Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells

Abstract

This study uses fluorescence microscopy combined with dynamic video imaging to examine the events associated with the rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R). In single non-pituitary human embryonic kidney 293 (HEK-293) cells, expressing either the rat or human TRH-Rs, TRH produced a rapid dose-dependent monophasic rise in [Ca2+]i. This Ca2+ transient was completely abolished by pretreatment of cells with the intracellular Ca2+ antagonists thapsigargin or cyclopiazonic acid, but not EGTA, the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine or the second-messenger-operated Ca2+ channel antagonist SK&F 96365. These results suggest that TRH causes the mobilization of Ca2+ from thapsigargin/cyclopiazonic acid-sensitive intracellular Ca2+ stores but not the influx of extracellular Ca2+. HEK-293 cells also failed to respond to KCl or the slow Ca(2+)-channel activator BAY K 8644, suggesting that they lack L-type VOCCs. Rat and human TRH-Rs are highly conserved except at the C-terminus where the sequence differs. The C-terminus is believed to be important in receptor desensitization. Despite differences in this region, rat and human TRH-Rs expressed in HEK-293 cells underwent rapid (within 1 min) desensitization. This desensitization was dose-dependent and did not involve receptor loss. Similarly the bradykinin receptor endogenous to HEK-293 cells also displays a rapid desensitization. We conclude that in TRH-R-expressing non-pituitary HEK-293 cells, TRH mobilizes intracellular Ca2+ resulting in a monophasic Ca2+ transient. The rat and human TRH-Rs as well as the endogenous bradykinin receptor also displayed rapid receptor desensitization.

Full text

Biochem.
J.
(1995)
311,
385-392
(Printed
in
Great
Britain)
Rapid
desensitization
of
the
thyrotropin-releasing
hormone
receptor
expressed
in
single
human
embryonal
kidney
293
cells
Lorraine
ANDERSON,*
Claire
L.
ALEXANDER,
Elena
FACCENDA
and
Karin
A.
EIDNE
MRC
Reproductive
Biology
Unit,
Centre
for
Reproductive
Biology,
37
Chalmers
Street,
Edinburgh
EH3
9EW,
Scotland,
U.K.
This
study
uses
fluorescence
microscopy
combined
with
dynamic
video
imaging
to
examine
the
events
associated
with
the
rapid
desensitization
of
the
thyrotropin-releasing
hormone
receptor
(TRH-R).
In
single
non-pituitary
human
embryonic
kidney
293
(HEK-293)
cells,
expressing
either
the
rat
or
human
TRH-Rs,
TRH
produced
a
rapid
dose-dependent
monophasic
rise
in
[Ca2+],.
This
Ca2+
transient
was
completely
abolished
by
pre-
treatment
of
cells
with
the
intracellular
Ca2+
antagonists
thapsigargin
or
cyclopiazonic
acid,
but not
EGTA,
the
voltage-
operated
Ca2+
channel
(VOCC)
antagonist
nifedipine
or
the
second-messenger-operated
Ca2+
channel
antagonist
SK&F
96365.
These
results
suggest
that
TRH
causes
the
mobilization
of
Ca2+
from
thapsigargin/cyclopiazonic
acid-sensitive
intra-
cellular
Ca2+
stores
but
not
the
influx
of
extracellular
Ca2
.
INTRODUCTION
Thyrotropin-releasing
hormone
(TRH)
is
a
hypothalamic
peptide
which
acts
on
the
anterior
pituitary
gland
to
induce
release
of
thyrotropin
and
prolactin.
The
TRH
receptor
(TRH-R)
is
a
member
of
the
G-protein-coupled
seven-transmembrane-
spanning
family
of
receptors
(GPCRs)
[1-3].
TRH-R
activation
causes
rapid
stimulation
of
the
phospholipase
C
(PLC)
pathway
via
its
Gq/Gll
G-protein,
producing
Ins(1,4,5)P3
[4,5]
and
1,2-
diacylglycerol.
Ins(l,4,5)P3
causes
a
transient
mobilization
of
Ca2+
from
internal
[6]
and
possibly
indirectly
from
external
[7]
sources,
whereas
diacylglycerol
increases
protein
phosphoryl-
ation
by
activating
protein
kinase
C
[8].
Given
that
changes
in
intracellular
Ca2+
concentration
([Ca2+],)
are
critical
in
the
regulation
of
a
variety
of
cellular
processes,
including
hormone
secretion,
it
is
important
to
understand
the
mechanisms
con-
trolling
these
events
in
different
cell
types.
TRH-induced
changes
in
[Ca2+],
have
been
measured
in
pituitary
and
non-pituitary
cell
types
expressing
TRH-Rs
[9-16].
These
spectrofluorimetric
studies
have
contributed
to
the
analysis
of
TRH-induced
changes
in
[Ca2+]1.
Interpretation
of
these
data
can,
however,
be
misleading,
as
these
studies
were
often
con-
ducted
in
heterogeneous
primary
pituitary
cultures
and/or
sus-
pension
cultures
of
established
pituitary
cell lines.
It
is
generally
believed
that
TRH
causes
a
biphasic
Ca2+
response
but
the
exact
origins
of
the
Ca2`
pools
involved
are
unclear.
Indeed
it
has
been
suggested
that
the
Ca2+
pools
mobilized
after
TRH-R
activation
are
cell-type
specific
[13].
In
pituitary
cell
types,
the
initial
Ca2+
spike
predominantly
involves
the
mobilization
of
Ins(1,4,5)P3-
HEK-293
cells
also
failed
to
respond
to
KCI
or
the
slow
Ca2+-
channel
activator
BAY
K
8644,
suggesting
that
they
lack
L-type
VOCCs.
Rat
and
human
TRH-Rs
are
highly
conserved
except
at
the
C-terminus
where
the
sequence
differs.
The
C-terminus
is
believed
to
be
important
in
receptor
desensitization.
Despite
differences
in
this
region,
rat
and
human
TRH-Rs
expressed
in
HEK-293
cells
underwent
rapid
(within
1
min)
desensitization.
This
desensitization
was
dose-dependent
and
did
not
involve
receptor
loss.
Similarly
the
bradykinin
receptor
endogenous
to
HEK-293
cells
also
displays
a
rapid
desensitization.
