From the Department of Molecular Pharmacology,
Diabetes & Metabolic Diseases Research Program, University Medical
Center, State University of New York, Stony Brook, New York 11794-8651, the § Howard Hughes Medical Institute, Vollum Institute,
Portland, Oregon 97201-3098, and the ¶ Department of Physiology & Biophysics, Diabetes & Metabolic Diseases Research Program, University
Medical Center, State University of New York,
Stony Brook, New York 11794-8661
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ABSTRACT |
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Signals mediated by G-protein-linked
receptors display agonist-induced attenuation and recovery involving
both protein kinases and phosphatases. The role of protein kinases and
phosphatases in agonist-induced attenuation and recovery of
Chronic stimulation of G-protein-linked receptors
(GPLRs)1 provokes attenuation
of the receptor-mediated signal, or desensitization (1). Protein
phosphorylation is a critical element of agonist-induced desensitization involving at least three prominent kinase activities (2), cyclic AMP-dependent protein kinase (protein kinase
A), calcium and phospholipid-sensitive protein kinase (protein kinase C), and members of the G-protein-linked receptor kinase family, like
the Recently, we reported cell-type-specific roles of various protein
kinases in agonist-induced desensitization using oligodeoxynucleotides to suppress these enzymes transiently (2). Suppression of protein kinase C, but not protein kinase A or Cell Culture--
Human epidermoid carcinoma cells (A431) were
grown in Dulbecco's modified Eagles' medium supplemented with 10%
fetal bovine serum (HyClone, Logan, UT), penicillin (60 µg/ml), and
streptomycin (100 µg/ml). A431 cells were transfected with the pLNCX
plasmid using Lipofectin® (Life Technologies, Inc.)
reagent according to the manufacturer's protocol. Stably transfected
A431 cells expressing antisense RNA to protein kinase A and protein
phosphatase 2A and 2B were screened for suppression of the target
enzyme as outlined below. At least three separate clones of stable
transfectants were selected and propagated for each antisense
construct. The stable transfectant clones routinely were maintained in
medium containing gentamycin (0.5 mg/ml; Life Technologies, Inc.).
Construction of the pLNCX Retroviral Vectors--
The antisense
sequences, 5'-TTGGCTTTGGCTAAGAATTCTTTCACGCTCTCC-3',
5'-TCGGCCAGCACCGCCTC-CAGGTCCGCCAT-3', and
5'-CTGCAGAAGGTGGGCTGCTTGAAGAAGCG-3' derived from the complementary
sequences of protein kinase A catalytic Immunoblotting Analysis--
Cells were harvested and
homogenized in 10 mM Hepes buffer, pH 7.4, 2 mM
MgCl2, 2 mM EDTA containing 10 µg/ml
leupeptin, 10 µg/ml aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride. Nuclei were collected by low speed
centrifugation. Fifty micrograms of post-nuclei fraction protein was
subjected to 10% SDS-PAGE, and the separated proteins were transferred
onto a nitrocellulose membrane. Expression of protein kinase C was
probed with antibody against the Protein Kinase A and C Assays--
Protein kinase A and C
activities were assessed by using commercially available assay kits
purchased from Life Technologies, Inc. The manufacturer's protocol was
followed. Protein kinase A activity is defined as the amount of
phosphate incorporated into a substrate peptide, Kemptide, in the
presence of 10 µM cyclic AMP minus that incorporated in
the presence of the protein kinase A inhibitor peptide (1 µM). The specific activity of protein kinase C is defined
as the difference between the amount of phosphorylation of an
acetylated peptide derived from myelin basic protein in the presence of
10 mM phorbol 12-myristate 13-acetate from the amount of
phosphorylation occurring in the presence of 20 µM
protein kinase C inhibitory peptide (protein kinase C Protein Phosphatase 2A and 2B Assays--
Protein phosphatase 2A
was isolated by immunoprecipitation with antibodies to the catalytic
subunit of PP2A (Transduction Laboratories, Lexington, KY). The PP2A
activity was measured using [32P]phosphorylated glycogen
phosphorylase A as a substrate and a commercially available kit
(13188-016; Life Technologies, Inc.). PP2A-specific activity was
defined as the activity sensitive to inhibition by 100 nM
calyculin A or 1 nM okadaic acid. For protein phosphatase
2B, two complementary techniques were employed for assay:
immunostaining with antibodies to the catalytic subunit of PP2B
(C26920; Transduction Laboratories, Lexington, KY) and identification
by the calmodulin-overlay assay. Calmodulin binding to the renatured
SDS-PAGE gel (overlay) was detected using antibodies to calmodulin. The
immunocomplexes then were made visible as described above. Both assays
provide comparable results.
