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From the
We have monitored agonist-induced
The
Despite these important advances,
significant gaps remain in our understanding of
Attempts to study
pCMV/
Specific immunoreactivity of
this antibody toward the receptor polypeptide was tested on immunoblots
by two approaches, as shown in Fig. 1. First,
These antibodies were used to study the influence of agonist
exposure on
The
agonist-induced redistribution was reversible. Exposure of cells to
norepinephrine for 1 h followed by removal of agonist and addition of
fresh growth medium containing prazosin restored the original diffuse
pattern of immunostaining within 15-30 min (Fig. 4). Similar
reversal of receptor redistribution was observed upon removal of
agonist from cultures treated with norepinephrine for 24 h (data not
shown).
The sustained and linear accumulation of
inositol phosphates stimulated by agonist presents an apparent conflict
with the rapid agonist-induced receptor internalization. To reconcile
this conflict we reasoned that the observed inositol phosphate response
might reflect multiple processes of receptor activation,
desensitization, or internalization. To test this hypothesis cells were
subjected to treatments which had been shown previously to alter the
subcellular distribution of the receptor, and the effects on
agonist-stimulated [
The development of an immunological probe for the
Taken together, these data lead to a working model
relating receptor activation, desensitization, and recycling. Salient
features of the model are summarized as follows. 1) Agonist activates
the receptor, generating inositol phosphate and diacylglycerol second
messengers. Robust agonist-elicited phosphoinositide metabolism is
demonstrated in Fig. 9; we have found functional coupling of this
response to the end point of elevated intracellular Ca
These results
offer insights into several aspects of the relationship between
A related consideration concerns the
relationship between receptor number and response. Our data suggest
that at steady state response a fraction of receptors will be
desensitized or inaccessible to agonist compared to naive cells. Thus
one may predict an initial transient activation of response followed by
a decline to the maintained steady state. Interestingly, Nahorski and
co-workers
(41) describe such a temporal sequence of activation
and rapid partial desensitization ( t
At present the signals
controlling
Significant
parallels exist between our data and extensive studies on agonist
regulation of
In conclusion, we demonstrate that agonist
activation of
We are indebted to Dr. Robert J. Lefkowitz for the
Volume 270,
Number 15,
Issue of April 14, pp. 8902-8909, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Adrenergic Receptor Subcellular
Distribution and Function (*)
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic
receptor (
AR) redistribution by immunocytochemical
procedures in concert with functional measurements of agonist-elicited
[
H]inositol phosphate (InsP) production in human
embryonal kidney 293 cells stably expressing AR cDNA
(HEK293/
). Anti-peptide antibodies directed against
the carboxyl-terminal decapeptide of the
AR were
prepared and shown to react specifically with
AR on
immunoblots and in situ in HEK293/
transfectants. Treatment of HEK293/
cells with
norepinephrine (10 µ
M) results in a rapid (5-15 min)
and striking internalization of cell surface receptor as visualized by
confocal immunofluorescence microscopy. Receptor redistribution is
sustained in the presence of agonist, rapidly reversed upon agonist
removal, and prevented by the
antagonist prazosin.
Receptor internalizes to endosomes, as shown by colocalization with
transferrin receptor, an endosomal marker. Activation of protein kinase
C (PKC) with phorbol 12-myristate 13-acetate (50 n
M) causes
receptor endocytosis similar to agonist; agonist-induced
internalization is blocked by the PKC inhibitor staurosporine (0.5
µ
M). In parallel experiments, agonist-induced
[H]InsP production is abolished by phorbol
12-myristate 13-acetate but potentiated by staurosporine. Inhibition of
receptor internalization with hypertonic sucrose attenuates
agonist-induced [H]InsP formation; this effect is
reversed by concomitant inhibition of PKC with staurosporine. These
results suggest that PKC-dependent phosphorylation occurring as a
consequence of AR stimulation induces receptor
desensitization and internalization. Internalized receptor is
reactivated and continuously recycled to the cell surface during
agonist exposure.
-adrenergic receptors
(AR)()
have long been recognized
to play a key role in regulation of blood pressure by the sympathetic
nervous system
(1) . Considerable progress has been made toward
a molecular description of the structures and signal transduction
mechanisms of -adrenergic receptors
(2) . The
primary structure of the -adrenergic receptors
identified by molecular cloning corresponds to the predicted
topographic model of the superfamily of G protein-coupled receptors,
which features a motif of seven transmembrane domains
(3) .