We
conclude
that
in
TRH-R-expressing
non-pituitary
HEK-293
cells,
TRH
mobilizes
intracellular
Ca2+
resulting
in
a
monophasic
Ca2+
transient.
The
rat
and
human
TRH-Rs
as
well
as
the
endogenous
bradykinin
receptor
also
displayed
rapid
receptor
desensitization.
sensitive
Ca2+
stores
and
the
longer-lasting
secondary
plateau
phase
relies
on
the
influx
of
extracellular
Ca2
.
In
contrast,
when
expressed
in
cells
of
non-pituitary
origin,
TRH
induces
a
monophasic
Ca2+
response
[13,16].
Receptor
activation
can
lead
not
only
to
stimulation
of
intracellular
events,
for
example
the
mobilization
of
Ca2+,
but
also
to
desensitization
of
these
responses.
Receptor
desensiti-
zation
is
potentially
a
physiologically
important
process,
as
it
provides
a
means
of
regulating
continuous
receptor
stimulation
[17].
Chronic
exposure
of
cells
to
TRH
results
in
the
internaliza-
tion
and
subsequent
loss
of
cell-surface
TRH-Rs
[18-20],
a
decrease
in
the
levels
of
TRH-R
mRNA
[21,22]
and
a
down-
regulation
of
the
Gq
and
Gil
G-proteins
[23].
In
contrast,
rapid
receptor
desensitization
does
not
involve
receptor
loss
but
rather
receptor
phosphorylation.
This
phosphorylation
results
in
an
uncoupling
of
the
receptor
from
its
cognate
G-protein
and
a
loss
of
subsequent
downstream
events.
Although
rapid
desensiti-
zation
of
a
number
of
GPCRs
has
been
demonstrated
[6,17,24],
it
remains
unclear
whether
or
not
the
G-protein-coupled
events
of
the
TRH-R
can
be
acutely
desensitized,
i.e.
within
the
first
few
minutes
of
receptor
activation.
The
present
study
aims
to
eliminate
the
problems
of
cell
heterogeneity
by
directly
monitoring
and
characterizing
TRH-
induced
changes
in
[Ca2+],
in
single
cells
of
TRH-R-expressing
clonal
non-pituitary
cell
lines.
We
demonstrate
that
TRH
causes
a
prompt
dose-dependent
monophasic
rise
in
[Ca2+],
in
HEK-293
cells
expressing
TRH-Rs.
By
using
a
variety
of
intra-
and
extra-
cellular
Ca2+
antagonists,
we
have
also
shown
that
this
response
does
not
involve
the
influx
of
extracellular
Ca2+
through
either
Abbreviations
used:
TRH,
thyrotropin-releasing
hormone;
TRH-R,
TRH
receptor;
BK,
bradykinin;
BK-R,
bradykinin
receptor;
[Ca2+]i,
intracellular
Ca2+
concentration;
VOCCs,
voltage-operated
Ca2+
channels;
SMOCCs,
second-messenger-operated
Ca2+
channels;
HEK-293,
human
embryonal
kidney
293;
TG,
thapsigargin;
CPZ
cyclopiazonic
acid;
GPCRs,
G-protein-coupled
receptors;
PLC,
phospholipase
C;
GTP[S],
guanosine
5'-[y-
thio]triphosphate;
Thi,
thienyl(alanine).
*
To
whom
correspondence
should
be
addressed.
385

386
L.
Anderson
and
others
voltage-
or
second-messenger-operated
Ca2+
channels
(VOCCs
or
SMOCCs),
but
rather
the
mobilization
of
Ca2+
from
thapsigargin/cyclopiazonic
acid
(TG/CPZ)-sensitive
intracellu-
lar
Ca2+
stores.
The
structure
of
the
TRH-R
is
highly
homologous
between
rat
[3],
mouse
[1]
and
human
[25]
except
for
the
stretch
of
amino
acids
at
the
extreme
end
of
the
C-terminus.
The
C-
termini
of
GPCRs
are
thought
to
be
important
in
the
events
associated
with
receptor
desensitization.
We
therefore
compared
acute
desensitization
events
in
cells
expressing
either
rat
or
human
TRH-Rs.
Desensitization
of
the
endogenously
expressed
bradykinin
(BK)
receptor
(BK-R)
was
also
examined.
TRH-Rs
from
both
species
as
well
as
the
endogenous
BK-R
exhibited
a
rapid
desensitization
of
the
agonist-induced
rises
in
[Ca2+]1.
MATERIALS
AND
METHODS
Materials
Tissue
culture
reagents,
media,
lipofectin
and
genetecin
were
supplied
by
Gibco,
Paisley,
Scotland,
U.K.
Glass
coverslips
(22
mm
x
0.175
mm)
were
supplied
by
Arnold
R.
Horwell
Ltd.,
West
Hampstead,
London,
U.K.
[3-Me-His2]TRH
and
guanosine
5'-[y-[35S]thio]triphosphate
([35S]GTP[S]
1245
Ci/mmol)
were
obtained
from
Dupont-NEN
Products,
Hertfordshire,
U.K.
All
other
drugs,
including
fura
2
penta-acetoxymethyl
ester
(fura
2/AM),
were
obtained
from
Calbiochem.
The
Bio-Rad
protein
assay
kit
was
obtained
from
Bio-Rad,
Richmond,
CA,
U.S.A.
Tissue
culture
Cell
lines
were
maintained
routinely
in
Dulbecco's
modified
Eagle's
medium
containing
10%
(v/v)
heat-inactivated
foetal
calf
serum,
glutamine
(0.3
mg/ml),
penicillin
(100
units/ml),
streptomycin
(100
units/ml)
and
geneticin
(800
mg/ml)
and
incubated
at
37
°C
in
a
humidified
atmosphere
of
5
%
(v/v)
CO2
in
air.
Cell
lines
expressing
TRH-Rs
Using
a
stable
transfection
protocol
[23],
the
full-length
rat
[3]
or
human
[25]
TRH-R
was
subcloned
into
the
eukaryotic
expression
vectors
pcDNA1
and
pcDNA3
respectively
and
expressed
in
HEK-293
cells.
Receptor-containing
clones
were
identified
using
a
functional
total
inositol
phosphate
assay
[26]
and
verified
with
a
TRH-R-binding
assay.
