Vol. 90, No. 4, 1979 October
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
29, 1979
Pages 1371-1378
RAPID DOWN-REGULATION OF PROLACTIN RECEPTORS
IN MAMMARY GLAND AND LIVER 3. Djiane, Laboratoire
H. Clauser and P.A. Kelly* de Physiologie de la Lactation 78350, Jouy-en-Josas, France,
I.N.R.A.,
*MRC Group in Molecular Endocrinology Centre Hospitalier de l'universite Lava1 Quebec GIV 4G2, Canada Received
August
10, 1979
SUMMARY : In contrast with the well known delayed stimulatory effect of prolactin onlevels of its own receptors, a rapid and reversible decline of prolactin receptors was observed in vivo, both with rabbit mammary glands and rat liver, after i.v. injection o-doses of prolactin. This "down regulation" could be clearly distinguished from mere occupation by,desaturating the occupied membrane receptors in vitro with 4M MgC12. The experiments lend strong support to the ubiquitous occurrence of down regulation and to the involvement of compartimentalization and ultimate destruction of the receptor/hormone complex. They further suggest that "down regulation" and "up regulation" are not merely antagonistic regulatory events, but partake in the mechanism of hormone action on the target cell.
INTRODUCTION tissues
:
Prolactin
including
of a large effect
mammary gland
number
liver4.
dissociate with
from
4M MgC12 (for
a short
of other
to the
receptors6.
short-term of evaluating
receptors, tion
is
of its
contending events
which
its
it
own receptor. that
This
down-regulation
might
be intimately
in vivo5
possible on its
studied in turn (and linked
in the
present
so far, would
possibly to the
lend
but, damage
to investigate
tissues,
ability
with
to up-regulate of inducing
some support
mechanism
the prolactir
a down-regula-
to recent
as well)
to
membranes
hormones
study
up-regulation very
techniques
irreversible
in target
capable
effect
mammary gland'
lactogenic
not cause
to its
rabbit
incubating
does
of
a stimulatory
and in vitro6
bound
receptors
in addition
both
involves
removes
agents,
in
number
inhibitory
own receptorL,
The latter
which
proved
hormones
of their
both
dissociating
prolactin,
most
level
receptors.
period),
in a large to the
has been observed
of prolactin if
like
on the
distributed
In contrast
and liverl.
receptor
Hence, action
are widely
we developed
prolactin
a number
goal
on its Recently,
unlike
the
of hormones
of prolactin
and rat
receptors
views7
are ubiquitous
of hormone
action.
0006-291X/79/201371-08$01.00/0 1371
Copyright All rights
@ I979 by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIAL AND METHODS : In this study, lactating, New Zealand rabbits and adult ovariectomized rats were used. Seven rabbits at 10 days of lactation were injected every 1.2 hours over a 36-hour period with 2 mg of the dopamine5agonist, 2-bromo-a-ergocryptine (CB-154), to lower circulating prolactin levels after which the animals were anesthesized with 50 mg/kg of sodium pentobarbital. Three mg bovine prolactin (bPRL) were injected intravenously and 2 g biopsies of mammary gland tissue were removed at the indicated times between 0 and 1 hour after prolactin injection. Animals were subsequently re-anesthesized at 6, 24 and 30 hours just prior to removal of biopsies. In another experiment, groups of 8 rats ovariectomized 3 days previously and treated every 12 hours, starting 24 hours before the onset of the study, with 500 ug of CB-154, were injected with 1 mg of bovine PRL and the animals were sacrificed by decapitation at various intervals from 0 to 36 hours after the PRL injection. In both the rabbit and rat study, CB-154 injections were continued every 12 hours8until the end of the experiment. Membranes were prepared as described previously . Four hundred (mamnar gland) or 300 (liver) ug of membrane protein were incubated with approximately 10 8 cpm vine prolactin labelled with 125I 689 . Specific using chloramine-T at a low concentration binding was calculated al the difference between binding in the absence and presence of excess unlabelled oPRL (1 ug). In vitro dissociation with 4M MgC12 was performed on parallel samples to remove the exogenous prolactin from its receptor . This allowed the measurement of free and total (MgC12-treated) prolactin binding sites. --RESULTS:
occupancy
As illustrated
in
of free
mamnary gland
rabbit
venous
injection.
lactin
at the corresponding
time
prolactin
present
circulating receptors
Table
Fig.1,
remained
were
incomplete
dissociation
membrane
preparations', between
concentrations between dissociation
prolactin
interval.
