Preferential Assembly of G- Complexes Directed by the g Subunits - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 176

lipid modification of the g component (Higgins and Casey 1996 ) , knockdown of the
g subunit would be expected to have functional consequences by affecting the
membrane localization or cellular levels of its a and b partners. Providing the first
experimental evidence for this hypothesis, an anti-antisense approach revealed a
requirement for the g 3 and g 4 subunits in somatostatin and muscarinic receptor
signaling, respectively, in rat pituitary cells (Kleuss et al. 1993 ) . However, these
results were limited by the unknown selectivity of the anti-sense oligonucleotides
and the inability to show knockdown of the targeted proteins. Subsequently, a
ribozyme approach demonstrated a role for the g 7 subunit in the assembly of the
G-a s b 1 g 7 combination that is required for b-adrenergic receptor signaling in human
HEK293 cells (Wang et al. 1997, 1999 ). However, these results were questioned
based on the small signaling defect relative to the marked reduction of the targeted
protein. Finally, and perhaps most compelling, a gene targeting approach identified
a critical role for the g 7 subunit in driving the assembly of a G-a olf b 2 g 7 complex
required for D 1 dopamine and A 2a adenosine receptor signaling in striatum
(Schwindinger et al. 2003, 2010 ). This requirement for the g 7 subunit was first
revealed by quantitative immunblot analysis (Schwindinger et al. 2003 ) and is now
confirmed by immunostaining of coronal slices prepared from control and Gng7 −/−
knockout brains. As shown in Fig. 10.1 , control slices exhibit prominent a olf stain-
ing that is confined to striatum and is mostly associated with neuropil (Panel a, left
side), consistent with its reported localization in the dendritic processes arising from
striatal neurons (Corvol et al. 2004 ). In stark contrast, G-g 7 knockout slices show
nearly complete loss of a olf staining (Panel a, right side), in agreement with previous
immunoblot results (Schwindinger et al. 2003 ) . Attesting to speci fi city, other
G-protein a subtypes are not suppressed in knockout brains (Schwindinger et al.
2003, 2010 ). Furthermore, other signaling components in the pathway are not
reduced in knockout brains, ruling out a developmental problem. As shown in
Fig. 10.1 , control and G-g 7 knockout slices exhibit comparable staining of the dop-
amine and adenosine regulated phosphoprotein-32 kD (DARPP-32) that is localized
to striatum and is associated with both the cell soma and underlying neuropil (Rajput
et al. 2009 ). Thus, the major effects of g 7 knockout are the specific and nearly stoi-
chiometric suppression of the b 2 protein (Schwindinger et al. 2003, 2010 ) , along
with the a olf protein shown above.
At this stage, the mechanism responsible for coordinating the assembly of this
striatal-speci fi c G- a olf b 2 g 7 heterotrimer is still speculative. Since reductions in b 2
and a olf protein levels occur despite normal levels of their mRNA transcripts in
Gng7 −/− knockout brains (Schwindinger et al. 2010 ) , a post-translational require-
ment for the g 7 subunit is the most likely explanation. This requirement may reflect
a direct role for the g 7 subunit in blocking sites on the b 2 and a olf proteins susceptible
to ubiquitination and proteolysis, or an indirect role in trafficking the G-a olf b 2 g 7
trimer away from the proteolytic machinery to the plasma membrane (Wang et al.
1999 ). Providing additional support for these possibilities, a more recent study
shows that loss of the g t1 subunit leads to mislocalization and proteolysis of the a t1
subunit (Lobanova et al. 2008 ) . Collectively, these in vivo studies identify the
assembly of distinct G-a b g heterotrimers that is controlled by their respective g
subunits at the post-translational level.