Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) which stabilizes the Gα_i/o subunits as an AGS3/Gα_i/o-GDP complex and presumably prevent the re-association with Gβγ subunits, thus enhancing Gβγ-mediated signaling. Recent studies have suggested that AGS3 is a molecular gateway for drug tolerance and dependence. Increased levels of AGS3 expression in the nucleus accumbens are associated with withdrawal of chronic cocaine or morphine administration. However, the signaling mechanism of AGS3 in regulating drug addiction has yet to be elucidated. It is hypothesized that AGS3 may modulate G protein-coupled receptor (GPCR) signaling such as those mediated through the Gα_i/o-coupled opioid receptors. In my current study, I found that overexpre...[ Read more ]

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) which stabilizes the Gα_i/o subunits as an AGS3/Gα_i/o-GDP complex and presumably prevent the re-association with Gβγ subunits, thus enhancing Gβγ-mediated signaling. Recent studies have suggested that AGS3 is a molecular gateway for drug tolerance and dependence. Increased levels of AGS3 expression in the nucleus accumbens are associated with withdrawal of chronic cocaine or morphine administration. However, the signaling mechanism of AGS3 in regulating drug addiction has yet to be elucidated. It is hypothesized that AGS3 may modulate G protein-coupled receptor (GPCR) signaling such as those mediated through the Gα_i/o-coupled opioid receptors. In my current study, I found that overexpression of AGS3 in HEK293 cells stably expressing the mu opioid receptor (MOR) led to inhibition of c-Jun N-terminal kinase (JNK) phosphorylation. The extracellular signal-regulated kinase (ERK) phosphorylation was, however, unaffected. AGS3 was also found to exhibit differential modulation of cAMP production in cells overexpressing MOR or kappa opioid receptor (KOR). On the other hand, it has recently been demonstrated in reconstitution experiments that the AGS3/Gα_i/o-GDP complex may act as a substrate of resistance to inhibitors of cholinesterase 8A (Ric-8A), a guanine exchange factor (GEF) for heterotrimeric Gα proteins. Since the ability of Ric-8A to activate Gα_i/o subunits that are bound to AGS3 in a cellular environment has not been confirmed, I thus examined the effect of Ric-8A on cAMP accumulation in HEK293 cells expressing different forms of AGS3 and Gα_i3. Co-immunoprecipitation assays indicated that full length AGS3 and its N- and C-terminal truncated mutants can interact with Ric-8A in HEK293 cells. Yeast two-hybrid assay further confirmed that Ric-8A can directly bind to AGS3S, a short form of AGS3 which is endogenously expressed in heart. However, Ric-8A failed to facilitate Gα_i-induced suppression of adenylyl cyclase, suggesting that it may not serve as a GEF for AGS3/Gα_i/o-GDP complex in a cellular environment. Moreover, I have shown in the study that AGS3 did not dissociate Gβ₁γ₂ from the endogenous Gα_i3 and AGS3 did not induce Gβγ-mediated PLCβ2 activation. By using fusion proteins of Gβ₁ fused to Gα_i3, I have demonstrated that Gβγ release is not a prerequisite for AGS3 binding to Gα_i3-GDP. Contrary to the established view, my results suggest that AGS3 may form a larger complex with the Gαβγ trimeric complex for signal transduction.