15 min after
from
prolactin the
of prolactin
from
(and unpublished
15 min and 1 to 6 hours of circulating
24 and 30 hours. rates
This
of the
saturating
its
20% of the
octuring
in vitro
an apparent
in vivo , even
in the
receptor
represent
prolactin
to the very
observed
observations),
between
of
be due to an inaccessibility
receptors
difference
pro-
concentrations
In contrast
Free
the intrainjected
or to some dissociation
prolactin.
of PRL may in reality
concentration
could
tissues.
to a maximal
15 min after
injection,
This
led
receptors
Although
125 I oPRL.
to bind
isolated
of 3 mg of prolactin
the circulating
to the circulating
membranes
begins
were free
of the receptors while
1 shows
injection
apparent
new receptors
with
of high
returned
in vitro
and
dissociation
presence
levels
slow
to normal
and in vivo recently
synthe-
assayed
following
sized.
in vitro
Somewhat
surprisingly,
desaturation
with
total
prolactin
4M MgC12 declined
1372
receptor progressively
levels
up to 6 hours
after
the
BIOCHEMICAL
Vol. 90, No. 4, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
FREE TOTAL
jmin
15min TIME AFTER
lh 6h INJECTION
Figure 1. - Effect of an intravenous injection of 3 mg bovine prolactin (NIH-P-BS, 32.2 IU/mg) on prolactin binding sites in rabbit mammary glands. Biopsies (2 g) were removed at the indicated times after PRL injection, from lactating rabbits (10 days) which had been injected with CB-154 for a 36h period (every 12h) prior to the PRL treatment. In vitro dissociation of prolactin from its receptor was performed as described(6)fly, the amount of crude membrane protein normally used for binding assays (400 ug for rabbit mammary gland or 300 pg for rat liver) was incubated with 0.5 ml of 4M MgC12 for 5-10 min after which 3.5 ml of cold Tris-HCl buffer (pH 7.4) containing 10 mM MgC12 and 0.1% BSA was added to each tube. Following centrifugation at 2200 x g for 15 min, the supernatantq were aspirated and the pellets resuspended in Tris-HCl buffer. Binding of { 2511 oPRL was measured by incubating duplicate tubes containing approximately 105 cpm labelled PRL. Specific binding was the difference in the cpn bound in the absence and presence of excess unlabelled hormone (1 ug oPRL) and was expressed as a percent of the total radioactivity added to the tube. Non-specific binding averaged 3-4% of the total cpm added. Binding was determined in H20-treated membranes to measure free receptors and MgC12-treated membranes to measure total receptors. Open bars (free receptors) show the occupation of receptors by the injected prolactin. Crosshatched bars represent total receptors. Binding is expressed as % specific binding per 400 ug membrane protein. Values are means fSEM of 7 animals.
intravenous difference
injection in
total
significant
(p
< 0.01)
difference
was
observed
down-regulation between
of
prolactin
prolactin as
6 hours.
binding determined
between
reflected 1 and
and
by The
the total
possibility
returned
to
between
time
by
Duncan-Kramer's
pattern
of
receptors. that
1373
0 and
at
24
6 hours multiple
occupation In
the
normal
30
was range receptors)
free
receptors
in
total
hours.
The
statistically
(free
addition, reduction
to
test
prolactin
10 and
. A the
increased recep-
Vol. 90, No. 4, 1979
BIOCHEMICAL
TABLE 1 - Serum levels after
of bovine prolactin
the intravenous
radioreceptor
injection
assay',
bPRL concentrations
AND BIOPHYSICAL
since in rats
in lactating
of prolactin. the specific (Table
RESEARCH COMMUNICATIONS
rabbits
Hormone levels
antiserum
2) was raised
were measured by
in a rabbit. bPRL (rig/ml)
0
leve7s
its
receptor
hormones"
< 10
1 min
15,956 -r 1995
5 min
11,270 -r 1043
15 min
4,415 f
496
1 hour
823 +
180
6 hours
77 +
37
24 hours
< 10
30 hours
< 10
was due to a reduced cannot as well
gradually
as such would
of the
be totally as for
tightens
regulation",
with
since
both
receptors
following
rat
(Fig.2).