The
cell
lines
expanded
from
these
clones
containing
either
the
rat
(293-E2
cell
line)
or
the
human
(293-hlO
cell
line)
TRH-Rs
were
then
used
for
further
study.
Measurement
of
[Ca2+],
Trypsin-treated
single
cells
were
plated
on
to
sterilized
glass
coverslips.
After
2
days,
attached
cells
were
washed
(x
2)
with
buffer
A
[127
mM
NaCl,
5
mM
KCI,
2
mM
MgCl2,
0.5
mM
NaH2PO4,
5
mM
NaHCO3,
1.8
mM
CaC12,
10
mM
Hepes
and
0.1
%
(w/v)
BSA,
pH
7.2].
Cells
were
loaded
in
this
buffer
with
fura
2/AM
(4,uM
final
concentration)
for
30
min
at
37
°C
in
a
5
%
(v/v)
CO2
humidified
incubator.
Unincorporated
dye
was
removed
by
washing
(
x
3)
with
buffer
A.
Coverslips
were
then
transferred
to
a
heated
stage
(37
°C)
of
an
inverted
Nikon
Diaphot
epifluorescence
microscope
with
a
x
40
oil
immersion
objective.
Cells
were
incubated
in
a
fixed
volume
of
buffer
A
(1
ml).
Drug
solutions
(5
ml)
were
added
directly
into
the
coverslip
chamber
and
1
ml
volumes
automatically
obtained
through
suction.
Dynamic
video
imaging
of
changes
in
[Ca2+]2
was
carried
out
in
single
cells
using
the
MagiCal
hardware
and
Tardis
software
Dukesway,
Team
Valley,
Gateshead,
Tyne
and
Weir,
U.K.
as
previously
described
[27].
Fluorescent
images
were
obtained
by
exposing
cells
to
filtered
340
and
380
nm
light
alternated
under
computer
control.
The
image
viewed
at
a
wavelength
of
510
nm
was
focused
on
to
the
face
of
an
intensified
charge-coupled
device
camera
(Photonic
Sciences).
Typically,
eight
images
were
averaged
at
each
wavelength
and
a
similar
number
were
collected
for
background
images
which
were
subsequently
subtracted
on
a
pixel-by-pixel
basis
from
the
imaged
samples.
These
images
were
held
in
memory
for
subsequent
processing
and
analysis.
Fluorescence
excitation
shifts
occur
when
fura
2
binds
Ca2+,
i.e.
the
excitation
efficiency
increases
at
340
nm
and
decreases
at
380
nm.
Ratios
of
values
obtained
at
340/380
nm
therefore
represent
changes
in
[Ca2+]1.
The
340/380
nm
ratio
was
calculated
on
averaged
video
frames
on
a
pixel-by-pixel
basis,
and
this
was
proportional
to
[Ca2+],.
During
non-imaging
periods
in
desensitization
experiments,
photobleaching
of
the
fura
2
dye
was
minimized
by
inserting
a
low-percentage
neutral-density
filter
between
the
light
source
and
the
filter
wheel.
Data
analysis
and
presentation
Software-based
image
analysis
allowed
quantification
of
[Ca2+]1
in
single
cells
versus
time.
ASCII
files
of
these
quantitative
data
were
used
to
derive
plots
of
the
mean
[Ca2+],
versus
time
for
single
cells
from
different
experiments.
A
minimum
of
ten
cells
was
analysed
from
each
experiment,
and
individual
treatment
regimes
were
carried
out
at
least
three
times.
Results
are
given
as
means
+
S.E.M.
Statistical
analysis
was
performed
using
Student's
t
test.
TRH-R-binding
assay
Monolayer
cultures
were
washed
with
PBS
(
x
2)
and
harvested
by
scraping.
The
cells
were
resuspended
in
20
mM
Tris/
HCl/2
mM
MgCl2,
pH
7.4,
and
membranes
prepared
after
homogenization
and
centrifugation
at
20000
g
for
30
min
at
4
°C.
The
membrane
pellet
was
resuspended
in
buffer
B
con-
taining
40
mM
Tris/HCl,
pH
7.4,
and
2
mM
MgCl2.
Ligand-
binding
assays
were
carried
out
with
[3H]
[3-Me-His2]TRH
in
buffer
B
(0.5
ml)
and
various
concentrations
of
unlabelled
peptide.
After
incubation
on
ice
for
I
h,
the
membranes
were
filtered
through
Whatman
GF-B
filters
and
washed
with
buffer
B
(
x
3).
All
assays
were
performed
in
triplicate.
Binding
parameters
were
determined
using
Scatchard
analysis.
Protein
concentrations
were
measured
using
a
Bio-Rad
protein
assay
kit
with
a
BSA
standard.
Binding
of
[NS]GTP[S]
to
membranes
[35S]GTP[S]
binding
to
membranes
prepared
from
293-E2
cells
was
measured
as
previously
described
[28].
Briefly,
reconstituted
293-E2
membranes
were
incubated
in
a
final
assay
volume
of
250,1u
in
buffer
C
[50
mM
Tris/HCl,
2
mM
EDTA,
10
mM
MgCl2,
2
mM
dithiothreitol,
200
mM
NaCl,
0.4
units/ml
adenosine
deaminase,
1
%
(v/v)
BSA]
containing
[35S]GTP[S]
(0.2
nM)
and
GDP
(10
,uM)
at
25
°C
for
45
min.
Samples
were
then
filtered
under
vacuum
through
Whatman
GF-B
filters
presoaked
with
buffer
D
(50
mM
Tris/HCl,
pH
7.4/5
mM
MgCl2).
Filters
were
washed
(
x
2)
with
4
ml
of
buffer
D,
digested
with formic
acid
and
counted
in
Emulsifier
SAFE
scintillation
fluid.
Non-specific
binding
was
determined
in
the
presence
of
provided
by
Applied
Imaging
(formerly
Joyce
Loebl.
Ltd.),
10#M
unlabelled
GTP[S].

Desensitization
of
thyrotropin-releasing
hormone
receptors
in
HEK-293
cells
0.1
nM
I;
1
nM
0.1
PM
400
1
0
120
240 360
480
600
0
120
lime
(s)
240
360
0
120
240
360
Figure
1
Effect
of
various
TRH
concentratons
(10
pM-1
pM)
on
[Ca2+],
in
293-E2
cells
Graphs
represent
an
average
plot
of
[Ca2+]j
measurements
versus
time
(s)
in
a
minimum
of
ten
cells
from
representative
experiments.