RII Overlay Assays of A Kinase-anchoring Proteins--
The
presence of AKAPs in A431 cells were detected by RII overlay assay as
described (15). Briefly, fifty micrograms of cell lysate or membrane
protein was separated by electrophoresis on an SDS-polyacrylamide gel
and electrotransferred to nitrocellulose. Filters were blocked with a
Tris-buffered saline solution containing 10% heat-inactivated horse
serum and incubated with RII Suppression via Antisense Oligodeoxynucleotides--
Antisense
and control missense oligodeoxynucleotides with the same base
composition, but in scrambled order, were synthesized and purified to
cell culture-grade (Operon, Alameda, CA), as described (2). Before
addition to cells, oligodeoxynucleotides were mixed at a ratio of 1:3
(w/w) with DOTAP (Boehringer Mannheim), a cationic diacylglycerol in
liposomal form which serves as a delivery vehicle. A431 cells were
treated with oligodeoxynucleotides (5 µg/ml) for 2 days prior to the
analysis of the expression of the target molecule. Cells in which
gravin was specifically suppressed by antisense oligodeoxynucleotides
were then analyzed for agonist-induced (isoproterenol) desensitization
and resensitization following a wash-out of agonist (2).
Immunoprecipitation--
The association of
Radioligand Binding Studies--
The number of
Desensitization and Resensitization of
Effect of Protein Phosphatase Inhibitors on Receptor
Resensitization--
Cells were preincubated in the presence or
absence of protein phosphatase inhibitors, such as FK506 (Fujisawa USA,
Deerfield, IL) and calyculin A (Boehringer Mannheim) for 15 min prior
to the addition of the agonist. Each inhibitor was included throughout the incubation for desensitization, washout, and resensitization, as
described elsewhere (2).
Data Presentation and Analysis--
Each of the experimental
protocols were performed, as indicated, with three to five separate
A431 clones. The values presented are means ± S.E., and the
autoradiograms are representative examples of data.
Agonist-induced desensitization of G-protein-linked receptors,
typified by studies of the -adrenergic receptors was explored by two complementary approaches,
antisense RNA suppression and co-immunoprecipitation of target
elements. Protein phosphatases 2A and 2B are associated with the
unstimulated receptor, the latter displaying a transient decrease
followed by a 2-fold increase in the levels of association at 30 min
following challenge with agonist. Protein kinase A displays a robust,
agonist-induced association with
-adrenergic receptors over the same
period. Suppression of phosphatases 2A and 2B with antisense RNA or
inhibition of their activity with calyculin A and FK506, respectively,
blocks resensitization following agonist removal. Recycling of
receptors to the plasma membrane following agonist-promoted
sequestration is severely impaired by loss of either phosphatase 2B or
protein kinase C. In addition, loss of protein kinase C diminishes
association of phosphatase 2B with
-adrenergic receptors. Overlay
assays performed with the RII subunit of protein kinase A and
co-immunoprecipitations reveal proteins of the A kinase-anchoring
proteins (AKAP) family, including AKAP250 also known as gravin,
associated with the
-adrenergic receptor. Suppression of gravin
expression disrupts recovery from agonist-induced desensitization,
confirming the role of gravin in organization of G-protein-linked
signaling complexes. The Ht31 peptide, which blocks AKAP
protein-protein interactions, blocks association of
-adrenergic
receptors with protein kinase A. These data are the first to reveal
dynamic complexes of
-adrenergic receptors with protein kinases and
phosphatases acting via an anchoring protein, gravin.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-adrenergic receptor kinase.
2-Adrenergic
receptors (
2AR) are substrates for protein kinase A,
protein kinase C, G-protein-coupled receptor kinase, as well as growth
factor receptors with intrinsic tyrosine kinase activity (3, 4). Study
of the complex roles of protein kinases in the functional regulation of
these multiply phosphorylated receptor substrates has been accelerated
through the use of loss-of-function mutant cells in which target
protein kinases have been suppressed by antisense oligodeoxynucleotides
(2) and dominant negative mutant kinase (5), in addition to protein
kinase inhibitors (6), receptor mutagenesis (7, 8), and reconstitution
of purified elements in vitro (9).
-adrenergic receptor kinase, amplified rather than attenuated agonist-induced desensitization in a
variety of cell types. In the current work, we explore the role of
protein kinases, phosphatases, and anchoring proteins in organizing
associations with the
-adrenergic receptor. Protein kinase C is
shown to be obligate for resensitization of GPLR, its action blocked by
protein kinase C deficiency and by the protein kinase C inhibitor
bisindolylmaleimide, and mimicked by FK506, a protein phosphatase2B
inhibitor. Protein kinase anchoring and scaffolding proteins have
emerged as central elements in many aspects of cell signaling (10-13).
