RGSZ1 and GAIP Regulate μ- but Not δ-Opioid Receptors in Mouse CNS: Role in Tachyphylaxis and Acute Tolerance

Abstract

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.

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

“…The blots were probed with affinity purified rabbit IgGs against: RGSZ1 and RGSZ2 (Garzón et al, 2004(Garzón et al, , 2005a, Mu-and Delta-opioid receptors (Garzón et al, 2005d); Gaz Helical domain (111-125) TGPAESKGEITPELL, Gaz C-Terminus domain (346-355) QNNLKYIGLC, Gai2 Helical domain (115-125) EEQGMLPEDLS (Sánchez-Blázquez et al, 1995;Garzón et al, 1997). Other antibodies used included: the mouse monoclonal antibody (IgM) to detect phosphoserines (Calbiochem, clone 1C8 525281); anti-GAIP C-terminus (goat) (sc-6207); anti-ubiquitin (Biomol, UG9510) (rabbit); anti-SUMO1 (Cell-Signaling, 4972) (rabbit); anti-SUMO2 C-terminus (sc-26973) (goat); anti-SUMO2/3 N-terminus (sc-26969) (goat).…”

“…injected 24 h later into morphine-primed mice. Analgesia was then determined by the tail-flick test at various post-morphine intervals (further details as in Garzón et al, 2004Garzón et al, , 2005a.…”

“…Neural RGS-Rz proteins isolated from synaptosomal membranes display different sizes (Garzón et al, 2004(Garzón et al, , 2005a and while this may be due to splicing of the RGSZ1 gene (Barker et al, 2001), RGSZ2 does not appear to be subjected to such events (Mao et al, 2004). Thus, the different isoforms of these RGS-Rz proteins are more likely generated by post-translational modifications such as glycosylation.…”

“…In fact, most of these protein isoforms can be found in the glycoprotein pool of neural membrane proteins and the action of N-and O-glycosidases decrease their apparent molecular weight. Nevertheless, glycosidase action does not generate a homogeneous population and a ladder pattern is still observed (Garzón et al, 2004(Garzón et al, , 2005a. Therefore, RGS-Rz proteins may be subjected to other modifications in nervous tissue that also accounts for their diversity of size, and that probably influence their biological activity.…”

“…Indeed, RGSZ1 and RGSZ2 proteins co-precipitate with Mu-opioid receptors in mouse brain and they antagonize effectors by retaining Mu-opioid receptor activated Gai2 and Gaz subunits. This action plays an important role in promoting opioid desensitization (Garzón et al, 2004(Garzón et al, , 2005a). This phenomenon is not consistent with their GAP activity and could be promoted by interactions with other proteins that consequently impair this activity.…”