TRH
added
at
t=
50
s
produced
a
monophasic
rise
in
[Ca2+]i.
P
<
0.001,
compared
with
control
response
(n
=
10).
RESULTS
Effect
of
TRH
on
[Ca2+],
In
HEK-293
cells
expressing
the
rat
TRH-R
(293-E2
cells)
Figure
1
shows
examples
of
averaged
traces
from
single
experi-
ments
consisting
of
at
least
ten
individual
293-E2
cells
illustrating
TRH-induced
changes
in
[Ca2+],.
The
application
of
various
concentrations
of
TRH
(10
pM-l
,uM)
to
293-E2
cells
produced
dose-dependent
changes
in
[Ca2+]1.
TRH
produced
a
rapid
transient
monophasic
increase
in
[Ca2+1],
and
maximal
responses
were
obtained
with
TRH
concentrations
of
10
nM
and
higher.
Within
10
s
of
the
application
of
1
,uM
TRH,
[Ca2+],
had
risen
from
49.4
+
2.7
nM
at
t
=
50
s
to
348.7
+
25.6
nM
at
t
=
57
s.
The
maximal
concentration
of
1
,#M
TRH
was
used
in
subsequent
experiments.
[Ca2+],
oscillations
were
also
often
observed
after
the
exposure
of
cells
to
concentrations
of
TRH
ranging
from
0.1
nM
to
1
,uM
(Figure
2).
The
origin
of
the
Ca2+
mobilized
by
TRH
was
investigated
using
a
variety
of
intra-
and
extra-cellular
Ca2+
antagonists.
Involvement
of
extracellular
Ca2+
In
the
TRH-induced
[Ca2+J,
response
In
293-E2
cells
Pretreatment
of
293-E2
cells
with
Ca2+-free
buffer
A
containing
the
Ca2+
chelator
EGTA
(2
mM;
6
min)
had
no
effect
on
the
[Ca2+],
response
to
TRH
(Figure
3a).
Similar
treatment
of
cells
400
-
300
+
~200
-I
X
/
j
0
0
120
240
360
480
600 720
840
960
Time
(s)
Figure
2
TRH-induced
(Ca2+1,
oscillations
In
293-E2
cells
This
trace
illustrates
a
typical
example
of
a
single
TRH-treated
(1
,tM;
t=
108
s)
oscillating
cell.
with
the
L-type
VOCC
blocker,
nifedipine
(1
1tM),
did
not
alter
the
response
to
TRH
(Figure
3b).
After
the
application
of
the
SMOCC
blocker,
SK&F
96365
(1
,tM),
293-E2
cells
responded
normally
to
TRH
(Figure
3c).
Collectively
these
data
suggest
10
pM
400
-
300-
200
100
*
i
-)
-W
0
10
nm
300-
200
100
0
387

388
L.
Anderson
and
others
SK&F
96365
TRH
(d)
TRH
(c)
720
0
120
240
360
480
600
720
TRH
(e)
Time
(s)
Figure
3
Effect
of
(a)
EGTA,
(b)
niedipine,
(c)
SK&F
96365,
(d)
TG
and
(e)
CPZ
pretreatment
on
the
TRH-induced
[Ca2+],
response
in
293-E2
cells
(a)
Cells
were
preincubated
for
6 min
in
Ca2+-free
buffer
A
with
2
mM
EGTA, and
TRH
(1
1sM;
t=
420
s)
was
added.
(b)
293-E2
cells
were
pretreated
with
nifedipine
(NIF;
1
,uM;
t=
108
s),
and
TRH
(1
ttM)
was
subsequently
added
at
t
=
420
s.
(c)
293-E2
cells
were
pretreated
with
SK&F
96365
(1
,uM;
t
=
10
s),
and
TRH
(1
1sM;
t
=
420
s)
was
then
added.
(d)
293-E2
cells
were
treated
with
TG
(1
,uM;
t=
108
s),
and
TRH
(1
,uM;
t=
688
s)
was
then
added.
(e)
293-E2
cells
were
pretreated
with
CPZ
(1
,uM;
t=
108
s),
and
TRH
(1
,uM;
t=
533
s)
was
then
added.
that
mobilization
of
extracellular
Ca21
is
not
involved
in
the
TRH-induced
Ca2+
response.
Neither
did
[Ca2+]1
increase
after
treatment
of
cells
with
the
L-type
VOCC
activator
BAY
K8644
or
depolarization
with
KCl
(2-50
mM;
results
not
shown).
Although
[Ca2+]
appeared
not
to
return
to
prestimulated
values
after
the
application
of
TRH
to
either
nifedipine-
or
SK&F
96365-pretreated
cells,
these
values
were
in
fact
not
significantly
higher
than
basal
prestimulated
[Ca2+].
Involvement
of
intracellular
Ca2+
in
the
TRH-induced
Ca2+
response
in
293-E2
cells
The
importance
of
intracellular
Ca2+
mobilization
in
the
TRH-
induced
Ca2+
response
was
investigated
using
the
structurally
unrelated
ATPase
inhibitors
TG
and
CPZ.
Both
compounds
are
thought
to
deplete
Ins(1,4,5)PJ-sensitive
Ca2+
stores
in
the
endoplasmic
reticulum.
Both
compounds
(1
#tM)
produced
a
slow
but
substantial
increase
in
[Ca2+]1
(Figures
3d
and
3e).
Although
the
subsequent
addition
of
TRH
to
these
cells
produced
a
temporary
fall
in
[Ca2+]1,
it
did
not,
however,
produce
the
expected
rise
in
[Ca2+]1
(Figures
3d
and
3e).
These
results
imply
that,
in
293-E2
cells,
the
mobilization
of
Ca2+
from
intracellular
pools
is
responsible
for
the
TRH-induced
Ca2+
response.
The
slight
decrease
in
[Ca2+]1
after
the
application
of
TRH
to
TG-
or
CPZ-pretreated
cells
is
consistent
with
the
idea
that
receptor
stimulation
can
lead
to
activation
of
Ca2+
pumping
from
cells,
an
effect
masked
under
normal
circumstances
[29].
