Biology Reference
In-Depth Information
nomenclature (
Ballesteros & Weinstein, 1995
)) corresponds to an aspartate residue
that is well conserved in GPCRs responding to biogenic amines and involved in their
binding site (
Strader et al., 1991
). D
100
A point mutation suppresses the lateral side
chain of this aspartate and, consequently, serotonin is unable to bind and activate the
mutated 5-HT
4
receptor (
Fig. 7.1
B;
Claeysen, Joubert, Sebben, Bockaert, &Dumuis,
2003
). However, this receptor remains fully activable by highly selective 5-HT
4
R
synthetic agonists, such as BIMU 8 (
Fig. 7.1
B), thus belonging to the RASSL family
(receptor activated solely by synthetic ligands) (
Conklin et al., 2008
). 5-HT
4
recep-
tors are rapidly desensitized and internalized by endocytosis after activation by an
agonist. Upon 5-HT exposure, D
100
A-5-HT
4
receptors stay at the plasma membrane,
whereas wild-type (WT) 5-HT
4
receptors enter inside the cell. However, if we form
dimers between WT and D
100
A protomers, these molecular complexes can undergo
endocytosis, providing a way to separate and to visualize D
100
A dimers from the
other populations of 5-HT
4
receptors expressed in the cell.
Prior to cell transfection, place glass coverslips in 12-well clusters and incubate
them with 300
for minimum 30 min at 37
C. Wash the slides
twice with 1 mL PBS before seeding the cells. Transfect HEK297 cells (10
7
cells)
with 300 ng of HA- or Myc-tagged 5-HT
4
receptors, alone or in combination. Resus-
pend them in 20 mL of DMEM-DFBS and plate 1 mL/well (500,000 cell/well). 24 h
later, replace the cell culture medium by DMEM alone. 48 h posttransfection, place
the clusters at 4
C in a cold room for 15 min. Remove the medium and incubate for
90 min at 4
C with the 500
m
L/well of PORN 1
L of the primary antibodies (Rb anti-HA, 1/300, and/or
Ms anti-Myc, 1/400) diluted in cold DMEM. Wash twice with cold DMEM. Place the
clusters back at 37
C in the cell culture incubator under routine parameters for
15 min. Remove cell medium and add 1 mL of stimulation medium (5-HT or BIMU8
and 10
5
M in DMEM) or DMEM alone as control for 30 min at 37
C. Add 145
m
L
of PFA 16% in each well (2% final concentration) and fix the cells 10 min at
37
C. Wash the cells three times for 10 min at room temperature with PBS-glycine.
Then, permeabilize the cell with 500
m
L of PBS-Triton for 5 min at room temp-
erature. Wash three times for 5, 10, and then 15 min with PBS-gelatin and incubate
with secondary antibodies coupled to fluorophores (e.g., GAR red, 1/1000, and
GAM green, 1/1000) for 1 h at room temperature in the dark. Wash three times
for 5, 10, and then 15 min with PBS and then mount the coverslips on glass slide
using VECTASHIELD. Image the samples using a confocal microscope. A typical
experiment is presented in
Fig. 7.3
.
Under basal conditions, WT-5-HT
4
receptors and D
100
A-5-HT
4
receptors
expressed alone or coexpressed were predominantly located at the cell surface
(
Fig. 7.3
A, D, G, J, and M). A marginal constitutive internalization, reflecting the
constitutive activity of 5-HT
4
receptors, was detected for both WT and mutant recep-
tors (e.g., some dotted labeling in
Fig. 7.3
A). Upon activation with an agonist (5-HT
or BIMU8), the WT-5-HT
4
receptors were internalized as shown by both an internal
dotted labeling and a decrease in cell surface labeling (
Fig. 7.3
B and C). In the pres-
ence of 5-HT, the D
100
A mutant remained at the cell surface (
Fig. 7.3
E), whereas it
internalized in the presence of BIMU8 (
Fig. 7.3
F). However, 5-HT, which does not
m
