(Received for publication, February 15, 1995; and in revised form, May 11, 1995)
From the
Persistent stimulation of the The mammalian Because it shares
physiologic agonists, G protein coupling, and a 54% amino acid homology
with the That the
293 cells were incubated at 37 °C
in 95% air, 5% CO
Figure 8:
Overexpression of
Figure 6:
Overexpression of GRKs augments both
agonist-induced phosphorylation and desensitization of the
Figure 5:
Stimulus-induced phosphorylation of the
Figure 4:
Stimulus-induced phosphorylation of the
To
assay the accumulation of cAMP in intact cells, transiently transfected
293 cells in 12-well dishes were labeled by incubation in 0.5 ml/well
of 30% medium B, 70% minimal essential medium with 2 µCi of
[ To assess ISO-stimulated cAMP
accumulation in 293 cells co-transfected with the 12
To test the possibility that the
Figure 1:
Short term desensitization of the
To the extent that the
Figure 2:
Homologous desensitization of the
Agonist-induced receptor
sequestration cannot explain the desensitization findings. Cells
treated identically to those assayed for desensitization were processed
for immunofluorescence with the monoclonal antibody 12CA5, which is
specific for the amino-terminal epitope-tagged If homologous
desensitization of the
Figure 3:
Immunoblots for
Agonist-induced phosphorylation of the To establish a role for one or more GRKs in
agonist-induced phosphorylation of the If a GRK-initiated
mechanism is important for desensitization of the To corroborate the transfected cell findings
of GRK-mediated
Figure 7:
Phosphorylation of the
The paradigm for
GRK-initiated receptor desensitization predicts that an arrestin
homologue should be important to the short term desensitization of a
GRK-regulated receptor(7) . To test this prediction, we
overexpressed To demonstrate that In this series of experiments, we have demonstrated for the
first time that rapid desensitization of the The importance of agonist-induced
phosphorylation in the desensitization of the With respect to rapid
desensitization and susceptibility to GRK-mediated phosphorylation, the
Previously,
Zhou and Fishman (5) used SK-N-MC neuroblastoma cells to
demonstrate short term (30 min) agonist-induced The In contrast to rhodopsin, the m Note Added in
Proof-Desensitization of the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic
receptor (
AR) engenders, within minutes, diminished
responsiveness of the
AR/adenylyl cyclase signal
transduction system. This desensitization remains incompletely defined
mechanistically, however. We therefore tested the hypothesis that
agonist-induced desensitization of the
AR (like that
of the related
AR) involves phosphorylation of the
receptor itself, by cAMP-dependent protein kinase (PKA) and the
-adrenergic receptor kinase (
ARK1) or other G protein-coupled
receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells
demonstrate receptor-specific desensitization of the
AR within 3-5 min. Both cell types also express
ARK1 and the associated inhibitory proteins
-arrestin-1 and
-arrestin-2, as assessed by immunoblotting. Agonist-induced
AR desensitization in 293 cells correlates with a 2
± 0.3-fold increase in phosphorylation of the
AR, determined by immunoprecipitation of the
AR from cells metabolically labeled with
P
. This agonist-induced
AR
phosphorylation derives approximately equally from PKA and GRK
activity, as judged by intact cell studies with kinase inhibitors or
dominant negative
ARK1 (K220R) mutant overexpression.
Desensitization, likewise, is reduced by only
50% when PKA is
inhibited in the intact cells. Overexpression of rhodopsin kinase,
ARK1,
ARK2, or GRK5 significantly increases agonist-induced
AR phosphorylation and concomitantly decreases
agonist-stimulated cellular cAMP production (p < 0.05).
Furthermore, purified
ARK1,
ARK2, and GRK5 all demonstrate
agonist-dependent phosphorylation of the
AR.
Consistent with a GRK mechanism, receptor-specific desensitization of
the
AR was enhanced by overexpression of
-arrestin-1 and -2 in transfected 293 cells. We conclude that
rapid agonist-induced desensitization of the
AR
involves phosphorylation of the receptor by both PKA and at least
ARK1 in intact cells. Like the
AR, the
AR appears to bind either
-arrestin-1 or
-arrestin-2 and to react with rhodopsin kinase,
ARK1,
ARK2, and GRK5.
AR
(
)is
coupled through G
to the activation of adenylyl cyclase and
is important in the regulation of heart rate and
contractility(1) , lipolysis by adipose tissue(2) , and
blood pressure homeostasis(3) , among other vital functions.