> 1000 rig/ml
dissociation Total
after
were binding
at 6 hours
compared
to distinguish
from
may involve
hormone of the
the
receptor
from
peptide interaction
hormone-receptor
process
referred
compartementalization
time-dependent
intravenous occupation
injection.
were
of available
has been shown for
the
tightening
to the
Maximal
still
of prolactin
receptors
it
prolactin
linkage
to as "downand processing
complex.
prolactin
tin
since
However,
events
similar
12 hours
of MgC12 to dissociate
dismissed,
time.
A pattern
levels
efficiency
catecholaminesl'that
be difficult
hormone-receptor
liver
times
to bPRL used to measure plasma
TIME
tot-
at various
injection occured
from
its
1 hour
at 15 min with
higher
1 min after
sites,
however, time
2, serum
after
receptor
injection,
receptor
0 (p < 0.05)
1374
was observed a return
levels thus
or new receptor
PRL injection, total
of the mammary gland
of prolactin
As seen in Table present
to either
occupation
possibly levels
were
or 1 min after
to normal
of bovine suggesting
synthesis
with
prolacapparent
or processing.
due to a protection significantly injection
reduced (p < 0.01).
Vol. 90, No. 4, 1979
TABLE venous
2 - Plasma injection
immunoassay
BIOCHEMICAL
levels of
using
of
bovine
prolactin.
an anti-bovine
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
prolactin Hormone
in
rats
levels
were
PRL antiserum
and
at
various
measured { I25Il
TIME
times
after
by a specific
intraradio-
bPRL.
bPRL
0
(rig/ml)
< 1
1 min
56,490
+ 1580
15 min
10,700
+
690
1,150
+
130
1 hour 6 hours
40
+
12 hours
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
29, 1979
Pages 1371-1378
RAPID DOWN-REGULATION OF PROLACTIN RECEPTORS
IN MAMMARY GLAND AND LIVER 3. Djiane, Laboratoire
H. Clauser and P.A. Kelly* de Physiologie de la Lactation 78350, Jouy-en-Josas, France,
I.N.R.A.,
*MRC Group in Molecular Endocrinology Centre Hospitalier de l'universite Lava1 Quebec GIV 4G2, Canada Received
August
10, 1979
SUMMARY : In contrast with the well known delayed stimulatory effect of prolactin onlevels of its own receptors, a rapid and reversible decline of prolactin receptors was observed in vivo, both with rabbit mammary glands and rat liver, after i.v. injection o-doses of prolactin. This "down regulation" could be clearly distinguished from mere occupation by,desaturating the occupied membrane receptors in vitro with 4M MgC12. The experiments lend strong support to the ubiquitous occurrence of down regulation and to the involvement of compartimentalization and ultimate destruction of the receptor/hormone complex. They further suggest that "down regulation" and "up regulation" are not merely antagonistic regulatory events, but partake in the mechanism of hormone action on the target cell.
INTRODUCTION tissues
:
Prolactin
including
of a large effect
mammary gland
number
liver4.
dissociate with
from
4M MgC12 (for
a short
of other
to the
receptors6.
short-term of evaluating
receptors, tion
is
of its
contending events
which
its
it
own receptor. that
This
down-regulation
might
be intimately
in vivo5
possible on its
studied in turn (and linked
in the
present
so far, would
possibly to the
lend
but, damage
to investigate
tissues,
ability
with
to up-regulate of inducing
some support
mechanism
the prolactir
a down-regula-
to recent
as well)
to
membranes
hormones
study
up-regulation very
techniques
irreversible
in target
capable
effect
mammary gland'
lactogenic
not cause
to its
rabbit
incubating
does
of
a stimulatory
and in vitro6
bound
receptors
in addition
both
involves
removes
agents,
in
number
inhibitory
own receptorL,
The latter
which
proved
hormones
of their
both
dissociating
prolactin,
most
level
receptors.
period),
in a large to the
has been observed
of prolactin if
like
on the
distributed
In contrast
and liverl.
receptor
Hence, action
are widely
we developed
prolactin
a number
goal
on its Recently,
unlike
the
of hormones
of prolactin
and rat
receptors
views7
are ubiquitous
of hormone
action.