Alternatively,
dye
compartmentalization,
which
can
occur
at
37
°C,
may
also
explain
this
observation.
Involvement
of
G-protein
coupling
in
the
TRH-induced
intracellular
Ca2+
response
Suramin
sodium
has
previously
been
shown
to
uncouple
G-
proteins
from
their
receptors.
The
effect
of
this
compound
on
Gq/G,1-linked
TRH
Ca2+
mobilization
was
examined
(Figure
4a).
Pretreatment
of
cells
with
suramin
sodium
(10
,uM)
signifi-
cantly
reduced
the
Ca2+
response
to
TRH.
[Ca2+],
rose
from
29.4+
8.6
nM
to
155.2+14.9
nM
at
t
=
422
s
compared
with
a
control
response
of
329.7
+
25.6
nM.
Despite
a
dose-dependent
reduction
in
total
binding,
neither
TRH-R
affinity
or
number
were
altered
by
treatment
with
suramin
sodium
(1-100
lM;
results
not
shown).
Suramin
did,
however,
abolish
both
TRH-
and
[3-Me-His2]TRH-stimulated
[35S]GTP[S]
binding
in
mem-
branes
prepared
from
293-E2
cells
(Figure
4b).
400-
300
200
100
i
-S
co
L0
0
400
TG
300-
200
100-
n
0
120
240 360
480
600
720
840 960

Desensitization
of
thyrotropin-releasing
hormone
receptors
in
HEK-293
cells
400
1
(a)
SS
TRH
2.0
0
E
n
1.8
n
0
.0
1.6-
U)
_
1.4J
, .
0
120
240
360
480
600
720
Time
(s)
(b)
TRH
[3-Me-His2I-TRH
cc
Ir
IX
)
2>
CC
ccC
L4
L
+
E e
+
U~~~~~
~~~~
a
Figure
4
Effect
of
suramin
sodium
pretreatment
on
(a)
TRH-induced
Ca2+
response
In
293-E2
cells
and
(b)
TRH-
and
[3Me-HisITRH-simulated
[S]GTP[SJ
binding
In
cell
membranes
prepared
from
293-E2
cells
(a)
Suramin
sodium
(SS)
pretreatment
(10
1sM;
t
=
108
s)
significantly
reduced
the
TRH-induced
[Ca2+]i
response
(1
FM;
t
=
422
s);
P
=
0.02,
compared
with
control
response
(n
=
5).
(b)
SS
(32
FM)
treatment
abolished
both
TRH
(1
/FM)-
and
[3-Me-His2]TRH
(1
FM)-stimulted
[35S]GTP[S]
binding
in
cell
membranes
prepared
from
293-E2
cells.
Results
are
representative
of
those
obtained
in
three
separate
experiments.
DesensitIzation
of
the
TRH-
and
BK-Induced
Intracellular
Ca2+
response
Exposure
of
293-E2
cells
to
a
1
min
pulse
of
TRH
caused
the
expected
transient
increase
in
[Ca2l],
(Figure
5a).
Subsequent
exposure
of
cells
to
further
1
min
pulses
of
TRH
at
various
intervals
(t
=
1000,
2400
and
3600
s;
Figure
5a)
failed
to
elicit
any
further
response.
Similar
results
were
obtained
when
cells
were
continuously
exposed
to
TRH
(results
not
shown).
This
lack
of
response
appeared
not
to
be
related
to
cell
viability.
Using
the
same
experimental
design,
cells
that
initially
showed
no
response
to
buffer
subsequently
responded
normally
to
TRH
(Figure
5b).
This
acute
desensitization
could
not
be
explained
by
changes
in
receptor
number
(Bmax
13.5
pM/mg
of
protein)
or
affinity
(Kd
4
nM)
after
TRH
exposure,
as
these
parameters
were
unaltered
in
control
buffer-treated
cells
compared
with
cells
treated
with
TRH.
The
desensitization
of
the
TRH-induced
Ca2+
response
also
appears
to
be
a
dose-related
phenomenon.
Low-
dose
TRH
pretreatment
(0.1
nM;
Table
1)
partially
desensitized
the
Ca2+
response
to
submaximal
concentrations
of
TRH
(0.1
nM-0.1
uM),
whereas
the
Ca2+
response
to
1
,uM
TRH
was
unaltered.
In
contrast,
pretreatment
with
higher
concentrations
of
TRH
(1
nM,
5
nM,
0.1
M)
completely
desensitized
cells
(results
not
shown).
Treatment
of
293-E2
cells
with
BK
(1
,uM;
Figure
6a)
caused
[Ca2+],
to
rise
from
8.5
+
1.3
at
t
=
47
s
to
145
+
17
nM
at
t
=
59
s.
This
response
was
monophasic
in
nature
and
resembled
the
low-
dose
TRH
responses
observed
in
this
cell
line.
Pretreatment
of
cells
with
a
potent
BK-R
antagonist
(sodium
adamantanecetyl-
D-Arg-[Hyp3,Thi5'8,D-Phe7]bradykinin)
abolished
the
BK-
but
not
the
TRH-induced
mobilization
of
intracellular
Ca2+
(results
not
shown).
As
the
characteristics
of
desensitization,
i.e.
the
rapidity,
extent
and
duration
of
desensitization,
appear
to
be
receptor-specific,
we
also
examined
desensitization
of
the
BK-
induced
[Ca2+],
response
in
293-E2
cells.
After
initial
treatment
with
BK
(1
,uM;
t
=
47
s;
Figure
6a),
cells
subsequently
failed
to
respond
to
BK
(1
,uM;
t
=
2195
s)
but
continued
to
respond
to
TRH
(1
,uM;
t
=
2500
s;
Figure
6a).
So,
although
the
BK-
induced
Ca2+
response
was
smaller
than
that
induced
by
TRH,
BK-R
desensitization
nevertheless
occurred.
These
results
further
imply
that
Ca2+
depletion
is
not
primarily
responsible
for
the
desensitization
phenomenon
observed
in
the
present
experiments.