Like many G
-coupled receptors (4) , the
AR demonstrates receptor-specific or homologous
desensitization(2, 5) ; persistent or repetitive
stimulation decreases the receptor's ability to activate adenylyl
cyclase. In contrast to desensitization of the
AR,
however, little is known about desensitization of the
AR at the molecular level.
AR(6) , the
AR might
be expected to share regulatory mechanisms with the
AR
as well. Occurring within seconds to minutes of agonist exposure, short
term desensitization of the
AR/G
/adenylyl
cyclase system involves phosphorylation of the receptor by two classes
of serine/threonine kinases (for review, see (4) ). The second
messenger-dependent kinases PKA and protein kinase C phosphorylate and
desensitize the
AR, but because they phosphorylate
multiple proteins, they mediate a generalized cellular
hyporesponsiveness known as heterologous desensitization(4) .
Second messenger-independent G protein-coupled receptor kinases
(GRKs)(7) , by contrast, initiate a two-step process of
homologous desensitization by phosphorylating only activated receptors.
Once phosphorylated, the receptors bind to inhibitory proteins, the
-arrestin isoforms(8, 9) , which uncouple the
receptors from G
. Like the
AR, the
AR possesses both a canonical site for PKA
phosphorylation in the carboxyl-terminal portion of its third
intracellular loop and a serine/threonine-rich cytoplasmic tail (6) , the region of the
AR phosphorylated by
GRKs(4, 10) .
AR may be
desensitized by a GRK-initiated mechanism is suggested by the
examination of myocardium from failing human hearts. In the lethal
condition of human chronic heart failure, depressed
-adrenergic
responsiveness accompanies a 2-3-fold up-regulation in the
expression of the
-adrenergic receptor kinase (
ARK1 or
GRK2)(11) . Despite this provocative association, however, the
only mechanistic investigation of short term
AR
desensitization published thus far suggests that only a PKA-dependent
mechanism, and not a GRK-dependent mechanism, effects
AR desensitization(5) . We therefore utilized
a combination of in vitro reconstitution and intact cell model
systems to assess directly the role of GRKs and
-arrestin isoforms
in both the agonist-induced phosphorylation and desensitization of the
AR.
Materials
All cell culture reagents were
procured from Life Technologies. Human embryonal kidney (293) cells and Spodoptera frugiperda (Sf9) cells were obtained from American
Type Culture Collection. H-89 was obtained from Biomol Research
Laboratories (Plymouth Meeting, PA). Nonidet P-40 and okadaic acid were
obtained from Calbiochem, and staurosporine was from Boehringer
Mannheim. Ascites containing the monoclonal antibody 12CA5 was obtained
from Berkeley Antibody Co. 3-Isobutyl-1-methylxanthine (IBMX),
forskolin, (-)-isoproterenol bitartrate, and the
AR/
AR antagonists nadolol and
(-)-alprenolol were obtained from Sigma. The
AR
antagonist ICI-118,551
HCl and dopamine
HCl were procured
from Research Biochemicals International.
[
P]Orthophosphate (carrier-free),
[
I]iodocyanopindolol,
[2,8-
H]adenine,
[
-
P]ATP,
[
-
P]ATP, [
H]cAMP, L-[
S]methionine/L-[
S]cyteine
(EXPRE
S
S protein labeling mix), and
[8-
C]cAMP came from DuPont NEN. Restriction
enzymes were obtained from Promega.