Substantial evidence indicates the importance of agonist and G
protein-regulated phospholipase C to generate phosphoinositide-derived
second messengers for Casignaling in response to
-adrenergic receptor activation
(2, 4) . The convergence of recent pharmacological
(5, 6, 7, 8) and molecular cloning
(9, 10, 11, 12) studies have revealed
the presence of isoforms of -adrenergic receptors. The
different structural subtypes of -adrenergic receptors
have been proposed to couple to distinct pathways of phosphoinositide
signaling and cellular Caregulation
(4, 13) .
receptor signaling. One area of key interest concerns the
disposition of the receptor in the cell membrane, and the influence of
agonist on receptor distribution, responsiveness, and metabolism.
Desensitization of AR responses by agonist has been
reported although the kinetics of these processes vary among different
systems
(14, 15, 16) . Controversy also exists
over whether agonists cause sequestration of the receptor from the
extracellular surface
(16, 17, 18) .
Furthermore, prolonged agonist exposure has been reported to decrease
total receptor number in some
(19, 20) but not all
systems
(14, 21) .
receptor distribution at the subcellular level have been limited
by the lack of specific structural probes of the receptor. In other
systems, immunological approaches have provided powerful tools for
studying receptor localization and organization at the cellular level,
as well as revealing important insights into the mechanisms of receptor
regulation
(22, 23, 24) . In the present study
we have developed a novel anti-peptide antibody directed against a
unique decapeptide sequence corresponding to the carboxyl terminus of
the -adrenergic receptor. The antibody specifically
recognizes the receptor on immunoblots and in situ in human
embryonal kidney 293 (HEK 293) cells transfected and selected for
stable expression of the cDNA for the
receptor
(HEK293/
). Using this experimental system we have
combined immunocytochemical localization of the receptor with
functional measurements of [
H]inositol phosphate
production to examine the mechanisms of agonist-induced receptor
redistribution and modulation of receptor responsiveness. The results
provide evidence for a cyclic mechanism of receptor activation,
desensitization, internalization, and recovery upon agonist
stimulation.
Materials
Cell culture media, reagents, and
normal goat and rabbit sera were from Life Technologies, Inc. Fetal
bovine serum was from Irvine Scientific (Irvine, CA), Bioproducts
Source (Indianapolis, IN), or Atlanta Biologicals (Norcross, GA).
[H]Inositol (20 Ci/mmol) and
[H]prazosin (70-80 Ci/mmol) were from
DuPont NEN. [H]Phenoxybenzamine (20 Ci/mmol) was
synthesized according to Kan et al. (25) by Amersham
Int. (Buckinghamshire, United Kingdom). It was stored at -20
°C under nitrogen. Purity was assessed by thin layer chromatography
on silica gel developed with toluene:methanol (85:15). Sephadex G-50,
(-)-norepinephrine-(+)-bitartrate,
L-phenylephrine-HCl, phorbol 12-myristate 13-acetate,
staurosporine, calphostin C, poly-
D-lysine, bovine serum
albumin, gelatin, keyhole limpet hemocyanin, superoxide dismutase, and
catalase were from Sigma. Prazosin-HCl was a gift from Pfizer (Groton,
CT). Phentolamine-HCl was a gift from Ciba-Geigy (Summit, NJ).
Quantitative protein assay reagents were from Pierce. Electrophoresis
reagents, goat anti-rabbit secondary antibodies, and other
immunochemical supplies were from Bio-Rad or Jackson Immunoresearch.
Mouse monoclonal anti-transferrin receptor antibodies were from
Amersham or Boehringer. Nitrocellulose (0.45 µm) was from
Schleicher and Schuell. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide
was from Calbiochem. Digitonin (lot 1191) was from Gallard-Schlesinger
(Carle Place, NY). Wheat germ agglutinin-Sepharose was from E-Y Labs
(San Mateo, CA). A cDNA clone of the -adrenergic
receptor (pBC12BI/
, Ref. 9) was generously provided
by Dr. Robert J. Lefkowitz, Duke University Medical School. Additional
reagents and supplies were obtained from conventional sources and were
the highest purity available.
Generation of Anti-peptide Antibodies
A peptide
with the sequence (C)-SNMPLAPGHF corresponding to residues
506-515 at the carboxyl terminus of the hamster
-adrenergic receptor
(9) was synthesized
commercially (Multiple Peptide Systems, La Jolla, CA). Purity was
higher than 90% as assessed by reverse phase high performance liquid
chromatography. Identity of the peptide was confirmed by mass
spectroscopy. The peptide was coupled to keyhole limpet hemocyanin with
m-maleimidobenzoyl- N-hydroxysuccinimide through the
terminal cysteine residue by Multiple Peptide Systems and rabbit
immunizations (two rabbits with boosts at 2-week intervals) were
performed by Bethyl Laboratories (Montgomery, TX). Antisera were
screened by standard enzyme-linked immunosorbent assay procedures
(26) against immobilized peptides, and gave titers up to
1:10,000. [
H]Phenoxybenzamine Labeling and Partial
Purification of -Adrenergic
Receptors-Reaction of
-adrenergic receptors
with [
H]phenoxybenzamine was performed
essentially as described previously
(27) . Briefly, DDT
MF-2 cells (0.5
10
cells/ml, 3
10
cells/preparation) were harvested, washed, and
resuspended in 1% of the original volume of phosphate-buffered saline
(PBS, 140 m
M NaCl, 5 m
M KCl, 10 m
M NaPO
, pH 7.4).
