Synthesis and Assembly of G Protein Dimers: Comparison of In Vitro and In Vivo Studies - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 163

assembly, CCT and PhLP1 were required for Gb5/RGS7 dimers, although PhLP1
function appears to be somewhat different from its role in assembly with dimers
containing Gb 1-G b4 (Howlett et al. 2009 ) (also see discussion below).
Overall, in vitro studies suggest two different influences on the specificity of Gb g
dimer formation (Dingus et al. 2005 ). One of these is the biochemical complemen-
tarity of Gg isoforms for forming productive complexes with Gb isoforms. This has
been noted by others (Wall et al. 1998 ), and given as the explanation as to why Gb 2
does not form dimers with Gg1. In particular, this provides a reasonable and obvious
explanation for why a Gb isoform would form dimers with some Gg isoforms but
not others. This may also account for the general failure of Gb5 to form dimers with
Gg, as recently proposed (Howlett et al. 2009 ) . However, since G b 5 G g dimers can
be observed functionally in cells (Fig. 9.2 , Column B) and stable dimers with Gg 2,
in particular, can be generated in insect cells (Fletcher et al. 1998 ) , but not in vitro
(Dingus et al. 2005 ), these results may alternatively suggest other determinants of
dimer formation besides biochemical complementarity.
The data (Dingus et al. 2005 ) (and Fig 9.3 ) also suggest that, in addition to
biochemical complementarity, there are other factors involved in Gb g dimer assembly
that are dependent upon the Gb isoform. This is seen most clearly in the extent of
dimer formation with all productive Gg combinations for a given Gb isoform, and
can be simply summarized as follows: Gb 1 = G b 4 = G b 2 > G b 3 > > G b5. For Gb 1
and Gb4, and, for Gb2 and those Gg with which it does make dimers, dimers are
formed at high (relative) levels in an in vitro translation system (Fig 9.2 and Ref
(Dingus et al. 2005 )). In contrast, dimers are made with lower efficiency with Gb 3,
very poorly with the Gb3s splice variant, and virtually not at all with Gb 5. These
observations point to some component of the translation mixture that affects the
ability of each Gb to form dimers. One example of this kind of component would be
an isoform-specific protein or co-chaperone, in addition to PhLP1, that is required
for efficient formation of dimers containing selective Gb. Alternatively, this could
suggest some regulatory process differentially affecting dimer formation with selec-
tive Gb isoforms. This hypothesis (Dingus et al. 2005 ) is strengthened by the obser-
vation that assembly of RGS dimers with Gb5 appear to use the same molecular
chaperone (CCT) and co-chaperone (PhLP1) as does formation of Gb g dimers with
other Gb isoforms, but by a different assembly mechanism (Howlett et al. 2009 ) .

9.11 G b Isoforms Bind Differently to the CCT

Macromolecular Complex

The interaction of Gb isoforms with CCT was investigated in an effort to explain
differences in the ability of Gb isoforms to form dimers (Fig. 9.4 ) (Dingus et al.
2005 ) . When in vitro translated Gb isoforms are immunoprecipitated, all CCT
subunits co-immunoprecipitate with Gb, although to varying degrees (Wells et al.
2006 ) ; the G b 1, G b2, and Gb4 isoforms bind CCT best, Gb3 less well, and the
Gb3s and Gb5 isoforms bind least. The order of the interactions can be summa-