The anchoring protein AKAP250, also known as gravin (14), is shown to
associate with the
-adrenergic receptor and be required for recovery
from agonist-induced desensitization to occur.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-subunit (16), protein
phosphatase 2A (17), and protein phosphatase 2B (18),
respectively, were engineered into the
HindIII/ClaI sites of the pLNCX retroviral vector
using standard recombinant DNA techniques (19). The pLNCX vector
contains the gene to confer neomycin resistance under the control of
the 5'- and 3'-long terminal repeats of the mouse Moloney virus, and
the expression of the antisense RNA is under the control of the
cytomegalovirus promoter. Antisense RNA to protein kinase A targets
both
- and
-isoforms of the catalytic subunit of protein kinase A
(16, 20).
-isoform of protein kinase C (Life
Technologies, Inc.).
peptide
19-36).
subunit for 2 h at room
temperature. After washing, the presence of AKAPs was detected using an
goat anti-RII
antibody, as described in immunoblotting analysis.
2-adrenergic receptor with other proteins was probed by
use of immunoprecipitation. Cells were harvested, and cell membrane was
prepared by homogenization. The membranes were solubilized in lysis
buffer (1% Triton X-100, 0.5% Nonidet P-40, 6.0 µM
dithiothreitol, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 100 µg/ml
bacitracin, 100 µg/ml benzamidine, 2 mM sodium
orthovanadate, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 40 mM sodium pyrophosphate, 50 mM KH2PO4, 10 mM sodium
molybdate, and 20 mM Tris-HCl, pH 7.4). Immunoprecipitation
was performed in the lysis buffer. The lysates were pre-cleared with
protein A/G-agarose for 90 min and then subjected to
immunoprecipitation for 2 h with antibodies specific either for
the
-adrenergic receptor (CM4) or for AKAP250 gravin. The primary
antibodies were linked covalently to a protein A/G-agarose matrix. The
variance of immunoprecipitation, sample loading, and/or immunoblotting
in these experiments in the aggregate was established at
10%.
2-adrenergic receptors was determined by radioligand
binding. Intact A431 cell clones were incubated with 0.5 nM
[125I]iodocyanopindolol (ICYP; NEN Life Science Products)
in the presence or absence of 10 µM propranolol at
23 °C for 90 min. The incubation buffer contained 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, and 150 mM NaCl. The affinity constants for ICYP and isoproterenol
(Sigma) binding were determined using crude membrane fractions (2). The
dissociation constant for ICYP binding was calculated by Scatchard plot
analysis with ICYP concentrations ranging from 0 to 1 nM (2).
2AR--
Two days prior to the analysis of
agonist-induced desensitization, the A431 cells were seeded in 96-well
microtiter plates at a density of 25,000-50,000 cells/well. Details of
the desensitization protocol have been published elsewhere (2). For the
actual assay, cells were washed and challenged with or without 1 µM isoproterenol for 30 min at 37 °C. At the end of
the first challenge, the cells were washed three times and then
incubated in 20 mM Hepes buffer, pH 7.4, containing 0.1 mM Ro-20-1724 (Calbiochem, San Diego, CA) and 0.5 units/ml
adenosine deaminase (Sigma) for 5 min prior to and again following a
second challenge with 1 µM agonist. For resensitization,
the cells challenged with isoproterenol for 30 min prior were washed
free of agonist and maintained in fresh buffer for an additional 30-60
min. Five minutes before the second challenge of the agonist, cells
were incubated again in the presence of Ro-20-1724 and adenosine
deaminase. The amount of cyclic AMP accumulated by cells in response to
agonist was measured in the naive cells (activation), in the cells
treated with agonist for 30 min prior to a second challenge with
isoproterenol (desensitized), and in cells treated with agonist for 30 min prior to a second challenge with isoproterenol and washed free of
agonist for 30-60 min (resensitized). The cyclic AMP accumulated by
the cells was determined as described (2, 21). "0%
desensitization" denotes a cyclic AMP response to a second challenge
with agonist that is equivalent in magnitude to that obtained in naive
cells in response to the first challenge. "100% desensitization"
represents the maximal level of desensitization obtained by a 30-min
pre-incubation of the cells with isoproterenol.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-adrenergic receptor, is sensitive to
chemical inhibitors of protein phosphatases (2). Inhibition of PP2A and
PP2B leads to enhanced desensitization, reflecting an attenuation of
the recovery phase (2). Direct analysis of the association of
-adrenergic receptors with protein phosphatases 2A and 2B was probed
by immunoprecipitating
-adrenergic receptors from detergent extracts
from human epidermoid carcinoma A431 cells and exploring the nature of
associated proteins either by immunoblotting or by direct assay of
phosphatase activity of the immunoprecipitates (Fig.