[
H]Phenoxybenzamine (70-100 n
M)
was added for 60 min at 30 °C with gentle agitation. The reaction
was quenched by adding 0.1%
-mercaptoethanol and incubating an
additional 30 min. Cells were diluted with 2-3 volumes of PBS,
washed by centrifugation, resuspended in one-tenth volume of PBS, and
sonicated on ice for 2 min. Membranes were collected by centrifugation
(20,000 g, 60 min, 4 °C). Solubilization of
membranes with digitonin and wheat germ agglutinin chromatography of
[
H]phenoxybenzamine-labeled receptor were
performed as described previously
(28) . Receptor samples were
desalted on Sephadex G-75 columns equilibrated with 50 m
M NH
-HCO
, concentrated, and stored at
-70 °C for use in immunoblotting or autoradiography
protocols.
Immunoblotting
Proteins were resolved by
electrophoresis through 10% SDS-PAGE minigels and electrotransferred
overnight (300 mA) to nitrocellulose in transfer buffer composed of 0.7
M glycine, 25 m
M Tris-HCl, pH 7.7
(29) .
Immunoblots were processed according to the manufacturer's
procedure (Bio-Rad). Briefly, nitrocellulose strips corresponding to
individual lanes from the gel were cut and incubated in blocking buffer
(500 m
M NaCl, 20 m
M Tris-HCl, pH 7.5) containing 3%
gelatin for 1 h, followed by anti-peptide antisera (1:500-1:1000
dilution) in blocking buffer containing 1% gelatin and 0.05% Tween 20
for 2 h. The strips were washed with blocking buffer and incubated with
goat anti-rabbit IgG conjugated with alkaline phosphatase as specified
by the manufacturer (Bio-Rad). Immunoblots were developed with
5-bromo-4-chloro-3-indoylphosphate and nitro blue tetrazolium. In some
experiments, parallel immunoblotting and autoradiography were performed
on [H]phenoxybenzamine-labeled receptor preparations. In this case replicate nitrocellulose
strips from the electrotransfer procedure either were immunoblotted as
described above, or were subjected to autoradiography by exposure with
Kodak X-Omat film with intensifying screens for 2 weeks at -70
°C.
Immunofluorescence Staining
HEK 293 cells stably
expressing receptor were subcultured in 35-mm dishes
containing poly-
D-lysine-coated coverslips. At subconfluence
(2-4 days), cells were processed for immunostaining. Treatments
of cultures with experimental agents were performed in growth medium
for the indicated times before processing. Treatment with
norepinephrine (NE) was always accompanied with propranolol (1
µ
M) to prevent activation of endogenous
-adrenergic
receptors, and with catalase (10 µg/ml) and superoxide dismutase
(10 µg/ml) to prevent catecholamine oxidation. Cells were fixed in
3.7% formaldehyde in PBS for 15 min. Fixed specimens were blocked for 2
h at room temperature with PBS containing 10% normal goat serum, 1%
bovine serum albumin, 0.05-0.1% Triton. For single antibody
labeling, specimens were incubated with receptor antibody
(1:500-1:1000 dilutions in blocking solution) at 4 °C
overnight, followed by fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG (Jackson) at a dilution of 1:400 in the blocking buffer
(1 h incubation at room temperature). In double immunofluorescence
labeling experiments, mouse monoclonal anti-transferrin antibody (1:100
dilution) was incubated with the fixed cells for 3 h at room
temperature followed by addition of anti-receptor antibody and
overnight incubation with both antibodies at 4 °C. Respective
antibody labelings were visualized with Lissamine rhodamine-goat
anti-mouse and fluorescein isothiocyanate-goat anti-rabbit IgGs.
Coverslips were mounted in Slow Fade mounting solution (Molecular
Probes). Immunofluorescence staining was examined by conventional
inverted fluorescence microscopy (Zeiss or Nikon) or by confocal
fluorescence microscopy with 1-µm optical sections. Confocal
fluorescence microscopy was performed at the Electron Microscopy and
Imaging Facility, Northwestern University School of Medicine, Chicago.