Plasmid Constructs
Recombinant DNA manipulations
were carried out by standard techniques(12) . The influenza
hemagglutinin nonapeptide epitope (YPYDVPDYA) for the monoclonal
antibody 12CA5 (13) was added to the amino terminus of the
human AR cDNA (6) with the polymerase chain
reaction. The 5` (sense strand) primer was
5`-CGCGGGGGATCCATGTACCCATACGACGTCCCAGACTACGCCGGCGCGGGGGTGCTCGTCCTG-3`,
with the start codon and nucleotides 4-24 of the native sequence
underlined; the 3` (template strand) primer was
5`-GGTCGGCGCTGGCCAGGGAC-3` (nucleotides 399-380). The resultant
polymerase chain reaction-generated 438-base pair fragment and the
expression vector pcDNA I (Invitrogen) were cut with BamHI and PstI and ligated to generate the construct pcDNA
I/12
5`. A 1.2-kilobase pair
AR cDNA
fragment produced by digestion with Bgl2 (Klenow-blunted) and PstI was then subcloned into pcDNA I/12
5`,
which had been prepared by digestion with XhoI
(Klenow-blunted) and PstI. The resulting construct was
designated 12
AR. A SacI (T
polymerase-blunted)/BglII fragment of the human
AR cDNA was subcloned into the baculovirus shuttle
vector pVL1393 (Invitrogen) to create pBac
. After
digestion with NcoI and SalI, the 12CA5
epitope-tagged
AR cDNA (14) was Klenow-blunted
and subcloned into pcDNA I to create
``12
AR.'' The cDNAs for bovine
ARK1(15) , bovine
ARK2(16) , and bovine
rhodopsin kinase (17) were subcloned into pcDNA I using HindIII, EcoRI/NdeI (Klenow-blunted), and HindIII/BamHI sites, respectively. The bovine GRK5
construct in pcDNA I (10) and the human D
-dopamine
receptor cDNA in pCMV5 (18) have been described previously. Rat
-arrestin-1 and
-arrestin-2 (9) were subcloned into
pcDNA I by blunting with T
polymerase after digesting with SacII/SalI and KpnI/SalI,
respectively. To create the K220R mutant of bovine
ARK1, we
amplified a 327-base pair AccI fragment with the the following
5` (sense strand) primer:
5`-CGCGGGGTCTACGGCTGCCGGAAGGCCGACACGGGCAAGATGTACGCCATGAGGTGTCTGGAC-3`
(the mutagenic codon is underlined). Cut with AccI, this
fragment and the (dephosphorylated)
ARK1/pBC12BI construct (15) were ligated. The
ARK1 K220R cDNA was then subcloned
into pcDNA I, as above. Dideoxy sequencing was used to confirm the
specificity of mutagenesis in all polymerase chain reaction-derived
fragments.
Insect Cell Culture and Infections
All recombinant
baculovirus operations were conducted according to standard
protocols(19) . Sf9 cells were co-transfected in monolayer
culture with pBac and BaculoGold® (PharMingen)
DNA, according to the manufacturer's instructions. The resultant
recombinant baculovirus (Ac
AR) was plaque-purified and
used to infect log-phase cultures of Sf9 cells in 100-ml spinner flasks
with a multiplicity of infection about 0.5. Forty-eight hours into
infection, the Sf9 cells expressed
5 pmol of
AR/mg of cell protein.
Mammalian Cell Culture and Transfections
Chinese
hamster fibroblast (CHW) cells were grown in 90% Dulbecco's
modified Eagle's medium, 10% fetal bovine serum, 100 µg/ml
streptomycin and 100 units/ml penicillin (medium A), as described
previously(20) . Cells were transfected with either the human
AR (20) or the human
AR (6) cDNAs in the expression vector pBC12BI (20) , and
stably transfected clones were selected as described
previously(20) . Cells used for these experiments expressed
either the
AR or
AR at
300
fmol/mg of membrane protein.
and grown in medium B (90% minimal
essential medium with Earle's salts, 10% fetal bovine serum, 100
µg/ml streptomycin, and 100 units/ml penicillin). The evening
before transfection, 4
10
cells were plated per
90-mm dish. These cells were transfected on day 1 by calcium
phosphate co-precipitation(21) . Each plate received 10 µg
of DNA total, comprising 0.25 µg of 12
AR along
with either a 4-fold molar excess of D
-dopamine receptor
plasmid, a 7-fold molar excess of GRK plasmid, a 10-fold molar excess
of
-arrestin plasmid, a 16-fold molar excess of
ARK1 K220R
plasmid, or a 3-fold molar excess of both
ARK1 and
-arrestin-2 plasmids; the balance of DNA comprised the empty
vector pcDNA I. For 12
AR experiments, each plate
received 10-15 µg of DNA comprising 5-10 µg of
12
AR and 2.5-5 µg of either
ARK1 or
-arrestin-2 plasmids, with the balance comprising just pcDNA I.
Cells were split on day 2 into assay dishes as follows: for
phosphorylation assays, 1
10
cells/9.6 cm
well of six-well dishes; for cAMP accumulation assays, 2
10
cells/3.8 cm
well of 12-well dishes. Assays
were performed on day 4.