(a)
TRH
400
-
300
-
200
100*
i
TRH
TRH
TRH
v
4-
c
(b)
Buffer
400
300
-
200
-
100
TRH
0
1000
2000
Time
(s)
L
3000
Figure
5
Effect
of
(a)
several
pulses
of
TRH
and
(b)
buffer
A
and
TRH
on
[Ca2+J,
In
293-E2
cells
(a)
293-E2
cells
initially
challenged
with
TRH
(1
FM;
t
=
98
s)
responded
with
an
increase
in
[Ca2+],
but
failed
to
respond
to
subsequent
regular
TRH
challenges
(1
FM;
t
=
1000,
2500
and
3600
s).
(b)
Application
of
buffer
A
(5
ml;
t
=
100
s)
had
no
effect
whereas
TRH
(1
FuM;
t=
2250
s)
produced
the
expected
increase
in
[Ca2+]i.
300
200
a
2
100*
A
-
4
I
I
I
6...-
m
%#P-
n
v
I
Li
389
m.'
.

390
L.
Anderson
and
others
When
cells
were
pretreated
with
TRH
(1
,#M;
t
=
47
s;
Figure
6b),
no
intracellular
Ca2+
mobilization
was
observed
after
the
application
of
either
BK
(1
,uM;
t
=
2230
s)
or
TRH
(1
sM;
t
=
2430
s).
Similar
results
were
observed
in
293-h20
cells
ex-
pressing
the
human
TRH-R
(Figures
6c
and
6d),
suggesting
that
this
pattern
of
desensitization
is
not
species-specific.
Again
this
desensitization
could
not
be
accounted
for
by
a
reduction
in
receptor
number
(Bmax
0.52
pM/mg
of
protein;
Kd
4
nM).
In
293-E2
cells
this
effect
was
dose-dependent,
as
pretreatment
of
cells
with
lower
TRH
concentrations
(0.1
nM;
t
=
110
s;
Figure
Table
1
DesensfflzatIon
of
the
TRH-induced
Ca2+
response
In
293-E2
cells
after
pretreatment
wIth
low-dose
TRH
293-E2
cells
initially
treated
with
TRH
(0.1
nM)
were
subsequently
challenged
with
various
concentrations
of
TRH
(0.1
nM-1
,uM).
Compared
with
control
responses,
low-dose
TRH
pretreatment
desensitized
subsequent
[Ca2+]
responses
to
low-dose
but
not
to
high-dose
TRH
treatment;
*P
<
0.05,
**P
<
0.001
(n
=
3).
[Ca2+]1
response
in
293-E2
cells
Control
[Ca2+];
after
pretreatment
response
in
293-E2
with
0.1
nM
TRH
[TRH]
cells
(nM) (nM)
0.1
nM
10
nM
0.1
,M
1
,uM
178
+
6
265
+121
242
±293
352
+144
103
+3*
141
+50*
139
+
42**
292
+89
6e)
only
partially
desensitized
the
[Ca2l],
response
to
BK
(1
#etM;
t
=
2125
s).
These
cells
were
then
treated
with
Ca2+-free
buffer
A
containing
EGTA
(2
mM;
5
min).
The
subsequent
addition
of
1
1tM
ionomycin
(Ca2+-free
salt)
produced
a
rapid
rise
in
[Ca2+]i,
suggesting
that
intracellular
Ca2+
stores
had
not
been
depleted.
DISCUSSION
Time-related
changes
in
[Ca2+]1
in
single
HEK-293
cells
expressing
either
the
rat
or
human
TRH-Rs
were
characterized.
Although
HEK-293
cells
do
not
normally
express
TRH-R,
this
cell
line
has
been
commonly
used
for
the
expression
of
a
variety
of
recently
cloned
GPCRs
[30,31].
The
application
of
TRH
produced
a
prompt
dose-dependent
monophasic
increase
in
[Ca2+]1
in
all
imaged
TRH-R-expressing
cells.
As
the
influx
of
extracellular
Ca2+
through
either
SMOCCs
or
L-type
VOCCs
were
not
involved
in
this
TRH-induced
Ca2+
response,
the
involvement
of
intracellular
Ca2+
was
examined.
Treatment
of
293-E2
cells
with
either
of
the
Ca2+-ATPase
inhibitors
TG
and
CPZ
resulted
in
a
transient
but
pronounced
rise
in
[Ca2+]1.
The
subsequent
addition
of
TRH
failed
to
provoke
a
Ca2+
response,
suggesting
that
Ins(1,4,5)P3-sensitive
Ca2+
stores
normally
released
during
TRH-
R
activation
are
absent
and
that
TRH-induced
Ca2+
mobilization
is
dependent
on
these
intracellular
stores.
These
data
are
in
accord
with
the
findings
of
a
recent
study
that
showed
that,
in
cell
lines
lacking
L-type
VOCC
activity
(for
example
HeLa
or
C6
cells),
TRH
similarly
generated
a
monophasic
Ca2+
response
which
was
also
blocked
by
TG
[13].
Physiological
concentrations
of
Ca2+-mobilizing
hormones
(b
BK
TRH
TRI
1 3
1000
2000
3000
0
(c)
BK
TRH
BK
1000
2000 3000
0
BK
TRH
1000
2000
3000
(e)
TRH
TRH
BK
TRH
BK
ION
10,
10
'0.
0
II
0
1000
2000
3000
0
1000
2000
3000
Time
(s)
Figure
6
Effect
of
BK
on
[Ca2+],
response
(a)
Effect
of
BK
pretreatment
on
[Ca2+]i
response
to
subsequent
BK
and
TRH
challenge
in
293-E2
cells.
Although the
application
of
BK
(1
,uM;
t
=
47
s)
produced
a
transient
rise
in
[Ca2+],
it
also
completely
desensitized
the
cells
to
the
effect
of
BK
(1
#M;
t
=
2195
s)
but
only
partially
desensitized
cells
to
the
effects
of
TRH
(1
1zM;
t
=
2500
s).
(b)
TRH
pretreatment
(1
FuM;
t
=
47
s)
abolished
[Ca2+]i
responses
to
both
BK
(1
FM;
t
=
2230
s)
and
TRH
(1
,M;
t
=
2430
s)
in
293-E2
cells.
(c)
Effect
of
BK
pretreatment
on
[Ca2+]1
response
to
subsequent
BK
and
TRH
challenge
in
293-h20
cells
expressing
the
human
TRH-R.