Stable Expression of
A 2.1-kilobase EcoRI
fragment containing the coding sequence of the hamster
-Adrenergic
Receptors in HEK 293 Cells
-adrenergic receptor (pBC12BI/
,
kindly provided by Dr. Robert J. Lefkowitz, Duke University Medical
School) was subcloned into pBluescript SK(+) (Stratagene, La
Jolla, CA). The pBluescript/
plasmid was used as the
source of a 2.1-kilobase HindIII- XbaI fragment that
was inserted at the HindIII and XbaI sites of the
eukaryotic expression vector pRc/CMV (Invitrogen). The construct,
pCMV/
, is capable of high levels of receptor
expression in mammalian cells.
was used to
transfect cultures of HEK 293 cells (adenovirus-transformed human
embryonal kidney cells, Ref. 30), seeded at 1-2
10
cells/10-cm dish by the calcium phosphate-mediated gene transfer
method of Chen and Okayama
(31) . One day following exposure of
cells to the DNA precipitate, cultures were re-plated at a
5-10-fold lower density in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum and 400
µg/ml G418. Cultures were maintained in selection medium for
2-4 weeks with fresh medium changes twice per week. Resistant
colonies were pooled and stock cultures of pooled transfectants stably
expressing receptors (10-20 pmol/mg membrane
protein by Scatchard analysis) were propagated in growth medium
containing G-418. Measurement of Agonist-elicited [
H]Inositol
Phosphate Production-Protocols for equilibration of HEK 293
cell cultures with [H]inositol, experimental
exposure to agonist, and analysis of [H]inositol
phosphate production were performed essentially as described previously
(32) . Briefly, cultures equilibrated 24-48 h with
[H]inositol (2-5 µCi/ml) were exposed
to physiological salt solution supplemented with LiCl (Li-PSS,
composition in millimolar: NaCl, 130; LiCl, 10; KCl, 5.4;
CaCl
, 1.8; MgCl, 1.6;
NaHPO
, 1.0;
D-glucose, 5.5; HEPES, 10;
pH 7.4) and specified agonist additions for indicated times at 37
°C. Addition of the -adrenergic agonist norepinephrine was
routinely accompanied by 1 µ
M propranolol and 10 µg/ml
each of superoxide dismutase and catalase. Reactions were terminated
with ice-cold 10% trichloroacetic acid. Samples were extracted with
ethyl ether, neutralized, and fractionated by anion exchange
chromatography (Dowex 18-formate). The columns were eluted with
water to remove [
H]inositol and then total
[H]inositol phosphates were collected by eluting
with 1.2
M ammonium formate in 0.1
M formic acid
according to published procedures
(32) .
Additional Procedures
Culture of DDTMF-2
(28) and HEK 293
(24, 30) cell lines
were performed as described previously. Receptor density was assessed
by [H]prazosin binding to intact cells or
membrane preparations by established procedures
(16) . Protein
determinations were performed by Bradford
(33) or bicinchoninic
acid assay (Pierce).
Anti-peptide antibodies were prepared in rabbits
against a unique amino acid sequence (amino acids 506-515) that
corresponds to the cytoplasmic carboxyl terminus of the cloned hamster
-Adrenergic Receptor Recognition
by Immunoblotting Using Anti-peptide
Antibodies
-adrenergic receptor. The antiserum reacted with high
specificity and sensitivity to the cognate peptide by enzyme-linked
immunosorbent assay (data not shown).
receptors from DDT
MF-2 cells were covalently
radiolabeled with [H]phenoxybenzamine,
solubilized with digitonin, and partially purified by wheat germ
agglutinin chromatography
(27) . Replicate samples of this
preparation were subjected to SDS-PAGE and examined by autoradiography
to visualize the radiolabeled receptor, or by immunoblotting. As shown
in Fig. 1 A, Lane 1,
[H]phenoxybenzamine labeled polypeptides of
approximate molecular mass = 80 kDa. The antiserum immunoreacted
with a band that co-migrated with the radiolabeled polypeptide
(Fig. 1 A, Lane 2). Previous studies have identified this
80-kDa peptide as the -adrenergic receptor. Labeling
was specifically blocked by the -adrenergic antagonist
prazosin (Ref. 27, and confirmed in these experiments, data not shown).
The diffuse labeling pattern arises from the glycosylation of the
receptor protein
(28) .
