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confirm specific coimmunoprecipitation of interacting proteins in transfected cells,
the immunoprecipitation should be performed in singly (transfected with only one
protein of interest, e.g.,
Fig. 17.2
B, lanes 6 and 7) and doubly (transfected with
two potential interacting partners) transfected cells. Specific interaction will be
detected only in cotransfected cells (expressing both proteins).
When dimerization between receptors is studied, an additional control of specific
interaction between two proteins in living cells can be added by mixing posttransfec-
tion cells that were transfected only with one protein of interest (e.g.,
Fig. 17.2
C, lane
10 indicated by
o
). In this case, no interaction should be detected between the two
proteins as they were not present in the same cell.
17.3
DISCUSSION
A better understanding of GPCR biogenesis can start with the unraveling of all the
proteins that interact with the GPCR of interest. We have focused on the coimmu-
noprecipitation technique to get an initial idea about protein-protein interactions,
and all methods described can also be applied to study GPCR dimerization.
The main issues are problems with GPCR solubilization, which can be hard;
therefore, we have discussed different methods to tackle this problem, and aggrega-
tion of GPCRs has to be occluded as it can lead to false-positive results. Extra con-
trols were suggested in this methodological overview.
The use of this coimmunoprecipitation technique to study GPCR complexes
would benefit enormously from the availability of good and specific antibodies in
order to isolate the endogenous receptor from primary cells and native tissues. Con-
ventional antibodies are large molecules, which can generate steric hindrance; thus,
antibodies are not necessarily the best tools for demonstrating and quantifying direct
receptor interactions. Nanobodies can be a good alternative since they are much
smaller and they can recognize epitopes usually not detected by conventional anti-
bodies (
Jahnichen et al., 2010; Rasmussen et al., 2011
). Therefore, they open new
perspectives in term of molecular recognition and specificity.
Next to this biochemical approach, the majority of oligomerization studies will
also include biophysical approaches such as fluorescence or bioluminescence reso-
nance energy transfer techniques (FRET or BRET) as reviewed before (
Milligan,
2013; Van Craenenbroeck, 2012
). Some criticisms suggest that GPCR dimerization
might be promoted at relatively high receptor expression levels and potentially be at
least partially an artifact of overexpression. However, studies of
b
2-adrenergic re-
ceptor dimerization have indicated that dimerization is unaltered over a wide range
of expression levels (
Mercier, Salahpour, Angers, Breit, & Bouvier, 2002
).
A major advantage of FRET in view of studying biogenesis of GPCRs is that it
can be combined with microscopic imaging and thus allow the study of dimerization
in different subcellular compartments (
Herrick-Davis, Weaver, Grinde, &
Mazurkiewicz, 2006
). A disadvantage of FRET is that the proteins of interest need
to be tagged with a large donor or acceptor protein.