AR expression ranged
from 0.8 to 2.5 pmol/mg of cell protein (see below); within a single
experiment, however,
AR expression among various
co-transfected cell types varied from control cells by <30%, except
where noted in the legend of Fig. 8.
-arrestins augments
desensitization of the
AR in a manner additive to that
of
ARK1. 293 cells were co-transfected with the
12
AR (stripedbars) and an
expression plasmid for either no cDNA (control),
-arrestin-1 (
arr1, n = 3),
-arrestin-2 (
arr2, n = 3),
ARK1 (n = 4), or
ARK1 along with
-arrestin-2 (n = 4). ISO-stimulated cAMP production in intact cells was
assayed as in Fig. 6C. 293 cells co-transfected with
the 12
AR (blackbars) and either
control (empty vector),
ARK1, or
-arrestin-2 plasmids were
also assayed (n = 3) for ISO-stimulated cAMP production
as above, except that cells were stimulated with 50 µM ISO
(
EC
in this
AR system). Conversion
of
H into cAMP in this figure is expressed as a percentage
of the cognate control cell value; mean ± S.E. is plotted. In
AR control cells, the values (mean ± S.D.) for
percent conversion of
H into cAMP were 0.04 ± 0.03
(basal) and 1.8 ± 1 (ISO-stimulated); untransfected cells gave 3
± 2% of
AR control cell ISO responses. In
AR control cells, basal values for percent conversion
of
H into cAMP were 0.12 ± 0.01, and ISO-stimulated
values were 1.3 ± 0.3. Untransfected cells gave 19 ± 4%
of
AR control cell responses. By immunoblotting,
overexpression levels of
ARK1 and
-arrestin-1 or -2 were
20 times endogenous levels in
AR experiments, and
10 times endogenous levels in
AR experiments. In
AR/
ARK1/
-arrestin2 experiments, cell lines
expressed the
AR at the following levels (pmol/mg):
1.2 ± 0.2 (
AR control cells), 2.1 ± .02
(
AR/
ARK1 cells), and 1.6 ± 0.3
(
AR/
ARK1/
-arrestin2 cells).
AR density over this range of
AR
expression levels showed no effect on ISO-stimulated cAMP accumulation
(data not shown). *, p < 0.01 (
-arrestin-1), p < 0.001 (
-arrestin-2) compared with control cells;
, p < 0.001 compared with control, and p < 0.05
compared with
-arrestin-1 or -2 cells;
, p <
0.001 compared with control and p < 0.05 compared with
ARK1 cells.
AR. 293 cells transiently co-transfected with the
12
AR and either the empty vector (Empty,
control) or an expression plasmid for one of 4 GRKs (
ARK1,
ARK2, GRK5, or rhodopsin kinase, RK) were metabolically
labeled with
P
and treated as in Fig. 5, except that 100 nM ISO was used to stimulate
the cells. A, an autoradiogram of immunoprecipitated
ARs is representative of four performed. Sample 11 was from untransfected cells. B, the radioactivity in the
immunoprecipitated
AR bands was quantitated, and
AR phosphorylation in ISO-treated cells (stimulated)
was normalized to
AR phosphorylation in unstimulated
control cells (basal) as follows: ((stimulated - basal)/(basal)).
The resulting values for
AR phosphorylation as
``-fold above control basal'' are plotted as the mean
± S.E. from n = 4 (
ARK1,
ARK2, and
GRK5) or n = 2 (RK) independent experiments. C, 293 cells from the same transfected cell pools used in B were labeled with [
H]adenine and
stimulated with 100 nM ISO, 50 µM forskolin, or
vehicle (for basal values) for 6 min as described under
``Experimental Procedures.'' ISO-stimulated cAMP production
is normalized to forskolin-stimulated cAMP production for each cell
line, and plotted as a percentage of the forskolin-normalized ISO
response in control cells. The values for percent conversion of
H into cAMP in control cells were 0.06 ± 0.02
(basal), 1.8 ± 0.3 (ISO-stimulated), and 2.8 ± 0.1
(forskolin-stimulated); forskolin-stimulated values for
GRK-overexpressing cells ranged from 95 to 103% of control.
Untransfected cells gave 6 ± 2% of
12
AR-transfected cell responses. Shown are results
(mean ± S.E.) from two independent experiments performed in
triplicate. Compared with control cells, *, p < 0.02, and
, p < 0.05.