0006-291X/79/201371-08$01.00/0 1371
Copyright All rights
@ I979 by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIAL AND METHODS : In this study, lactating, New Zealand rabbits and adult ovariectomized rats were used. Seven rabbits at 10 days of lactation were injected every 1.2 hours over a 36-hour period with 2 mg of the dopamine5agonist, 2-bromo-a-ergocryptine (CB-154), to lower circulating prolactin levels after which the animals were anesthesized with 50 mg/kg of sodium pentobarbital. Three mg bovine prolactin (bPRL) were injected intravenously and 2 g biopsies of mammary gland tissue were removed at the indicated times between 0 and 1 hour after prolactin injection. Animals were subsequently re-anesthesized at 6, 24 and 30 hours just prior to removal of biopsies. In another experiment, groups of 8 rats ovariectomized 3 days previously and treated every 12 hours, starting 24 hours before the onset of the study, with 500 ug of CB-154, were injected with 1 mg of bovine PRL and the animals were sacrificed by decapitation at various intervals from 0 to 36 hours after the PRL injection. In both the rabbit and rat study, CB-154 injections were continued every 12 hours8until the end of the experiment. Membranes were prepared as described previously . Four hundred (mamnar gland) or 300 (liver) ug of membrane protein were incubated with approximately 10 8 cpm vine prolactin labelled with 125I 689 . Specific using chloramine-T at a low concentration binding was calculated al the difference between binding in the absence and presence of excess unlabelled oPRL (1 ug). In vitro dissociation with 4M MgC12 was performed on parallel samples to remove the exogenous prolactin from its receptor . This allowed the measurement of free and total (MgC12-treated) prolactin binding sites. --RESULTS:
occupancy
As illustrated
in
of free
mamnary gland
rabbit
venous
injection.
lactin
at the corresponding
time
prolactin
present
circulating receptors
Table
Fig.1,
remained
were
incomplete
dissociation
membrane
preparations', between
concentrations between dissociation
prolactin
interval.
15 min after
from
prolactin the
of prolactin
from
(and unpublished
15 min and 1 to 6 hours of circulating
24 and 30 hours. rates
This
of the
saturating
its
20% of the
octuring
in vitro
an apparent
in vivo , even
in the
receptor
represent
prolactin
to the very
observed
observations),
between
of
be due to an inaccessibility
receptors
difference
pro-
concentrations
In contrast
Free
the intrainjected
or to some dissociation
prolactin.
of PRL may in reality
concentration
could
tissues.
to a maximal
15 min after
injection,
This
led
receptors
Although
125 I oPRL.
to bind
isolated
of 3 mg of prolactin
the circulating
to the circulating
membranes
begins
were free
of the receptors while
1 shows
injection
apparent
new receptors
with
of high
returned
in vitro
and
dissociation
presence
levels
slow
to normal
and in vivo recently
synthe-
assayed
following
sized.