Figure 1:In vitro recognition of
-adrenergic receptor by immunoblotting with
anti-peptide antibodies. A,
[
H]phenoxybenzamine-labeled DDTMF2
cell membranes, partially purified on wheat germ agglutinin-Sepharose,
were subjected to SDS-PAGE (40-50 fmol of receptor/lane),
electrotransferred to nitrocellulose, and visualized by autoradiography
( Lane 1) and immunoblotting with antiserum directed against
the carboxyl-terminal decapeptide sequence from the receptor ( Lane 2), as indicated under
``Experimental Procedures.'' Positions and apparent masses
(in kDa) of molecular weight standards are indicated. B, crude
membrane extracts from wild type HEK 293 cells ( Lane 1), or
cells transfected and stably expressing the
receptor
( Lane 2) were immunoblotted with the
antiserum.
Specific immunoreactivity of the
antiserum was further demonstrated by immunoblotting against crude
membrane fractions prepared from HEK 293 cells transfected to stably
express the receptor. The antibody reacted with
polypeptides of approximate molecular mass = 80 kDa present in
membrane extracts from transfected cells (Fig. 1 B, Lane
2), but absent in membranes from untransfected cells (Fig. 1 B,
Lane 1). Furthermore, the immunoreactivity of antibody
506-515 was completely abolished by prior absorption with its
corresponding peptide (data not shown).
Agonist-induced Redistribution of
The capability
of the antibodies to recognize the receptor in situ was tested
by immunofluorescence staining of pooled populations of HEK 293 cells
selected for stable -Adrenergic Receptors
AR expression. The pattern of
immunostaining obtained with this antibody appears diffusely and
uniformly distributed over the cell surface (Fig. 2 A).
Specificity of the immunostaining was demonstrated by the following
criteria (data not shown). First, preabsorption of the antiserum with
the corresponding peptide abolished the specific staining. Second, no
positive immunofluorescence was observed when the anti-peptide
antiserum was substituted with preimmune serum. Third, no
immunofluorescence was observed in untransfected HEK 293 cells.
Furthermore, no immunostaining was observed if the cells were not first
permeabilized with detergent allowing access of the antibody to the
cell interior. This latter result is consistent with the proposed
cytoplasmic location of the peptide epitope recognized by the antibody.
receptor distribution. As shown in Fig.
2 B, exposure of HEK 293/
cells for 3 h to
experimental growth medium containing 10 µ
M norepinephrine
produced a striking redistribution of the staining pattern of the
receptor and the appearance of highly non-uniform aggregates of intense
immunostaining. The
receptor antagonist prazosin (1
µ
M) blocked agonist-induced redistribution of
immunostaining (Fig. 2 C), confirming that the
redistribution was specifically mediated by agonist occupancy of the
receptor.
Figure 2:
Agonist induced redistribution of the
-adrenergic receptor. HEK 293 cells stably expressing
the
receptor were treated with growth medium
containing specified additions for 3 h, and processed for
immunofluorescence microscopy using antiserum directed against the
receptor as described under ``Experimental
Procedures.'' A, control, no additions; B, 10
µ
M norepinephrine; C, 10 µ
M norepinephrine plus 1 µ
M prazosin. Scale
bar, 10 µm.
The kinetics of agonist-induced redistribution are
indicated in Fig. 3. Redistribution was evident within 5 min of agonist
exposure, the earliest interval examined, essentially complete by 15
min, and sustained during agonist exposure intervals up to 24 h.
Localization of Internalized Receptors
In order to
visualize receptor localization at higher spatial resolution, the
staining of control and agonist-treated cells was examined by confocal
immunofluorescence microscopy. Scanning of sequential optical sections
(1-µm thickness) of individual cells was performed. Fig. 5 shows
representative control and agonist-treated images obtained at
comparable focal planes relative to the attachment surface. Control
cells (Fig. 5 A) displayed immunostaining on the cell
periphery, consistent with localization of the receptor at the cell
surface. NE-treated cells (Fig. 5 B) showed non-uniform
and bright patches of immunofluorescence localized internally
throughout the cytoplasm; some aggregates also appeared to be
associated with the cell surface. These images clearly demonstrate that
the -adrenergic agonist elicits a profound redistribution of
receptor within these cells, and they strongly suggest that a
significant fraction of receptors translocate from the cell surface to
an intracellular location.
Figure 5:
Agonist-induced
-adrenergic receptor redistribution observed with
confocal microscopy. Confocal microscopy images (optical section at
z = 2 µm above attachment surface) are shown of:
A control cell, or B, NE-treated cell (10
µ
M). Treatment interval was 3 h. Scale bar, 10
µm.