AR: (effects of GRK inhibition). A, 293 cells
were co-transfected with the 12
AR, and one of three
expression plasmids: one without a cDNA insert (empty), one
for
ARK1, or one for a dominant negative mutant (K220R) of
ARK1. After metabolic labeling with
P
,
cells were challenged with control (-) or 10 µM ISO-containing (+) medium for 3 min and then washed and
solubilized. The autoradiogram was produced as in Fig. 4. B, 293 cells were co-transfected with the
12
AR and either the empty vector (control) or the
K220R
ARK1 mutant plasmid. Experiments (n = 4 for
ISO, n = 2 for TPA and forskolin) were carried out as
in Fig. 4, except that cells were exposed to 1 µM okadaic acid immediately before the stimulation with forskolin or
TPA. The difference between stimulated and basal
AR
phosphorylation is plotted as a percentage of this difference observed
in control cells (mean ± S.E.). *, p < 0.001 for the
comparison with control cells.
AR: (effects of PKA inhibition). 293 cells transiently
transfected with the 12
AR were metabolically labeled
with
P
in the absence (control) or
presence (+H-89) of 20 µM H-89. Cells were
then challenged with control (None) or stimulus-containing
medium for varying times: 3 min with ISO (10 µM ISO); 10 min with TPA (3 µM TPA), cAMP (3 mM dibutyryl cAMP), and Forsk (50 µM forskolin with 0.5 mM IBMX). Receptors were
immunoprecipitated and resolved by SDS-polyacrylamide gel
electrophoresis. A, the autoradiogram from a single experiment
is shown. Samples 6 and 12 derive from untransfected cells, and the
positions of molecular weight markers are shown by arrows. B, three replications of this experiment are summarized, with
AR phosphorylation (mean ± S.E.) expressed as
(((stimulated/basal) - 1)
100) for control (darkbars) and H-89-treated (stripedbars)
cells. With or without H-89 preincubation, basal levels of cellular
AR phosphorylation were
indistinguishable.
Desensitization and Adenylyl Cyclase Assays
CHW
cells at 90% confluence were incubated for 2 h in medium A without
serum, and then exposed at 37 °C to this medium containing 100
µM ascorbate with (desensitized cells) or without (control
cells) 1 µM(-)-isoproterenol (ISO) for the indicated
times. Cell dishes were then transferred to ice and washed 4 times with
20 ml of ice-cold phosphate-buffered saline (PBS). Cells were next
scraped into 5 mM TrisCl, pH 7.4 (25 °C), 2 mM EDTA, and membranes were prepared as described
previously(20) . Adenylyl cyclase activity of membrane
preparations from control and desensitized cells was assayed, and data
analysis was conducted as described previously (20) .
H]adenine/ml overnight or 4 µCi of
[
H]adenine/ml for 3 h. Assays were performed at
37 °C in room air, in medium C (1 mM IBMX, 100 nM
AR antagonist ICI-118,551 in minimal essential
medium with Earle's salts). Paired 12-well dishes (ISO-pretreated
and control) were handled together, and each comprised four sets of
triplicate wells. Labeling medium was aspirated, and cells were exposed
to medium C (0.5 ml/well) with or without 10 µM ISO or 2
mM dibutyryl cAMP for the indicated times (exposure 1).
Afterward, media were aspirated, and the cells were washed with 1
ml/well of medium C. Cells were then challenged (or rechallenged) with
0.5 ml/well medium C with or without 10 µM ISO, 10
µM dopamine, or 50 µM forskolin for 4 min
(exposure 2). Subsequently, dishes were transferred to ice, and cells
were lysed by the addition of 0.5 ml/well of 2
stop solution
(0.2 mM cAMP and 9 nCi/ml [
C]cAMP in 5%
(w/v) perchloric acid). The cAMP accumulated in the cells and media was
quantitated chromatographically by the method of Salomon (22) and expressed as the percent incorporation of
H into cAMP (
H in cAMP per well / total uptake
of
H per well). [
C]cAMP counts were
used to normalize results for column yield, which averaged
50%.
The intra-assay coefficient of variation on triplicate determinations
was
10%. The cAMP produced in response to stimulation during
exposure 2 (see above) was calculated as the cAMP accumulation in
stimulated cells minus the cAMP accumulation in unstimulated cells from
the same 12-well dish. Data were normalized to values obtained on the
control dish of each pair (i.e. the dish treated with control
medium during exposure 1).