1A). In the absence of
exposure to the
-adrenergic agonist isoproterenol,
immunoprecipitates of
-adrenergic receptors subjected to
SDS-polyacrylamide gel electrophoresis and stained with antibodies to
PP2B
reveal the presence of phosphatase PP2B in association with the
-adrenergic receptor (0-min desensitization time, Fig. 1A). A characteristic doublet band of PP2B with molecular
mass values of 57 and 61 kDa are detected with antibodies to PP2B
, whereas staining either with a non-immune serum or with antibodies to
unrelated antigens reveals no such bands (data not shown).
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Fig. 1.
Association of protein phosphatase 2B with
-adrenergic receptor: agonist stimulation of a dynamic
association. Lysates were prepared from A431 cells treated with
isoproterenol (10 µM) for periods up to 30 min. Cell
lysates were incubated with antibodies to
-adrenergic receptors
(CM-04), and then the immunocomplexes recovered by adsorption to
protein A agarose beads. The immunocomplexes were subjected to
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and stained either with antibody specific for the catalytic subunit of
PP2B (panel A) or with calmodulin followed by an
anti-calmodulin antibody (calmodulin-overlay assay, panel
B). The immunocomplexes were made visible by the chemical
luminescence method. The data presented are representative of at least
four separate determinations performed with separate cell
lysates.
Challenging A431 cells with isoproterenol stimulates agonist-induced
desensitization (2, 21, 22) and results in a decline of PP2B associated
with the -adrenergic receptors that was prominent within 5 min of
the challenge (Fig. 1A). Within 10 min of agonist challenge,
the amount of PP2B associated with
-adrenergic receptors began to
return toward normal. By 30 min of challenge with isoproterenol, levels
of PP2B associated with
-adrenergic receptors nearly doubled compared with that observed in the naive cells (Fig. 1A).
Use of the calmodulin-overlay assay as an alternative to detect the 57- and 62-kDa forms of phosphatase PP2B confirms (i) the association of
PP2B with
-adrenergic receptors in the naive cells, (ii) the decline
in PP2B associated with the receptor upon early challenge with agonist,
and (iii) the enhanced association of PP2B with the receptor at 30 min,
a time when recovery from agonist-induced desensitization occurs (1,
2).
The relative levels of PP2A, PP2B, and protein kinase A (PKA)
associated with -adrenergic receptors was determined in A431 cells
using this same strategy (Fig. 2).
Summation of data from several independent assays confirms the results
displayed in Fig. 1, i.e. PP2B associates with
-adrenergic receptors in the naive cells, is lost transiently early
during agonist treatment, and associates in greater abundance as
desensitization progresses to 30 min. Unlike the case for PP2B
determinations, antibodies capable of detecting either PP2A or protein
kinase A were found to be unsuitable for the purpose of detection of
either protein in the immunoprecipitates. For measurement of PP2A
associated with
-adrenergic receptors, PP2A activity was measured in
immunoprecipitates of
-adrenergic receptors using
[32P]phosphorylated glycogen phosphorylase a
as a substrate. PP2A activity was readily measurable in
immunoprecipitates of
-adrenergic receptors from naive cells. Upon
challenge with agonist, the amount of PP2A associated with the receptor
declined by 20-25% at 5 min, and returned to control pre-challenge
levels within the next 5 min, and remained rather constant over the
next 20 min. Thus, both protein phosphatases PP2A and PP2B associate
with the
-adrenergic receptors in naive cells and display a partial
dissociation from the receptor early after challenge with an
agonist. For PP2A the agonist-induced dissociation is transient and
returns to normal, while PP2B actually displays increased association
with the receptor as the time course continues to the 30-min challenge
with agonist. For protein kinase A, kinase activity is found in
association with
-adrenergic receptors of naive A431 cells and
increases approximately 3-fold over control levels at 30 min after
challenge with isoproterenol (Fig. 2).
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In an effort to explore further the role of PP2A and PP2B in
agonist-induced desensitization and resensitization, stable
transfectant clones of A431 cells were created that harbor pLNCX
antisense RNA-expressing vectors (2). pLNCX antisense constructs,
characterized previously (2, 23), were engineered to include 30-base
pair sequences antisense to the catalytic subunit of either PP2A or PP2B. Each of these antisense expression vectors is driven by the
cytomegalovirus promoter, harbors the neomycin resistance gene, and
stably transfects clones of A431 cells, selected with the neomycin
analog G418 (2, 25). Clones stably transfected with pLNCXASPP2A vector
display an 85-95% suppression of PP2A levels (Fig.
