Biology Reference
In-Depth Information
BiP, calnexin, calreticulin, and ERp57. Besides these classical chaperones, other
proteins have been characterized typically interacting with specific GPCRs and play-
ing a role in GPCR biogenesis; for example, receptor activity-modifying protein 1
(RAMP1) facilitates maturation of the calcitonin receptor-like receptor by interac-
tion with the immature receptor in the ER (
Luttrell, 2008
).
Dimerization
A general role for GPCR dimerization in quality control and ER export has not yet
been established although a plethora of studies have showed that GPCR dimerization
starts in the ER. Dimerization of the metabotropic
g
-aminobutyric acid (GABA
B
)
receptor and
b
2-adrenergic receptors was shown to be a prerequisite for its expres-
sion at the plasma membrane (
Margeta-Mitrovic, Jan, & Jan, 2000; Salahpour et al.,
2004
). 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
the interaction partner, suggesting that these GPCRs are interacting early in receptor
biogenesis, as reviewed by
Dupre, Hammad, Holland, and Wertman (2012)
.
Interaction with “gatekeepers”
A new concept was raised that next to the quality-control checkpoints in the ER,
there is also another mechanism involved in the progression of GPCRs along the bio-
synthetic pathway executed by so-called gatekeepers. The role of gatekeepers seems
to be the control of GPCR trafficking from the ER to the plasma membrane upon
receiving a specific external stimulus (
Shirvani, Gata, & Marullo, 2012
). This
new concept might represent an adaptive mechanism to specific physiological con-
straints, such as the sustained activation of the receptor.
For a detailed description of these interaction studies we refer to the cited pub-
lications, the main idea is that all these experiments make use of coimmunoprecipi-
tation approaches as this method is ideally suited to identify interactions between
proteins and is often the experiment of choice to start testing initial hypotheses.
Tissues or cells endogenously expressing proteins of interest or cells artificially
overexpressing the (epitope-tagged) proteins are used. We want to stress that the
functional outcome of receptor activation often depends on the cellular context,
and, therefore, it is important to study the GPCR complexes in endogenous tissue.
Due to a shortage of high-affinity selective anti-GPCR antibodies, most studies
predominantly rely on coimmunoprecipitation experiments of epitope-tagged
GPCRs, transiently expressed in heterologous cell lines. The cell line used is often
HEK293T and as the transfection of this cell line is an important preliminary step, we
will spend some time describing the best methods to transiently transfect HEK293
cell lines resulting in high transfection efficiency.
Coimmunoprecipitation is widely used to detect GPCR-GPCR interactions bio-
chemically. For this coimmunoprecipitation technique, an antibody, targeting a
known protein, is used to isolate (immunoprecipitate) a protein complex, containing