AR
and either a GRK or a
-arrestin, each distinct co-transfected cell
type was plated in a single row of triplicate wells in 12-well dishes.
Cells were labeled as described above, but after aspiration of labeling
medium and before stimulation, cells were washed with 1 ml/well of 37
°C minimal essential medium. Subsequently, cells were treated with
0.5 ml/well of medium C for 7 min at 37 °C and then challenged with
an additional 0.5 ml/well of medium C with or without various
concentrations of ISO or 100 µM forskolin for 6 min.
Reactions were terminated by the addition of 1 ml/well of 2
stop solution, and cAMP was assayed as described above. Pilot ISO
concentration/response experiments with these conditions demonstrated
the EC
to be
100 nM and the maximally
effective concentration to be
1 µM ISO.
AR experiments were conducted in a similar manner,
except that endogenous 293 cell
ARs were antagonized
with 20 µM nadolol instead of 100 nM ICI-188,551.
Immunoblotting
CHW cells were washed twice in PBS,
and scraped into 0 °C lysis buffer (10 mM TrisCl, pH
7.4 (25 °C), 2 mM EDTA with the following protease
inhibitors: 0.1 mM PMSF; 10 µg/ml benzamidine, leupeptin,
and soybean trypsin inhibitor; 5 µg/ml aprotinin; 1 µg/ml
pepstatin A). Following cell disruption (20) , the supernatant
fraction taken after a 15-min, 100,000
g spin was
designated as the cytosolic fraction. Whole cell extracts of 293 cells
were prepared in a similar fashion, without centrifugation.
Immunoblotting was performed with previously characterized
antisera(9, 23) , essentially as described previously (9) except that chemiluminescent detection of immune complexes
was performed with ECL reagents and Hyperfilm-ECL (Amersham Corp.),
according to the manufacturer's instructions. -Fold
overexpression of proteins in transfected cells was quantitated by end
point dilution of transfected cell homogenates.
Intact Cell Phosphorylation
Assays were performed
at 37 °C, in phosphate-free Dulbecco's modified Eagle's
medium, 20 mM HEPES, pH 7.4, with antibiotics as above (medium
D). Cells were washed with 2 ml of medium D/well. Labeling was then
conducted for 40 min in a CO incubator with 0.5 ml of
medium D/well containing 100 µCi of
P
/ml.
In PKA inhibition experiments, this labeling medium contained 0.04%
dimethyl sulfoxide with or without 20 µM H-89. Next, to
each well was added 0.5 ml of medium D containing vehicle with or
without the indicated stimulating compound, and incubation continued
for 3 min (with ISO) or 10 min (with forskolin, dibutyryl cAMP, and
TPA). In
ARK1 K220R experiments, 0.25 ml of medium D containing 3
µM okadaic acid (1 µM final concentration)
was added to labeled cells immediately before forskolin or TPA
stimulation (and 7 min before ISO stimulation) to inhibit cellular
phosphatases. In experiments designed to inhibit both PKA and protein
kinase C, 0.5 ml of medium D containing 0.1% dimethyl sulfoxide with or
without 1 µM staurosporine was added to each well during
the terminal 10 min of labeling. Stimulations were terminated by
transferring dishes to ice, adding 2 ml of ice-cold PBS/well, washing
once with PBS, and adding 0.5 ml/well of RIPA+ buffer (150 mM NaCl, 50 mM Tris
Cl, pH 8.0 (25 °C), 5 mM EDTA, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1%
(w/v) SDS, with 10 mM NaF and 10 mM disodium
pyrophosphate as phosphatase inhibitors, and protease inhibitors as
above, in lysis buffer). Solubilized cells and debris were transferred
to tubes in a total volume of 0.8 ml. After 1 h on ice, debris was
separated from soluble material at 200,000
g for 15
min at 4 °C in a TLA 100.2 rotor (Beckman). From the supernatant
fraction, 730 µl were transferred to 1.5-ml conical tubes. Two
15-µl aliquots were removed for protein assay (Bio-Rad DC protein
assay kit). The remaining volume was processed for immunoprecipitation,
each step of which was conducted at 0-4 °C. Pre-clearing was
effected by adding to each tube 100 µl of 10% (v/v) protein
A-Sepharose beads (Pharmacia Biotech Inc.) in 3% (w/v) bovine serum
albumin/RIPA+ and rotating for 1 h. After the beads were pelleted
at 10,000
g for 15 s, the supernatant fluid was
transferred to a new tube containing protein A beads (as above) and 12
µg of the monoclonal IgG 12CA5. After 2 h on an inversion wheel,
beads were pelleted, and the supernatant fluid was removed. The beads
were next washed 3 times with 1 ml of RIPA+ and aspirated to
dryness with a 28-gauge needle. Subsequently, 60 µl of 1
Laemmli sample buffer (24) was added to each sample. Immune
complexes were dissociated by heating to 65 °C for 10 min and
resolved by SDS-polyacrylamide gel electrophoresis in 10% gels. Gel
lanes were loaded with the volume of sample buffer necessary to give
equivalent amounts of
AR from each sample, as follows.