3). Glycogen phosphorylase a
is dephosphorylated by either protein phosphatase type 1 or PP2A. Using
radiolabeled [32P]phosphorylated glycogen phosphorylase
a as a substrate, the sum of these two activities in
whole-cell extracts was found to be reduced >75% in the clones stably
transfected with the pLNCXASPP2A vector (data not shown). The
suppression of PP2A expression (immunoblotting) by antisense RNA and
corresponding loss of activity suggest that PP2A is largely responsible
for the read-out using phosphorylase a as the substrate for
detection of PP2A in immunoprecipitates of -adrenergic receptors
from A431 cells (Fig. 3). Similar clones deficient in PP2B expression
were sought using the pLNCXASPP2B form of the vector, with surprising
results. Despite considerable experience with pLNCXAS constructs to
suppress the expression of a variety of targets (2, 25, 26), we were
unable to identify clones in which PP2B levels are suppressed in excess of 50-60% even after several independent rounds of transfection (Fig.
3). This observation leads us to speculate that suppression of PP2B
beyond the 50-60% range may not be compatible with cell viability.
Alternatively, some PP2B isoform not sensitive to the antisense RNA may
be expressed in A431 cells. Notably, the growth rate of the stably
transfected A431 cell clones with 50% the complement of PP2B was
reduced by severalfold. Assay of PP2B activity in whole-cell extracts
of the PP2B-deficient clones was performed, and the amount of PP2B
activity was found to be similarly reduced to about 50-60% of the
control levels (data not shown).
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Agonist-induced desensitization was measured in the A431 clones stably transfected to suppress either PP2A or PP2B. The availability of chemical inhibitors for either PP2A or PP2B as well as clones in which levels of expression of either PP2A or PP2B are reduced provided two complementary strategies with which to study the functional significance of protein phosphatases PP2A and PP2B in agonist-induced desensitization. The amount of desensitization measured in either the clones expressing the empty vector or control A431 cells was set at 100% for these studies. A431 clones deficient in PP2A display a modest increase in the amount of agonist-induced desensitization in response to a challenge with 10 µM isoproterenol, 137 ± 09% of the control levels (mean ± S.E.; n = 4). Treating cells with 10 nM calyculin A, a selective inhibitor for PP2A, yields agonist-induced desensitization, which is unaltered (105 ± 12%, mean ± S.E.; n = 4). The optimal concentration (100 nM) of calyculin A insuring maximal inhibition of PP2A activity could not be employed in the current studies. Calyculin A at 100 nM provoked cell death and detachment of the A431 cells. Thus, suppression of PP2A expression leads to a modest increase in the amount of desensitization, whereas partial inhibition of PP2A with 10 nM calyculin does not alter the extent of the desensitization.
A431 cells lacking >50% of the PP2B complement display normal levels of desensitization, 92 ± 8% of control levels (mean ± S.E.; n = 4). Treating cells with the PP2B inhibitor FK506 (100 ng/ml), in contrast, more than doubles (210 ± 06%, mean ± S.E.; n = 4) the amount of agonist-induced desensitization over that observed in the control, untreated A431 cells. The inability of the antisense RNA to suppress the expression of PP2B more than 50-60%, whereas the FK506 effectively blocks PP2B activity under the same conditions, provides an explanation for the apparent dichotomy in the inhibitor versus antisense data. In the desensitization paradigm employed for these studies, loss or inhibition of PP2A enhances the extent of desensitization only slightly, whereas inhibition of PP2B enhances markedly the extent of agonist-induced desensitization.
Earlier studies implicated PP2A in agonist-induced desensitization (1). Since protein phosphatases would likely have a more prominent role in re-sensitization rather than desensitization per se, the re-sensitization of the cyclic AMP response of A431 cells following a challenge with 1 µM isoproterenol was evaluated in cells for which levels of PP2A and PP2B were suppressed by either antisense RNA or through chemical inhibition of their activities (Fig. 4). A431 clones harboring the empty vector alone were employed as controls. Stable transfectant clones were treated with isoproterenol for 30 min to achieve full desensitization (1, 2). During the last 15 min of incubation with agonist, the A431 clones were challenged with either vehicle alone, calyculin A (10 nM), or FK506 (100 ng/ml). Cells were then washed free of agonist repeatedly with fresh buffer supplemented with or without one of the phosphatase inhibitors maintained at the same concentration. Resensitization was measured 60 min after washout of the agonist, a time at which full recovery from desensitization occurs.
