GPCR Oligomerization: Contribution to Receptor Biogenesis - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 45

Table 3.1 Advantages and disadvantages of the described techniques in the study of GPCR-GPCR
interactions

Advantages Disadvantages

Co-immunoprecipitation In vivo applications possible (provided

that a good antibody is available)

Solubilization of GPCRs
can be difficult

Straight forward to study deletion
mutants

Aggregation of GPCRs
have to be occluded

FRET Study intact cells Need for tagged proteins

Imaging microscopy possible Auto fl uorescence

Photobleaching
Cell damage

BRET Study intact cells Need for tagged proteins

Real-time analysis Weak signal makes

imaging dif fi cult

Convenient to determine affinity
of interacting proteins

Proximity ligation assay In vivo applications possible Need for good antibodies

3.2.1 Co-immunoprecipitation

Co-immunoprecipitation is used to detect protein - protein interactions biochemically.
Tissues or cells endogenously expressing proteins of interest or cells artificially
over-expressing the (epitope-tagged) proteins are used. Due to a shortage of
high affinity selective anti-GPCR antibodies, most studies predominantly rely on
co-immunoprecipitations of differentially epitope-tagged GPCRs, transiently co-
expressed in heterologous cell lines.
For this co-immunoprecipitation technique, an antibody that targets a known
protein is used to isolate (immunoprecipitate) a protein complex from a lysate of a
cell line or a tissue. Polyacrylamide gel electrophoresis and Western blotting can be
performed to identify members of this complex using antibodies specific to epitopes
of those proteins. Co-immunoprecipitation has been used to detect both homo- and
hetero-oligomerization of many GPCRs. The first data was published in 1996, indi-
cating dimerization of the b 2 adrenergic receptor (Hebert et al. 1996 ) and of the
dopamine D 2 receptor (Ng et al. 1996 ). Since then, a growing number of studies
describing both homo- and hetero-oligomerization of the three different classes of
GPCRs have been published.
The most critical step in this technique is the solubilization of the GPCRs.
Solubilization with detergents is usually required; this process can lead to aggre-
gation of the receptors, due to the hydrophobic nature of the a-helices of the
GPCR. In order to rule out a false positive result due to aggregation, a control
treatment involves mixing lysates from distinct cell populations, each expressing
only one form of the GPCR. In this set-up, no interaction may occur. Extra care
has to be taken to avoid contaminating the lysate with membrane fragments: ultra
centrifugation or passage through 0.22 mM filters are techniques which have