Synthesis and Assembly of G Protein Dimers: Comparison of In Vitro and In Vivo Studies - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 164

Fig 9.4 Association of Gb isoforms with CCT subunits. FLAG-G b isoforms were synthesized
in rabbit reticulocyte lysate and immunoprecipitated with anti-FLAG antibody beads.
Immunoprecipitates were analyzed for the indicated CCT subunits by immunoblotting to obtain
the relative association of CCT subunit with each Gb. Modified from Wells et al. ( 2006 )
rized as follows: Gb1 = Gb4 > Gb2 > Gb3 > > Gb3 s > Gb5. The extent to which a
given Gb isoform binds CCT correlates well with the ability of that Gb to make
dimers (compare Figs. 9.2 and 9.4 ), as the Gb isoforms that best bind to CCT,
Gb 1, G b4, and Gb2, are the ones that are best able to make dimers, and make
dimers with all or (in the case of Gb2) most Gg isoforms. This correlation suggests
that the folding of Gb on CCT is associated with dimer formation.
The WD40 repeat 2 of Gb1 has been proposed to be the region of the protein that
is most important for the binding of Gb to CCT (Kubota et al. 2006 ) . This region of
the proteins is hydrophobic and very highly conserved in Gb 1, G b 2, G b3, and Gb 4,
but less so in Gb5. For actin and tubulin, the interaction with CCT is sequence-
specific and ionic, but for other proteins, a hydrophobic interaction may occur
(Brackley and Grantham 2009 ) . G b5 is the Gb isoform that is the most different
from the other isoforms. It shares only about 50% homology with other Gb isoforms,
while Gb1 thru 4 have 80% or more, and this may explain the weaker interaction of
Gb5 with CCT and also with PhLP1.
Additionally, there is evidence that PhLP1 also has a preference for some Gb
isoforms (Howlett et al. 2009 ). This may in fact be responsible for the observed
difference in Gb isoforms for interacting with CCT. In both co-immunoprecipitation
studies and a BRET co-localization assay, Gb1 and Gb2 were shown to bind to PhLP1
preferentially to Gb3 (Dupre et al. 2007 ). In one of these studies (Howlett et al. 2009 ) ,
but not the other (Dupre et al. 2007 ) , G b4 was essentially equivalent to Gb1 and Gb 2,