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A431 stably transfected clones harboring the empty vector (EV) and naive, wild-type A431 cells displayed 100% resensitization by 60 min after wash-out of agonist (Fig. 4). For clones made deficient in PP2A with antisense RNA, the extent of resensitization was reduced to approximately half of that observed for A431 clones harboring the empty vector (Fig. 4). This sharp decline in resensitization in the PP2A-deficient cells provides an explanation for the apparent increase in desensitization, as measured on the "supply" side of the equation. Partial inhibition of PP2A with 10 nM calyculin A, much like PP2A-deficiency, reduced the extent of resensitization, but to a lesser extent (20-25%) than that provoked by suppression of PP2A (Fig. 3). The inability of calyculin A inhibition to mimic the full effects observed in PP2A-deficient cells likely reflects the suboptimal concentration of the PP2A inhibitor employed for these studies, as discussed above.
Suppression of PP2B activity markedly attenuated resensitization of the agonist response (Fig. 4). Resensitization in PP2B-deficient cells under these conditions is ~60% of the control level. Chemical inhibition of PP2B with FK506, like PP2B deficiency, attenuates resensitization, displaying levels of resensitization less than 50% of control. The enhanced agonist-induced desensitization observed in the presence of the FK506 likely reflects the reduction in resensitization accompanying inhibition of PP2B. The chemical inhibitor is more effective than antisense in both suppressing PP2B activity as well as attenuating resensitization. In view of the inability of the viable, antisense RNA-producing cells to suppress PP2B more than 50% of control values (Fig. 3), the more pronounced effect of the chemical inhibitor seems predictable (Fig. 4).
Receptor sequestration was assayed in order to define to what extent,
if any, do PP2A and PP2B exert their influence on agonist-induced desensitization/resensitization through receptor sequestration. The
hydrophilic -adrenergic antagonist ligand CGP-12177 was employed to
measure receptor sequestration (Fig. 5).
Each of A431 clones under study display equivalent levels of
-adrenergic receptor expression, as measured by the binding of
iodocyanopindolol (data not shown). In the absence of treatment with
agonist, the amount of CGP-12177 binding measured in the intact cells
is equivalent among all A431 clones (data not shown). To provoke
sequestration, cells were challenged with 10 µM
isoproterenol for 30 min in the standard desensitization protocol and
amount of CGP-12177 binding measured in the whole cells. Deficiency of
either PP2A or PP2B had no significant effect on the extent to which
CGP-12177 binding declined in response to agonist-induced
desensitization. Each of the clones displayed a reduction in the amount
of CGP-12177 bound ranging from 18% to 26%, typical for 30-min
agonist-induced receptor sequestration in control A431 cells (1,
2).
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Assay of CGP-12177 binding capacity at 60 min of resensitization
following a wash-out of agonist revealed several features of protein
phosphatase action. EV control clones treated with agonist, washed free
of agonist, and allowed to recover for 60 min displayed >95% of the
CGP-12177 binding capacity of naive, untreated cells (Fig. 5). The
suppression of PP2A by antisense RNA (Fig. 3) attenuated
resensitization (Fig. 4), but did not alter recovery of CGP-12177
binding, which returned to ~110% of that observed in the naive,
untreated cells. Partial inhibition of PP2A with 10 nM
calyculin A yielded identical results, i.e. inhibition of
PP2A failed to alter the ability of the cells to return to levels of
CGP-12177 binding observed in naive, untreated cells (data not shown).
Partial suppression of PP2B by antisense RNA (Fig. 3), in contrast,
blocked the recovery from receptor sequestration (Fig. 5), as well as
resensitization of the -adrenergic receptor response (Fig. 4).
Inhibition of PP2B with FK506 also blocked recovery of the
agonist-induced decline in CGP-12177 binding by 60 ± 11% (Fig.
5). The results from assay of these two aspects of receptor function
and cycling are in agreement and suggest that PP2B plays a key role in
the recovery of
-adrenergic receptor from agonist-induced desensitization.
Previously, protein kinase C deficiency was shown to provoke enhanced
agonist-induced desensitization (2), much like either PP2B deficiency
or inhibition of PP2B with FK506 shown here (Figs. 4 and 5). CGP-12177
binding studies of protein kinase C-deficient, A431 stable
transfectants reveal a similar pattern for protein kinase C deficiency
as for either partial suppression of expression or chemical inhibition
of PP2B (Fig. 5). A431 cells pre-treated for 15 min and thereafter in
the presence of the protein kinase C inhibitor bisindolylmaleimide (300 nM, not shown) yielded identical data to those obtained
with the protein kinase C-deficient cells (Fig. 5). PP2B associated
with -adrenergic receptors in the absence of agonist, dissociating
during the early period of agonist-induced desensitization, but
rebounding in association with the receptor as the desensitization
continues (Figs. 1 and 2). Loss of either protein kinase C or PP2B
shared many of the same features, leading to enhanced agonist-induced
desensitization, to sustained loss of function following agonist
removal ("resensitization"), and to an inability of the
-adrenergic receptors to recover from agonist-induced receptor sequestration.
