THESIS
2014
xii, 134 pages : illustrations (some color) ; 30 cm
Abstract
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...[
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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 Gβ
1γ
2 from the
endogenous Gα
i3 and AGS3 did not induce Gβγ-mediated PLCβ2 activation. By using fusion
proteins of Gβ
1 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.
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