine treatment, the impairment of RGS9 proteins facilitated increases in the response to the delivered opioid. This was only effective for 2–3 h after the subcutaneous implantation of an oily morphine pellet; later, tolerance developed. To reduce the impact of the chronic morphine acting on opioid receptors, other RGS proteins presumably substitute the GTPase‐activating function of RGS9 on morphine‐activated Gα‐GTP subunits. The desensitization of mu‐opioid receptors appears to be a cell membrane‐limited process facilitated by RGS9′s sequestering of agonist‐segregated Gα subunits.
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.
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