For A431 cells, deficiency of protein kinase A (1, 2) attenuates
whereas deficiency of protein kinase C potentiates agonist-induced
desensitization (26). The effect of protein kinase C deficiency on the
association of protein kinase A with the -adrenergic receptors was
explored. Whereas agonist treatment enhanced the amount of protein
kinase A associated with the
-adrenergic receptor (Fig. 2),
deficiency of protein kinase C disrupted the association of protein
kinase A with the
-adrenergic receptors (Fig.
6A). Association of either
PP2A or PP2B with
-adrenergic receptors, in contrast, was unaltered
in the protein kinase C-deficient cells, being 105 ± 12% for
PP2A and 97 ± 6% for PP2B association (mean ± S.E.,
n = 4), respectively. This interplay between protein kinase A and protein kinase C suggested the possible participation of
either anchoring or scaffold proteins in agonist-induced
desensitization (10-13).
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Anchoring and scaffold proteins have been discovered that participate
in cell signaling (10-13). The AKAPs represent a diverse family of
proteins that assist in organizing signaling elements (10-16). The
expression of AKAP proteins in A431 cells was probed using antibodies
to two prominent members of the family, AKAP79 and AKAP250 (gravin).
Antibodies to AKAP79 failed to identify any immunoreactive species with
a mass of approximately 70-90 kDa (data not shown). Staining blots of
cellular proteins of A431 cells with the RII subunit of protein
kinase A, the technique employed in the discovery of AKAPs (15),
revealed one potential AKAP at Mr 175,000, which
is a proteolytic product of a Mr 250,000 AKAP
(Fig. 7). Subsequent staining of A431
cell proteins with antibodies to other AKAPs reveal the identity of the
AKAP250 to be the protein gravin (13). As an additional test of a
possible role of AKAPs in the organization of the
-adrenergic
receptors and protein kinase A, the interaction between the
-adrenergic receptors and protein kinase A was probed with Ht31, a
24-amino acid, conserved amphipathic helix peptide common to all AKAP
family members (12). Ht31, which blocks protein-protein interactions of
AKAPs, effectively blocked the ability of the
-adrenergic receptors
to bind protein kinase A (Fig. 6B). Ht31 blocked the association of protein kinase A with
-adrenergic receptors in naive
cells (Fig. 6B), as well as in cells treated with
-adrenergic agonist (data not shown). The Ht31 peptide in which a
prolyl residue has been inserted to disrupt the AKAP binding motif
(Ht31-Pro) was without effect on PKA association with the
-adrenergic receptors, whereas the Ht31 peptide essentially
abolished PKA association with the
-adrenergic receptors. Inclusion
of either the active Ht31 or inactive Ht31-Pro peptide yielded a small
(<10%) reduction in the PKA activity measurements.
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To probe further the involvement of AKAP250 (gravin),
immunoprecipitations of both -adrenergic receptors as well as of
gravin were performed and the immunoprecipitates subjected to
SDS-polyacrylamide gel electrophoresis and staining with antibodies to
either
-adrenergic receptors or gravin (Fig. 7, A-C).
Subcellular fractionation of A431 cells followed by SDS-PAGE and
staining with the RII subunit of protein kinase A (RII-overlay assay)
revealed prominent staining of gravin in the subcellular cell
membrane-enriched, "pellet" (P) fraction collected by low speed
centrifugation (Fig. 7A). The supernatant (S) fraction, in
contrast, was essentially devoid of immunoreactive gravin. Gravin
(AKAP250) was rapidly nicked to a dominant Mr
175,000 species, which was stained prominently by the RII subunit of
PKA. The amount of the Mr 250,000 and 190,000 species of gravin was variably determined by some non-suppressible proteolytic activity in cell homogenates. In most preparations, the
Mr 175,000 form of gravin was the major form and
appears to be the limit cleavage product under these conditions.
Immunoblotting of the pellet fraction with anti-gravin antibodies
revealed gravin and its major proteolytic species. Thus, gravin is
essentially confined to the membrane-enriched, pellet fraction, whether
identified by either the RII overlay or immunoblotting with antibodies
to gravin.
Staining of immunoprecipitates of -adrenergic receptors of A431 cell
extracts with antibodies to gravin revealed prominent staining of
AKAP250, providing compelling evidence of gravin association with
-adrenergic receptors (Fig. 7B). Similarly, staining of immunoprecipitates of gravin revealed prominent staining of
-adrenergic receptor, found in association with gravin (Fig.
