Journal of the American Chemical Society
ARTICLE
R7-1(ꢀ) (50-TCGATCCTTGTACAGCTTCCTTTGGAACTCTCC-
TTGCAGGGACTCGCGCACGA-30) and R7-2(ꢀ) (50-GCCCCGCC-
GCCGCCAGCAGCCACAGCATCAGCGCCCGGAGGCCCATGG-
TGGC-30). An XhoIꢀEcoRI fragment of pBS-R7-FLAG-B2R was
inserted into the same digestion site of pCI-neo plasmid (Promega) to
yield pCI-R7-FLAG-B2R.
Construction of the Expression Plasmid for EGFP-Fused
B2R. An EGFP fragment was obtained by PCR from pEGFP-N1
(Clontech) using a pair of primers, forward (50-AGGATCCAATGGT-
GAGCAAGGGCGAGGAGC-30) and reverse (50-TGGATCCGCCT-
TGTACAGCTCGTCCATGCC-30), and subcloned into the BamHI
site of pBS-R7-FLAG-B2R to yield pBS-R7-FLAG-EGFP-B2R. An
XhoIꢀEcoRI fragment from pBS-R7-FLAG-EGFP-B2R was inserted
into pCI-neo plasmid (Promega) to yield pCI-EGFP-B2R.
Fluorescein Labeling of B2R and the BFQR Assay on the
Surface of HEK 293 Cells. HEK 293 cells stably expressing human
B2R fused to an R7-FLAG sequence were maintained in DMEM with
10% fetal bovine serum (FBS), penicillin (100 units/mL), and strepto-
mycin (100 μg/mL) at 37 ꢀC and 5% CO2. Before labeling, the cells
were washed twice with HBS buffer (107 mM NaCl, 6 mM KCl, 1.2 mM
MgSO4, 2 mM CaCl2, 11.5 mM glucose, 20 mM HEPES, pH 8).
Labeling was conducted in HBS buffer containing AGD catalyst 10 or 11
(10 μM) and acyl donor 12 (20 μM) for 30 min at 37 ꢀC. As control, the
labeling experiments in the presence of HOE 140 (10 μM) or in the
absence of catalyst 11 were also conducted. After labeling, the cells were
washed with HBS buffer three times to remove the unreacted acyl donor
and catalyst. The cells were then observed by CLSM. The BFQR assay
using quencher-ligand 19 was performed as described in the correspond-
ing figure captions.
To estimate the fluorescein labeling efficiency, we used fluorescein-
modified B2R ligand 20 as a fluorescent marker. After labeling and
washing procedures, the fluorescence intensity of the labeled, cell-surface
B2R (Flabel) was measured by CLSM. Subsequently, 20 was added to the
cell culture medium (at a final concentration of 1 μM) to stain the whole
cell-surface B2R, and the total fluorescence intensity (Ftotal) was
measured. The labeling efficiency was then determined by the eq 1:
with anti-B2R antibody and anti-rabbit IgG-HRP conjugate (both
Santa Cruz Biotechnology).
Evaluation of the B2R-Mediated Ca2+ Influx Activity. The
B2R-mediated Ca2+ influx activity was measured according to the method
previously reported.37 HEK 293 cells transiently expressing B2R were
plated onto poly-L-lysine-coated glass coverslips. B2R labeling was carried
out using AGD catalyst 11 and acyl donor 12 as described above. The
postlabeled cells were added with 1 μM Fura-2/AM (Dojindo) and
incubated at 37 ꢀC for 40 min in HBS buffer (pH 7.4, containing 2 mM
Ca2+). The coverslips were set in a perfusion chamber mounted on the
stage of the microscope. One micromolar bradykinin, the B2R agonist,
was applied to the cells by perfusion as a stimulus of B2R-mediated Ca2+
signaling. Fluorescence changes of Fura-2 inside the cells were recorded
and analyzed with a video image analysis of system (Aqua Cosmos;
Hamamatsu Photonics). As control, HEK 293 cells transfected with
pCI-neo vector were subjected to the Ca2+ measurement.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures S1ꢀS11, Tables S1ꢀS2,
b
and experimental details of the synthesis. This material is available
’ AUTHOR INFORMATION
Corresponding Author
Present Addresses
‡Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
§INAMORI Frontier Research Center, Kyushu University, 744
Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
’ ACKNOWLEDGMENT
We thank Dr. Tomohisa Ogawa (Tohoku University) for the
E. coli strain JM109/pTV-Con II. Y.K. and H.N. acknowledge the
Japan Society for the Promotion of Science Research Fellowships
for Young Scientists.
Labeling efficiency ð%Þ ¼ Flabel=ðFtotal ꢀ FlabelÞ ꢁ 100
ð1Þ
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Biotin Labeling of B2R. Biotin labeling was carried out as
described above using AGD catalyst 11 (10 μM) and acyl donor 13
(10 μM) for 30 min at 37 ꢀC. After washing, the cells were collected and
lysed with RIPA lysis buffer (pH 7.6, 25 mM Tris, 150 mM NaCl, 0.1%
SDS, 1% Nonidet P-40, 1% Deoxycholic acid) containing 1% protease
inhibitor cocktail set III (Novagen) at 4 ꢀC for 30 min. After centrifuga-
tion, the supernatant was recovered. The biotinylated B2R was purified
by SoftLink Soft Release Avidin Resin (Promega) and eluted with 5 mM
biotin in RIPA buffer according to the manufacture’s protocol. The
eluent was mixed with 2ꢁ sample buffer (pH 6.8, 125 mM Tris HCl,
3
20% Glycerol, 4% SDS, 0.01% Bromophenol blue, 100 mM DTT).
The samples were resolved by 10% SDSꢀPAGE and electrotrans-
fered onto an Immun-Blot PVDF membrane (Bio-Rad). The
biotinylated product was detected with streptavidinꢀhorseradish
peroxidase conjugate (SAv-HRP, Invitrogen) using Chemi-Lumi
One (Nacalai Tesque). The immunodetection of B2R was performed
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dx.doi.org/10.1021/ja204422r |J. Am. Chem. Soc. 2011, 133, 12220–12228