Abstracts - Association for Chemoreception Sciences

P O S T E R S
#P194 POSTER SESSION IV: CHEMOSENSORY
TRANSDUCTION AND SIGNALING
Calcium sensing receptor agonists induce response in taste cells
Yutaka Maruyama, Reiko Yasuda, Motonaka Kuroda, Yuzuru Eto
Institute of Life Sciences, Ajinomoto Co., Inc. Kawasaki, Japan
Many researches have identified the signaling of basic tastes such
as sweet and umami. However, the mechanisms underlying the
generation of palatability are not clear. Taste enhancer (is called
“kokumi“) for salty, sweet and umami is one of the important
factors for palatability of foods, and we recently reported that
calcium sensing receptor (CaSR) is a kokumi receptor (Ohsu et al.
J. Biol. Chem. 2010). Interestingly, CaSR agonists enhance the
basic tastes, although they do not induce any taste when they are
applied alone. In this study, we figured out the receptor cells for
taste enhancers, and their physiological properties. For this
purpose, we used Calcium Green-1 loaded mouse taste cells in
lingual tissue slice and confocal microscopy. Taste enhancers
applied focally around taste pore induced increase of intracellular
Ca 2+ concentration ([Ca 2+ ]i) in a subset of taste cells. These
responses were inhibited by pretreatment with CaSR inhibitor,
NPS-2143. Taste enhancer-induced responses did not require
extracellular Ca 2+ . In addition, a part of taste enhancer-responding
cells also responded to depolarizing stimulation with 50 mM KCl.
These observations indicate that CaSR-expressing taste cells are
primary detector of taste enhancers, and these cells are type III
and non-type III taste cells.
#P195 POSTER SESSION IV: CHEMOSENSORY
TRANSDUCTION AND SIGNALING
The interaction between PKD1L3 and PKD2L1 through their
transmembrane domains is required for localization of
PKD2L1 protein at taste pore in taste cells of circumvallate
and foliate papillae
Yoshiro Ishimaru 1,2 , Yuka Katano 1 , Kurumi Yamamoto 1 ,
Masato Akiba 1 , Richard W. Roberts 2 , Tomiko Asakura 1 ,
Hiroaki Matsunami 2 , Keiko Abe 1
1
The University of Tokyo Tokyo, Japan, 2 Duke University
Durham, NC, USA
Polycystic kidney disease 1 like 3 (PKD1L3) and polycystic
kidney disease 2 like 1 (PKD2L1) have been proposed to form
heteromers to function as sour taste receptors in mammals. Here
we show that PKD1L3 interacts with PKD2L1 through their
transmembrane domains, but not through the coiled-coil domain,
by co-immunoprecipitation experiments using a series of deletion
mutants. The deletion mutants lacking the region critical for the
interaction were not transported to the cell surface but retained in
the cytoplasm, whereas PKD1L3 and PKD2L1 proteins are
expressed at the cell surface when both are transfected. Calcium
imaging analysis revealed that neither the coiled-coil domain nor
EF-hand domain located in the C-terminal cytoplasmic tail of
PKD2L1 is required for response upon stimulation with acid
solution. Finally, PKD2L1 protein was not localized to the taste
pore but robustly distributed throughout the cytoplasm in taste
cells of circumvallate and foliate papillae in PKD1L3 (-/-) mice, in
which the genomic region encoding transmembrane 7 to 11 was
deleted, whereas it was localized to the taste pore in wild-type
mice. Collectively, these results suggest that the interaction
between PKD1L3 and PKD2L1 through their transmembrane
domains is essential for proper trafficking of the channels to the
cell surface in taste cells of circumvallate and foliate papillae as
well as in cultured cells. Acknowledgements: Grant-in-Aid for
Young Scientists from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan, a grant from The Kao
Foundation for Arts and Sciences, and a grant from Nestlé
Nutrition Council
#P196 POSTER SESSION IV: CHEMOSENSORY
TRANSDUCTION AND SIGNALING
Expression and characterization of ligand-binding domain of
T1R1 taste receptor
Maud Sigoillot, Elodie Maîtrepierre, Loïc Briand
Centre des Sciences du Goût et de l’Alimentation (CSGA)
Dijon, France
Umami is the typical taste induced by monosodium glutamate,
which is thought to be detected by a heterodimeric G-protein
coupled receptors, T1R1 and T1R3. The most unique feature of
umami taste is its potentiation by purine nucleotide
monophosphates (IMP, GMP), which also elicit umami taste by
their own. Zhang et al. (Proc. Natl Acad. Sci. USA, 2008) have
recently proposed a cooperative ligand-binding model involving
T1R1 N-terminal domain (NTD), where L-glutamate (L-glu)
binds close to the hinge region, and purine nucleotides bind to an
adjacent site close to the opening of the Venus flytrap domain. To
further understand the structural basis of umami stimuli
recognition by T1R1, a large amount of purified T1R1 NTD is
suitable for biochemical and structural studies. Here, we report
the successful expression and purification of the soluble NTD of
human T1R1. The protein was expressed as insoluble aggregated
protein (inclusion bodies) expressed in high level in Escherichia
coli. The protein was solubilized and in vitro refolded using
suitable buffer and additives. We then measured the binding of
umami stimuli to T1R1 NTD using fluorescence spectroscopy.
Fluorescent assay demonstrated that T1R1 NTD is properly
refolded and able to bind L-glu with physiological relevant
affinity. The mode of interaction was specific since T1R1 NTD
did not bind sweet stimuli like fructose, sucralose or glycine. In
addition, we observed that L-glu binding affinity is enhanced in
presence of IMP. To further validate our results, we have
generated single amino-acid changes in L-Glu and IMP binding
sites. In summary, our expression system will allow large scale
production of active protein suitable for structural and functional
studies. Acknowledgements: This work was supported by INRA
and Burgundy council.
92 | AChemS Abstracts 2010 Abstracts are printed as submitted by the author(s)