Biology Reference
In-Depth Information
Kaupmann et al., 1998
). Other class C GPCRs, for example, the metabotropic glu-
tamate receptors and the calcium-sensing receptor, function as homomers.
Indeed, the receptors of the TAS1R family are prime examples for GPCR dimer-
ization. Expression analysis of the receptors in taste buds gave early hints about their
possible structure and function: TAS1R3 is coexpressed with either TAS1R1 or
TAS1R2, but neither receptor seems to be expressed alone at significant levels
(
Nelson et al., 2001
). Functional analysis of the receptors in heterologous
expression systems then showed that the combination of TAS1R3 with TAS1R1
is an umami taste receptor, detecting
L
-glutamate (human receptors) or a variety
of
L
-amino acids (mouse receptors), while TAS1R2/TAS1R3 serves as a general
sweet taste receptor and is activated not only by natural sugars but also by artificial
sweeteners and sweet-tasting
D
-amino acids and proteins (
Li et al., 2002, Nelson
et al., 2001, 2002
). Studies using knockout mice subsequently confirmed these re-
sults
in vivo
; both respective subunits of the sweet and umami taste receptor are nec-
essary for functional detection of the molecules and the behavioral attraction of the
animals towards sweet and umami tastants (
Zhao et al., 2003
). Furthermore, actual
active dimerization of the receptors could be shown—at least
in vitro
—using two
different techniques: coimmunoprecipitation (CoIP) and bioluminescence resonance
energy transfer (BRET) (
Jiang et al., 2004; Nelson et al., 2002
). All of these findings
taken together make a very strong case for TAS1Rs as obligate heteromeric recep-
tors, and TAS1R3 remains until now the only known GPCR that was shown to be an
essential partner in functionally distinct receptors with essentially nonoverlapping
ligand profiles.
Mammals have another family of taste receptors, the TAS2Rs, with
25 members
in humans (
Shi, Zhang, Yang, & Zhang, 2003
). Many evidences obtained from
in vitro
and
in vivo
studies showed that these receptors are bitter taste receptors that detect po-
tentially toxic substances and induce aversive behaviors towards them (see, e.g.,
Behrens, Reichling, Batram, Brockhoff, & Meyerhof, 2009; Yarmolinsky et al.,
2009
). Indeed, most of the receptors of this family have been deorphanized by
now. While some receptors seem to be specialized receptors detecting only few sub-
stances, many of them show a broad ligand spectrum, a characteristic that might ex-
plain how thousands of bitter-tasting substances can be detected with a rather small set
of receptors (
Meyerhof et al., 2010
). The question if also these receptors form protein
assemblies remained, however. To answer this question, we investigated TAS2R olig-
omerization with two different experimental approaches and found that TAS2Rs can
indeed form receptor-receptor complexes (
Kuhn, Bufe, Batram, & Meyerhof, 2010
).
Oligomerization seemed to be a general feature of these receptors as we found that
90% of all possible binary receptor combinations showed positive results.
In this chapter, we present the experimental protocols for analysis of oligomer-
ization of TAS1Rs and TAS2Rs in greater detail compared to what we reported in
our previously published study (
Kuhn et al., 2010
). As the number of receptors to be
investigated is large (there are 25 different TAS2Rs and 325 different possibilities for
combinations of two receptors!), we want to put special emphasis on the adaption of
the used methods for higher throughput.