7B). Immunoprecipitations performed with antibodies to
gravin and extracts from Chinese hamster ovary (CHO-K) clones that lack
-adrenergic receptors revealed no such staining or associations
(data not shown).
The functional significance of gravin in agonist-induced desensitization/resensitization in A431 cells was explored. Antisense oligodeoxynucleotides were employed to suppress gravin production. Treatment with oligodeoxynucleotides antisense (anti), but not missense (mis), to gravin for 2-3 days suppresses the expression of gravin by >90% in A431 cells (Fig. 7C). Agonist-induced desensitization was examined in the cells treated with and without oligodeoxynucleotides antisense to gravin. The progress and extent of the agonist-induced desensitization in response to 1 µM isoproterenol was equivalent in all of the cells, including those treated with oligodeoxynucleotides antisense to gravin (Fig. 8). To assess the role of gravin in resensitization, the cells were washed free of agonist and studied over the next 60 min (Fig. 8). Although altering neither the time course nor the extent of the desensitization, suppression of gravin provoked a dramatic loss of resensitization in A431 cells at 30, 45, and 60 min following the wash-out of agonist (Fig. 8). Thus, deficiency in gravin profoundly delayed the ability of the A431 cells to recover from agonist-induced desensitization.
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Consistent with earlier observations (2), the association of protein
kinase A with the -adrenergic receptors was found to be dependent
upon the expression of protein kinase C. Association of protein kinase
A with
-adrenergic receptors was not detectable in A431 cells made
deficient of protein kinase C. Similar studies in other systems have
revealed a central role for protein kinase C in the association of
AKAPs (13, 26). The AKAP gravin was shown to be associated with
-adrenergic receptors physically and functionally. Taken together,
these data provide support for the formation of signaling complexes in
which protein kinases and phosphatases that regulate a G-protein-linked
receptor are maintained in proximity to the target protein (Fig.
9). Recent work in the mitogen-activated
protein kinase cascade in yeast and mammalian systems implicates
scaffold complexes in signaling (27-29). In neurons, AKAP proteins
have been shown to coordinate three multifunctional enzymes: PKA, PKC,
and PP2B (27). Herein we provide evidence for the existence of
signaling complexes for a prominent member of the G-protein-linked
receptor superfamily, the
-adrenergic receptor. According to this
scheme, the
-adrenergic receptor is localized to the AKAP gravin, in
association with PKA (sensitive to Ht31 inhibition) and PKC (PKA fails
to associate in the PKC-deficient cells) as well as with PP2B. Thus,
much like the organization in yeast and in neurons, G-protein-linked
receptors are organized into complexes with multifunctional
enzymes.
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The current work illuminates also several key features of
agonist-induced desensitization, the resensitization that follows, and
receptor sequestration. Protein phosphatase 2B plays an obligate role;
partial deficiency of this protein phosphatase or inhibition of PP2B by
FK506 disrupts all three parameters. Both protein kinase A and PP2B
physically associate with -adrenergic receptors, as analyzed through
co-immunoprecipitation, and display a dynamic association. The ability
of protein kinase C expression or deficiency to influence
agonist-induced desensitization and protein kinase A association with
-adrenergic receptors provoked a search for possible AKAPs.
Importantly,
-adrenergic receptors are shown to physically associate
with AKAP250 (gravin). The suppression of gravin by antisense
oligodeoxynucleotides is shown to disrupt resensitization. These
results provide compelling evidence to support the notion that
G-protein-linked receptors, such as the
-adrenergic receptor, that
activate adenylyl cyclase participate in macromolecular complexes that
are composed minimally of protein kinases and phosphatases in
association with a prominent member of the AKAP family of
anchor/scaffold proteins, gravin. These associations are functionally
relevant, as disruption of specific interactions among these molecules
influences agonist-induced desensitization, resensitization, and/or
receptor sequestration.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Pharmacology-HSC,
SUNY, Stony Brook, NY 11794-8651. Tel.: 516-444-7873; Fax:
516-444-7696; E-mail: craig{at}pharm.som.sunysb.edu.
The abbreviations used are:
GPLR, G-protein-linked receptor; 2AR,
2-adrenergic receptor; ICYP, [125I]iodocyanopindolol; PAGE, polyacrylamide gel
electrophoresis; AKAP, A kinase-anchoring protein; EV, empty vector; CHO, Chinese hamster ovary; PKA, protein kinase A, PKC, protein kinase
C; PP2A and PP2B, protein phosphatases 2A and 2B, respectively.
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REFERENCES |
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