The RGSZ2 Protein Exists in a Complex with μ-Opioid Receptors and Regulates the Desensitizing Capacity of Gz Proteins

Abstract

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.

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).…”

“…Equal loading was verified in parallel blots generated with the same immunoprecipitated samples (Garzón 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.…”