Biomedical Engineering Reference
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Fig. 3.3
Proximity ligation assay to investigate GPCR dimerization.
(
a
) Schematic representa-
tion of in situ proximity ligation assay detection. (
b
) Cells expressing GPCR1 and GPCR2 are
fixed and two specific antibodies from a different species are added followed by incubation with
specific oligo-conjugated secondary antibodies and proximity ligation assay reagent. The detected
dimers are represented by the fluorescent rolling circle products (
red clusters
). Nuclei are stained
with DAPI (
blue
). Adapted from (Borroto-Escuela et al.
2011
)
3.2.4
The Use of Mutants: Dominant Negative
and ER Trapping Studies
Mutants of GPCRs have been constructed by inserting an ER-retention motif,
causing them to be trapped in the ER. Most of these mutants will prevent ER-exit
of interacting GPCRs (see further), suggesting that these GPCRs are interacting
early in receptor biogenesis. Another approach to investigate whether or not oli-
gomers of the GPCR of interest form in the ER is through the use of folding
mutants. Given the importance of effective GPCR dimerization in cellular quality
control prior to ER exit, GPCRs that fold incorrectly might interfere with the cell
surface delivery and function of wild-type GPCRs, indicating oligomerization of
these GPCRs (Salahpour et al.
2004
; Wilson et al.
2005
). However, it is also
possible that a folding mutant can be rescued by a wild type receptor upon oli-
gomerization, resulting in PM expression of both receptors (Van Craenenbroeck
et al.
2011
; Hague et al.
2004
) .
Another approach to study dimerization during receptor biogenesis is the use of
dominant negative proteins that are involved in the trafficking of the receptor to the
PM. By using dominant negative Rab and Sar GTPases, which are involved in
ER-to-Golgi, intra-Golgi and/or trans-Golgi-to-plasma membrane transport, the
assembly of the b
2
-adrenergic receptor prior to PM trafficking was demonstrated
(Dupre et al.
2006
) .
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