Biology Reference
In-Depth Information
both homomeric and heteromeric receptor complexes. The employed techniques,
coimmunoprecipitations and bioluminescence resonance energy transfer (BRET),
are based on different principles and complement each other well and therefore pro-
vided compelling evidences for TAS2R oligomerization. Furthermore, we have
adapted the protocols to include a number of controls and for higher throughput
to accommodate the investigation of a large number of receptors and receptor com-
binations. Here, we present the protocols in detail.
INTRODUCTION AND RATIONALE
Our sense of taste serves us in our daily lives to advise us on which foods are safe and
worth consuming and turns eating into pleasure. On the molecular level, the different
taste modalities—sweet, umami, bitter, salty, and sour—are detected by different
taste receptors expressed on the tongue, ionotropic receptors and metabotropic recep-
tors. Taste receptors of the TAS1R (also referred to as T1R) family, mediating sweet
and umami taste, and those of the TAS2R (or T2R) family, mediating bitter taste,
belong to the superfamily of G protein-coupled receptors (GPCRs) (
Yarmolinsky,
Zuker, & Ryba, 2009
). Both families are unrelated, however; the
25 members
of the human TAS2R family have short N-termini and are distantly related to opsins,
whereas the three different TAS1Rs are class C GPCRs, have long N-termini, and are
related to metabotropic glutamate and GABA
B
receptors.
While it was initially common belief that class A (rhodopsin-like) and class B
(secretin receptor family) GPCRs act as singular units, over the past 10-15 years,
many reports demonstrated that GPCRs might not (only) function as monomeric re-
ceptors but that they can also assemble with one another and form receptor dimers or
even higher-order oligomers with potentially dramatic and important implications
(see, e.g.,
George, O'Dowd, & Lee, 2002
). However, many of these discoveries were
made in
in vitro
systems and the functional and physiological significance of these
findings remained debated. But at least for some receptors, a functional significance
and/or relevance
in vivo
is evident. Recently,
in vivo
functional dimerization could be
demonstrated for the luteinizing hormone receptor. Mice with either a binding-
deficient mutant receptor or a signaling-deficient mutant receptor have only small
gonads and are sterile. In contrast, when mice are heterozygous for both mutants,
receptor dimerization will lead to receptor complementation and the phenotype of
the mice is rescued; they have normal-sized gonads and are fertile (
Rivero-M¨ ller
et al., 2009
).
Many studies have also established that for class C GPCRs, homo- or hetero-
oligomerization is indispensable for receptor function (
Pin, Galvez, & Prezeau,
2003
). For example, GABA
B
receptors are formed by GABA
B
R1 and GABA
B
R2 class C GPCRs. The assembly of both receptor subunits was shown to take place
in neurons
in vivo
and is essential for the targeting of the dimeric GABA
B
receptors
to the plasma membrane and for ligand binding and signaling (
Jones et al., 1998,