Although
the
application
of
TRH
(1
FM;
t
=
47
s)
produced
a
transient
rise
in
[Ca2+]i,
it
markedly
desensitized
cells
to
the
effect
of
BK
(1
FM;
t=
2080
s)
but
not
TRH
(1
FM;
t
=
2263
s).
(d)
Effect
of
TRH
pretreatment
on
[Ca2+]i
responses
to
TRH
and
BK
in
293-h20
cells.
TRH
pretreatment
(1
F%M;
t
=
47
s)
abolished
subsequent
effects
of
TRH
(1
uM;
t
=
2286
s)
and
BK
(1
FM;
t
=
2523
s)
on[Ca2+]i.
(e)
Low-dose
TRH
pretreatment
(0.1
nM;
t
=
110
s)
partially
desensitized
the
[Ca2+]i
response
to
BK
(1
FM;
t=
2125
s).
Ca2+-free
buffer
A
containing
EGTA
(2
mM;
5
min)
was
then
added,
followed
by
1
FM
ionomycin
(ION).
(a)
BK
I
.i
30u
300-
(d)
200
100
0
2
C4
C)
401
30
20
10
_
anti
I

Desensitization
of
thyrotropin-releasing
hormone
receptors
in
HEK-293
cells
have been
shown
to
produce
repetitive
and
periodic
Ca2l
spikes/oscillations.
These
oscillations
are
believed
to
play
an
important
role
in
cellular
signal-transduction
processes
[15,161.
In
the
present
study,
TRH-induced
Ca2l
oscillations
were
occasionally
observed.
As
these
oscillations
were
an
apparently
random
occurrence
in
only
a
small
proportion
of
the
cells
imaged,
we
were
unable
to
investigate
the
source
of
Ca2+
involved
in
these
responses.
Similar
observations
have,
however,
been
demonstrated
in
HeLaR
cells
transfected
with
the
mouse
TRH-
R
[13].
These
responses
were
independent
of
extracellular
Ca2+
and
more
likely
arose
from
Ca2+
release
and
reuptake
at
intracellular
Ca2+
storage
pools.
Indeed
the
Ca21
oscillations/
spikes
observed
in
both
this
and
the
present
study
are
charac-
teristic
of
those
produced
by
the
cytoplasmic
oscillator
[32]
which
are
typically
insensitive
to
extracellular
Ca2
.
TRH-R
belongs
to
a
family
of
hormone
and
neurotransmitter
receptors
whose
actions
are
mediated
via
the
activation
of
G-proteins.
Suramin
sodium
has
previously
been
shown
to
antagonize
the
interaction
between
receptors
and
the
G-proteins
that
regulate
adenylate
cyclase
[33,34]
or
PLC
activity
[35,36].
It
is
believed
to
intercalate
directly
with
the
receptor-G-protein
complex
thereby
forming
an
a-helix
which
in
some
way
uncouples
the
receptor
from
its
G-protein.
In
the
present
study
the
partial
inhibition
by
suramin
of
the
TRH-induced
rise
in
[Ca2+]1
could
reflect
an
interaction
of
the
compound
with
either
the
TRH-R
or
its
associated
G-protein(s).
Owing
to
the
lack
of
a
specific
TRH-
R
antagonist,
the
effects
of
suramin
on
TRH
binding
and
TRH-
stimulated
[35S]GTP[S]
binding
were
investigated
instead.
The
dose-dependent
inhibition
of
both
TRH
and
[3-Me-His2]TRH-
induced
[35S]GTP[S]
binding
in
293-E2
cells
by
suramin
sodium
without
altering
either
TRH-R
affinity
or
number
suggests
that
suramin
acts
directly
at
the
site
of
receptor-G-protein
coupling
to
prevent
any
interaction
between
these
two
proteins.
Similar
data
have
been
reported
for
the
polyanionic
compound
L-
451,167
in
CHO
cells
expressing
the
cc2-adrenoceptor
[34].
Some
controversy
surrounds
the
desensitization
of
the
G-
protein-coupled
events
of
the
TRH-R.
Whereas
some
groups
have
shown
that
TRH
can
maintain
second-messenger
pro-
duction
at
a
constant
level
for
periods
of
up
to
1
h
[20,37],
others
have
shown
a
marked
reduction
in
the
rate
of
second-messenger
production
within
minutes
of
exposure
to
the
peptide
[4,38,39].
The
methodology
employed
to
measure
the
events
of
receptor
desensitization,
experimental
design,
data
interpretation
and
varying
cell
types
may
all
contribute
to
these
conflicting
results.
To
assess
desensitization
more
directly
at
the
level
of
the
single
cell,
we
examined
the
effects
of
TRH
pretreatment
on
the
TRH-
induced
Ca2+
response
in
293-E2
cells.
After
either
a
1
min
pulse
or
continuous
exposure
of
cells
to
a
high
concentration
of
TRH,
293-E2
cells
at
first
responded
with
the
expected
monophasic
rise
in
[Ca2+]i.
Subsequent
exposure
of
these
cells
to
TRH
at
various
intervals
for
a
peri-od
of
up
to
1
h
failed
to
promote
Ca2+
mobilization.
Desensitization
of
the
TRH-induced
Ca2+
response
was
dose-dependent,
as
low-dose
TRH
pretreatment
reduced,
whereas
high-dose
pretreatment
completely
abolished,
Ca2+
responses
to
subsequent
TRH
treatment.
Although
these
data
suggest
that
TRH-Rs
expressed
in
HEK-293
cells
undergo
rapid
desensitization,
this
desensitization
could
be
attributed
to
the
depletion
of
internal
Ca2+
stores.
We
therefore
challenged
TRH-
treated
cells
with
ionomycin
in
buffer
containing
EGTA.
Under
these
conditions
ionomycin,
but
not
TRH,
was
able
to
mobilize
intracellular
Ca2+.
These
results
indicate
that
intracellular
Ca2+
stores
have
not
been
totally
depleted.
It
should
also
be
noted
that
fura-2-labelled
cells,
which
showed
no
initial
response
to
buffer,
were
capable
of
responding
normally
to
TRH
for
up
to
1
h.
This
ability
could
account
for
the
TRH-induced
desensitization
observed
in
the
present
study.