Internalization of many receptors and
integral membrane proteins occurs by endocytosis to a common endosomal
compartment from which further intracellular sorting or recycling to
the cell surface proceeds
(34) . To determine whether
receptor trafficking occurs by a similar pathway we
performed immunofluorescence colocalization of the internalized
receptors with transferrin receptor, a classic endosomal marker. Each
antigen was labeled with its corresponding primary antibody and
visualized with fluorescein isothiocyanate-(
) or
Lissamine rhodamine-(transferrin) conjugated secondary antibodies,
respectively. Confocal images obtained from control cells showed that
receptor is mainly localized to the cell surface
while transferrin receptors reside in internal vesicles (Fig. 6, A and B). After 1 h NE exposure, the internalized
receptor colocalizes with transferrin receptor in
endosomes (Fig. 6, C and D). This result
indicates that
receptor translocates to endosomes
during agonist exposure. In addition, agonist-induced receptor
internalization was completely prevented by pretreatment of cells with
hyperosmotic sucrose solutions (0.45
M), a procedure which has
been shown previously to inhibit receptor-mediated endocytosis (Ref.
35, data not shown). In sum, these results are consistent with an
endocytic pathway of
receptor internalization.
Figure 6:
Co-localization of
-adrenergic receptor and transferrin receptor. Cells
were treated (1 h) with growth medium containing indicated
norepinephrine additions and then were fixed, permeabilized, and
labeled simultaneously with rabbit polyclonal antiserum directed
against the
receptor (1:1000 dilution) and a mouse
monoclonal antibody specific for transferrin receptor (1:100 dilution).
Immunofluorescence detection was achieved with fluorescein
isothiocyanate-goat anti-rabbit IgG (
receptor) and
Lissamine rhodamine-goat anti-mouse IgG (transferrin receptor)
secondary antibodies. Confocal images are shown of a control cell
( A and B) and a NE (10 µ
M) treated cell
( C and D) labeled with
AR ( A and C) and transferrin receptor ( B and
D) antibodies.
Role of Protein Kinase C in
Recent studies suggest that protein kinase
C-mediated phosphorylation may regulate -Adrenergic Receptor
Internalizationreceptor
internalization
(36) . Using immunocytochemical procedures we
tested the involvement of protein kinase C in agonist-induced receptor
internalization. The characteristic receptor internalization induced by
norepinephrine is shown in Fig. 7 A. Inhibition of protein
kinase C by staurosporine (0.5 µ
M) completely blocks the
NE-induced receptor redistribution (Fig. 7 B). Moreover,
activation of protein kinase C by PMA (50 n
M) induces receptor
internalization to endosomes. Fig. 8, A and B,
respectively, show colocalization of
-adrenergic
receptors and transferrin receptors in cells treated with PMA. The
kinetics of PMA-induced receptor internalization are comparable to
those observed with agonist, and the effects of PMA are blocked by
staurosporine (data not shown). The possibility that these
pharmacologic agents may exert actions in addition to their effects on
protein kinase C should also be considered. However, the body of data
outlined above which show the parallels between the actions of agonist
and PMA, and the inhibition of their effects by staurosporine, strongly
suggest that protein kinase C-dependent phosphorylation plays an
important role in the mechanism of agonist-induced receptor
internalization in this system.
Activation and Desensitization of
Experiments were next designed to
investigate the relationship between Receptor Response
receptor
redistribution and functional responsiveness. Activation of
-adrenergic receptors by norepinephrine elicits a
sustained and robust accumulation of [
H]inositol
phosphates in cultures equilibrated with
[H]inositol and stimulated in the presence of
LiCl to prevent inositol phosphate degradation (Fig. 9 A).
Under these conditions, accumulation of
[H]inositol phosphates is linear over at least 60
min of agonist exposure.
H]inositol phosphate
production were measured. Results are shown in Fig. 9 B.
Activation of protein kinase C by PMA abolishes agonist-stimulated
[H]inositol phosphate production. Conversely,
inhibition of protein kinase C by staurosporine (2 µ
M,
Fig. 9B) or calphostin C (0.5 µ
M, data not
shown) enhances agonist-stimulated inositol phosphate production.
However, the linear accumulation of [H]inositol
phosphates suggests that receptor reactivation must also occur during
prolonged agonist exposure. Recent studies on -adrenergic
receptors suggest that receptor internalization is necessary for
recovery following agonist-induced desensitization
(37) . We
therefore tested the role of receptor
internalization as a determinant of receptor responsiveness by
measuring agonist-stimulated [
H]InsP production
in solutions containing hyperosmotic sucrose to block receptor
internalization. As further shown in Fig. 9 B, this
treatment attenuates agonist-stimulated [H]InsP
production. Moreover, concurrent inhibition of protein kinase C with
staurosporine in the presence of sucrose reverses the attenuation so
that agonist responses again are potentiated relative to naive
cultures. The elevation of basal InsP production by sucrose treatment
does not affect the calculated responses, since all data were
normalized relative to basal and agonist-stimulated responses of
control cultures. These results indicate that activation of PKC which
is predicted to occur as a consequence of agonist stimulation inhibits
receptor responsiveness. Receptor internalization during agonist
stimulation is necessary to sustain the functional response, suggesting
that dynamic cycling of receptors between cell surface and internal
compartments is required for the regeneration of active receptor.