in vitro
Somewhat
surprisingly,
desaturation
with
total
prolactin
4M MgC12 declined
1372
receptor progressively
levels
up to 6 hours
after
the
BIOCHEMICAL
Vol. 90, No. 4, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
FREE TOTAL
jmin
15min TIME AFTER
lh 6h INJECTION
Figure 1. - Effect of an intravenous injection of 3 mg bovine prolactin (NIH-P-BS, 32.2 IU/mg) on prolactin binding sites in rabbit mammary glands. Biopsies (2 g) were removed at the indicated times after PRL injection, from lactating rabbits (10 days) which had been injected with CB-154 for a 36h period (every 12h) prior to the PRL treatment. In vitro dissociation of prolactin from its receptor was performed as described(6)fly, the amount of crude membrane protein normally used for binding assays (400 ug for rabbit mammary gland or 300 pg for rat liver) was incubated with 0.5 ml of 4M MgC12 for 5-10 min after which 3.5 ml of cold Tris-HCl buffer (pH 7.4) containing 10 mM MgC12 and 0.1% BSA was added to each tube. Following centrifugation at 2200 x g for 15 min, the supernatantq were aspirated and the pellets resuspended in Tris-HCl buffer. Binding of { 2511 oPRL was measured by incubating duplicate tubes containing approximately 105 cpm labelled PRL. Specific binding was the difference in the cpn bound in the absence and presence of excess unlabelled hormone (1 ug oPRL) and was expressed as a percent of the total radioactivity added to the tube. Non-specific binding averaged 3-4% of the total cpm added. Binding was determined in H20-treated membranes to measure free receptors and MgC12-treated membranes to measure total receptors. Open bars (free receptors) show the occupation of receptors by the injected prolactin. Crosshatched bars represent total receptors. Binding is expressed as % specific binding per 400 ug membrane protein. Values are means fSEM of 7 animals.
intravenous difference
injection in
total
significant
(p
< 0.01)
difference
was
observed
down-regulation between
of
prolactin
prolactin as
6 hours.
binding determined
between
reflected 1 and
and
by The
the total
possibility
returned
to
between
time
by
Duncan-Kramer's
pattern
of
receptors. that
1373
0 and
at
24
6 hours multiple
occupation In
the
normal
30
was range receptors)
free
receptors
in
total
hours.
The
statistically
(free
addition, reduction
to
test
prolactin
10 and
. A the
increased recep-
Vol. 90, No. 4, 1979
BIOCHEMICAL
TABLE 1 - Serum levels after
of bovine prolactin
the intravenous
radioreceptor
injection
assay',
bPRL concentrations
AND BIOPHYSICAL
since in rats
in lactating
of prolactin. the specific (Table
RESEARCH COMMUNICATIONS
rabbits
Hormone levels
antiserum
2) was raised
were measured by
in a rabbit. bPRL (rig/ml)
0
leve7s
its
receptor
hormones"
< 10
1 min
15,956 -r 1995
5 min
11,270 -r 1043
15 min
4,415 f
496
1 hour
823 +
180
6 hours
77 +
37
24 hours
< 10
30 hours
< 10
was due to a reduced cannot as well
gradually
as such would
of the
be totally as for
tightens
regulation",
with
since
both
receptors
following
rat
(Fig.2).
> 1000 rig/ml
dissociation Total
after
were binding
at 6 hours
compared
to distinguish
from
may involve
hormone of the
the
receptor
from
peptide interaction
hormone-receptor
process
referred
compartementalization
time-dependent
intravenous occupation
injection.
were
of available
has been shown for
the
tightening
to the
Maximal
still
of prolactin
receptors
it
prolactin
linkage
to as "downand processing
complex.
prolactin
tin
since
However,
events
similar
12 hours
of MgC12 to dissociate
dismissed,
time.
A pattern
levels
efficiency
catecholaminesl'that
be difficult
hormone-receptor
liver
times
to bPRL used to measure plasma
TIME
tot-
at various
injection occured
from
its
1 hour
at 15 min with
higher
1 min after
sites,
however, time
2, serum
after
receptor
injection,
receptor
0 (p < 0.05)
1374
was observed a return
levels thus
or new receptor
PRL injection, total
of the mammary gland
of prolactin
As seen in Table present
to either
occupation
possibly levels
were
or 1 min after
to normal
of bovine suggesting
synthesis
with
prolacapparent
or processing.
due to a protection significantly injection
reduced (p < 0.01).
Vol. 90, No. 4, 1979
TABLE venous
2 - Plasma injection
immunoassay
BIOCHEMICAL
levels of
using
of
bovine
prolactin.
an anti-bovine
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
prolactin Hormone
in
rats
levels
were
PRL antiserum
and
at
various
measured { I25Il
TIME
times
after
by a specific
intraradio-
bPRL.
bPRL
0
(rig/ml)
< 1
1 min
56,490
+ 1580
15 min
10,700
+
690
1,150
+
130
1 hour 6 hours
40
+
12 hours