Although
in
general
the
rat,
human
and
mouse
TRH-Rs
show
high
sequence
homology,
considerable
sequence
variation
exists
at
the
C-terminus
of
these
receptors
[25].
Compared
with
the
human
TRH-R,
the
long
form
of
the
rat
TRH-R
has
an
extended
terminus
containing
an
extra
13
amino
acids
including
an
additional
potential
phosphorylation
site.
The
terminal
region
of
a
variety
of
receptors
including
TRH-R
have
been
associated
with
receptor-G-protein-coupled
events
and
receptor
desen-
sitization
[40].
We
therefore
examined
whether
the
truncated
C-terminus
of
human
TRH-R
altered
the
ability
of
the
receptor
to
desensitize.
Despite
sequence
differences,
the
TRH-induced
Ca2+
response
in
293-h20
cells
expressing
human
TRH-R
was
similarly
desensitized.
Interestingly,
in
a
recent
study
by
Pedersen
et
al.
[39],
desensitization
of
mouse
TRH-R
was
shown
to
be
cell-type-
specific.
Although
the
internalization
and
eventual
loss
of
TRH-Rs
are
associated
with
long-term
exposure
to
TRH
[18-20],
the
rapid
desensitization
observed
in
the
present
study
cannot
be
accounted
for
by
receptor
loss,
as
receptor
number
and
affinity
were
unaltered
after
the
acute
exposure
of
cells
to
TRH.
A
possible
explanation
for
this
rapid
desensitization
is
receptor
phosphoryl-
ation.
The
occupation
of
the
receptor
with
its
agonist
is
believed
to
induce
receptor
phosphorylation
at
multiple
sites
resulting
in
an
uncoupling
of
the
receptor
from
its
cognate
G-protein.
This
is
certainly
the
case
for
the
widely
studied
,-adrenoceptor
[41].
Agonist-dependent
phosphorylation
of
the
PLC-linked
mus-
carinic
M3
[42],
substance
P
[43]
and
cholecystokinin
[44]
receptors
has
also
been
demonstrated.
The
time
course
measured
for
this
receptor
phosphorylation
is
indeed
rapid
enough
to
account
for
the
acute
desensitization
of
the
TRH-induced
Ca2+
response.
Although
rapid
receptor
desensitization
appears
to
be
a
feature
common
to
PLC-linked
receptors,
the
level
of
desensitization
has
been
reported
to
vary
from
receptor
to
receptor
[45,46].
BK-Rs
are
expressed
in
normal
rat
kidney
fibroblasts
[47]
and
in
the
kidney-derived
HEK-293
cells.
In
293-E2
and
293-hlO
cells,
BK
produced
a
transient
monophasic
rise
in
[Ca2+],.
This
appears
to
be
a
receptor-specific
effect,
as
a
potent
BK-R
antagonist
abolished
the
BK-
but
not
the
TRH-induced
mobilization
of
intracellular
Ca2
.
We
subsequently
carried
out
a
series
of
experiments
comparing
the
level
and
patterns
of
desensitization
of
the
endogenously
expressed
BK-R
and
exogenously
expressed
TRH-R
in
293-E2
cells.
High-dose
TRH
treatment
of
cells
resulted
in
desensitization
of
both
the
TRH-
and
BK-induced
Ca2+
response.
In
contrast,
high
concentrations
of
BK
desensitized
the
BK-
but
not
the
TRH-induced
Ca2+
response.
Therefore
despite
similar
intracellular
signalling
pathway,
it
initially
appears
that
these
two
receptors
have
different
desensitization
mechanisms.
However,
it
is
also
possible
that
this
might
simply
reflect
differences
in
the
efficiency
of
receptor-G-
protein
coupling.
It
has
been
suggested
that
there
is
a
regulatory
negative
feedback
loop
between
cytosolic
Ca2+
and
PLC
activity
[48].
The
mobilization
of
intracellular
Ca2+
could
therefore
influence
rapid
desensitization
by
virtue
of
its
ability
to
feedback
negatively
on
PLC
activity.
Perhaps
the
extensive
Ca2+
mobilization
induced
by
TRH
results
in
a
marked
negative
regulatory
effect
on
PLC
activity
thereby
desensitizing
the
responses
to
both
TRH
and
BK.
Compared
with
TRH,
BK
is
less
effective
in
mobilizing
intracellular
Ca2+
and
may
therefore
have
a
smaller
negative
regulatory
effect
on
PLC
activity
such
t-hat
the
subsequent
exposure
to
a
more
vigorous
stimulus,
i.e.
TRH,
is
still
capable
of
mobilizing
Ca2+.
It
has
been
proposed
that
within
any
given
cell
there
is
a
limited
pool
of
G-proteins
391
demonstrates
that
neither
dye-quenching
nor
reduced
cell
vi-

392
L.
Anderson
and
others
shared
by
a
variety
of
endogenous
receptors.
It
is
possible
that
TRH-R
stimulation
and
subsequent
receptor
uncoupling
limits
the
G-protein
pool
available
to
couple
to
other
endogenously
expressed
receptors,
in
this
case
the
BK-R.
The
problems
often
associated
with
the
measurement
of
agonist-induced
changes
in
[Ca2+]1
in
heterogeneous
populations
of
cells
were
eliminated
in
the
present
study
by
combining
dynamic
video
imaging
with
fluorescence
microscopy
to
measure
acute
time-related
changes
in
[Ca2+],
in
single
HEK-293
cells
expressing
either
the
rat
or
human
TRH-Rs.
These
cells
are
non-
pituitary
in
origin
and
apparently
lack
L-type
VOCC
activity.
TRH
produces
a
prompt
dose-dependent
rise
in
[Ca2+]1
in
these
cells,
an
effect
solely
dependent
on
the
mobilization
of
TG/CPZ-
sensitive
intracellular
Ca2+
stores.
We
have
also
clearly
demon-
strated
a
rapid
desensitization
of
the
rat
and
human
TRH-Rs
as
well
as
the
endogeneously
expressed
BK-R.
We
thank
Professor
D.
W.
Lincoln
for
support,
J.
Zabavnik
for
generating
the
293-
E2
cell
line
and
Dr.
P.
L.
Taylor,
J.
V.
Cooke
and
A.
McGregor
for
assistance
with
TRH-R-binding
assays.
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