Figure 9:
Activation and desensitization of
agonist-elicited [H]inositol phosphate production
in HEK 293 cells. A, time course of agonist-elicited
[H]inositol phosphate production. Cultures were
equilibrated with [H]inositol, stimulated in
Li-PSS containing the indicated agonist additions for the specified
times, and [H]inositol phosphates were measured
as described under ``Experimental Procedures.'' , 10
µ
M phenylephrine;
, no added agonist. B,
regulation of receptor responsiveness by protein kinase C or receptor
internalization. Cultures equilibrated with
[
H]inositol were incubated with Li-PSS containing
the indicated additions for 20 min pre-treatment interval (1-ml
incubation volume). Replicate cultures were then stimulated for a
further 20 min by addition of phenylephrine (10 µ
M final
concentration, hatched bars) or vehicle ( open bars)
to the pre-treatment solution. Quantitation of
[H]InsP production was performed as described
under ``Experimental Procedures.'' Data are corrected for
basal [H]InsP production and normalized to
agonist-stimulated [H]InsP production obtained in
control cultures incubated with Li-PSS containing no further additions
during the pre-treatment interval. Treatments: PMA, 100 n
M;
STP, staurosporine, 2 µ
M; SUC, 0.45
M sucrose.
-adrenergic receptor has enabled us to visualize the
receptor with unprecedented subcellular resolution, and to elucidate
mechanisms of agonist-induced internalization. Alterations in receptor
responsiveness were observed in parallel functional measurements of
agonist-elicited [
H]InsP production. Evidence is
presented suggesting the involvement of protein kinase C in both
receptor internalization and desensitization which occur during agonist
exposure.
using fura 2 methodology.
()
2) Protein
kinase C-mediated phosphorylation consequently causes feedback
inhibition of receptor response. Pharmacologic activation of PKC by PMA
inhibits receptor response to agonist. Conversely, inhibition of PKC
with staurosporine during agonist stimulation potentiates receptor
response. 3) Agonist induces receptor endocytosis by a mechanism
involving protein kinase C-mediated phosphorylation. Internalized
receptor co-localizes with transferrin receptor, a
classic marker for endosomes. Receptor internalization is prevented by
treatment of cells with hyperosmotic sucrose solution, which has been
shown to block receptor-mediated endocytosis in other systems.
Furthermore, activation of protein kinase C by PMA causes receptor
internalization to endosomes, mimicking the effects of agonist.
Conversely, inhibition of PKC with staurosporine blocks agonist-induced
receptor internalization. 4) Receptor reactivation and recycling to the
cell surface occur continuously during agonist exposure. Interruption
of receptor internalization with hyperosmotic sucrose attenuates the
sustained agonist response, consistent with an accumulation of
desensitized cell surface receptor resulting from protein kinase C
activation (see above). Simultaneous inhibition of protein kinase C
relieves this inhibition by reducing the extent of receptor
desensitization, suggesting that desensitized receptor is regenerated
during internalization and subsequently returned to the cell surface.
This result argues that the effect of sucrose treatment is specific for
blocking receptor internalization rather than nonspecific uncoupling of
receptor responsiveness. Upon removal of agonist, the initial cell
surface disposition of the receptor is rapidly restored, further
supporting the reversible nature of this process.
-adrenergic receptor activation and desensitization.
First, our model suggests that steady state receptor
response will reflect a balance between the rates of receptor
desensitization and recovery. The reactivation component of the model
provides a means to reconcile observations of agonist-induced
alterations in receptor properties with the relatively sustained
physiological responses to -adrenergic stimulation
which are observed in vivo (14, 38, 39) . Variability in receptor
responsiveness among different experimental systems could then result
from alterations in this balance. Specifically, our data point to a
role for protein kinase C-mediated phosphorylation in the process of
receptor desensitization. Variations in protein kinase C activation
(arising, for example, from heterogeneous expression of specific PKC
isoenzymes, alterations in diacylglycerol metabolism, or availability
of key PKC substrates) would in turn affect the rate or extent of
receptor desensitization, and contribute to the observed discrepancies
among different experimental systems. Consistent with the proposed role
of PKC in AR regulation, previous studies have shown
that PMA treatment causes AR phosphorylation,
modulation of agonist affinity, and uncoupling of the receptor from
cellular responses
(16) . A recent report
(40) demonstrates that staurosporine treatment to inhibit PKC
potentiates AR-mediated InsP production and
contractile responses in rat aorta smooth muscle, similar to the
results in this study.
<
30 s) of recombinant M3 muscarinic receptor coupling to
phosphoinositide metabolism in transfected Chinese hamster ovary cells;
partial desensitization of this receptor is accompanied by its
phosphorylation. Moreover, agonist-induced alteration in receptor
accessibility or responsiveness further predicts that an apparent
excess of total receptors will be measured relative to the magnitude of
the sustained response, whereas the initial rate or latency of response
activation will more closely reflect the total number of receptors.
Parallel measurements of receptor number and agonist-stimulated
intracellular Ca
elevations in individual astroglial
cells are consistent with this prediction
(42) . HEK 293 cells
express
receptors in excess over physiological
systems, and caution is always advisable in extrapolating results from
expression systems to intact tissue. However, apparent receptor reserves have been documented in a number of
preparations
(43) . Our results provide a rationale to
understand their physiological function.
receptor internalization are not well
known. Using radioligand binding measurements to assess receptor
redistribution in Chinese hamster ovary cells transfected with
receptor cDNA, Zhu and Toews
(36) report
that agonist or PMA cause receptor internalization which is blocked by
staurosporine. Our studies which monitor the receptor directly with an
immunological probe demonstrate a role of PKC-mediated phosphorylation
in receptor internalization in this system. However, the site(s) of PKC
phosphorylation and the molecular interactions which control receptor
internalization are unknown. Mutagenesis studies of
adrenergic
receptors have identified a tyrosine-containing sequence
(NP XXY) which is conserved among AR and
similar to the internalization signal (NP XY) of constitutively
recycling receptors
(44) . It is tempting to speculate that
PKC-mediated phosphorylation triggers receptor
internalization by facilitating the interaction of this NP XXY
locus with the endocytic machinery of the cell.
-adrenergic receptor responsiveness and disposition,
as follows. 1) Agonist exposure in a variety of systems leads rapidly
to receptor desensitization, characterized by reduced ability of
the cell surface receptor to couple functionally to the stimulatory G
protein, G
, and to activation of adenylyl cyclase
(45) . 2) Phosphorylation of -adrenergic receptors by the
second messenger activated cAMP-dependent protein kinase, as well as by
specific -adrenergic receptor kinases, appears important for
receptor desensitization
(45) . Although the corresponding
receptor kinases have not been described, our data do
not rule out this possibility. Moreover, we show here evidence
supporting involvement of protein kinase C-mediated phosphorylation in
AR receptor desensitization and internalization. 3)
-Adrenergic receptor desensitization is accompanied by rapid and
reversible sequestration of the receptor
(46, 47) .
Elegant immunocytochemical procedures of von Zastrow and Kobilka
(24) have demonstrated that -adrenergic receptor
sequestration occurs by an endocytic pathway. 4) -Adrenergic
receptor sequestration is necessary for reactivation of desensitized
receptor
(37) , possibly by a dephosphorylation mechanism
(48) . Significantly, recycling of reactivated receptors appears
to occur continuously in the presence of agonist
(49) . 5)
-Adrenergic receptor desensitization, sequestration, and
down-regulation are independently regulated processes rather than
occurring as an obligatory sequence of events
(44, 50) .
In additional studies we find no decrease in AR
immunoreactivity or [
H]prazosin binding activity
following 24 h agonist exposure, demonstrating that internalization
occurs independently of down-regulation (data not shown). These overall
similarities suggest that members of the family of G protein-coupled
receptors may share comparable cycles of activation, desensitization,
and internalization; yet exhibit significant disparities in their
temporal responses.
receptors leads to a continuous cycle
of receptor desensitization, internalization, and recovery. The
availability of this defined and functionally coupled expression system
combined with novel immunochemical probes should allow further progress
in understanding the molecular interactions underlying
receptor cycling. It will be of interest to extend these
approaches to examine the organization and regulation of
-adrenergic receptors in vascular tissues under normal
and pathophysiological conditions.
AR, -adrenergic receptor; HEK cell,
human embryonal kidney cell; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; NE, norepinephrine; PMA, phorbol
12-myristate 13-acetate; InsP, inositol phosphate; Li-PSS,
physiological salt solution supplemented with LiCl; PKC, protein kinase
C.
-adrenergic receptor cDNA clone used in this study,
to Drs. Brenda Russell and Herb Proudfit for use of fluorescence
microscopy instrumentation, to Drs. John Reece and Palmer Taylor for
helpful discussions, to Dr. Kelly Ambler for critically reading the
manuscript, and to Fran Fries for assistance with cell culture.
Adrenergic Receptor (Ruffolo, R. R., Jr., ed) pp. 269-272, Humana Press, Clifton, NJ
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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