The
AR density (pmol/mg of whole cell protein) of each
transfected cell line was determined on a nonradioactive aliquot of
each transfected cell population and was multiplied by the value for
RIPA+-solubilized protein in each immunoprecipitation tube; the
resultant receptors/tube values were then normalized and used to adust
gel loading volumes. The gels were stained, dried, and subjected to
autoradiography on DuPont Reflection film with an intensifying screen
at -80 °C for 48-72 h. The same gels were analyzed
quantitatively with a Molecular Dynamics PhosphorImager. Identical
rectangles were drawn about each receptor band, and the signal
intensity within each rectangle was integrated. Specific radioactivity
in the receptor bands was determined as the difference between
radioactivity in the receptor rectangle and radioactivity in an
identical rectangle drawn in lanes of untransfected parental cells.
Automatic background subtraction was not used.
Stoichiometry of
Biosynthetic labeling and
immunoprecipitation (25) were employed to determine the
stoichiometry of AR
Phosphorylation
AR phosphorylation in intact 293
cells. Briefly, transfected cells were labeled in medium B containing
dialyzed fetal bovine serum, with either L-[
S]methionine/L-[
S]cyteine
(50 µCi/ml met) for 48 h or with
P
(0.5
mCi/ml) for 7 h. Cells were stimulated (or not) with 10 µM ISO, washed twice with PBS, and then processed for
immunoprecipitation as above, except that fluorography was used to
image the
S-labeled proteins(24) . Gel bands
corresponding to the
AR were cut, processed, and
counted in a liquid scintillation counter as described
previously(25) . The total counts contained in the
AR bands were corrected by subtracting nonspecific
counts obtained from the cognate bands of gel lanes derived from
untransfected cells. The specific activities of
S-labeled
proteins and cellular [
P]ATP were determined as
described previously(25) .
Membrane Phosphorylation Assays
Sf9 cell plasma
membrane fragments were prepared as described previously (26) from AcAR-infected cells, yielding
50-80 pmol of
AR/mg of protein. GRKs were
purified as described previously (10, 16, 26) from Sf9 cells infected with
recombinant baculoviruses for
ARK1,
ARK2, and
GRK5(10, 26) . Phosphorylation reactions were
conducted at 30 °C for 15 min, with
1 pmol of
AR in Sf9 membranes/tube, exactly as described in (26) .
Radioligand Binding and Immunofluorescence
To
determine the AR or
AR expression,
transfected 293 cell homogenates, Sf9 cells or membranes, or CHW cell
membranes (20) were subjected to saturation binding with 0.7
nM [
I]iodocyanopindolol, as described
previously(20) . The protein concentration of cell homogenates
was estimated by the method of Bradford(24) , using bovine
-globulin as a standard. Immunofluorescence and flow cytometry
were used to quantitate 12
AR expression and
12
AR sequestration and were performed essentially as
described previously(27) . Cells with fluorescence
2
standard deviations above the mean of untransfected cells were
considered positive. By this criterion, 12
AR
transfection efficiencies in these experiments ranged from 35 to 50%.
Statistical Analysis
ISO concentration/response
curves for adenylyl cyclase activity were generated by an iterative
nonlinear least squares technique(14) . The t test for
the comparison of two independent means was used, with pooled estimates
of common variances(28) . Throughout the text, one-tailed p values are given.
AR is
desensitized by a GRK-initiated mechanism, we first sought to compare
the pattern of short term
AR desensitization with that
of the
AR, which is known to be regulated by at least
a
ARK1-initiated
mechanism(4, 29, 30, 31) . In CHW
cells stably transfected with either the
AR or the
AR, a 5-min exposure to a saturating dose of the
AR agonist ISO engenders substantial desensitization of the
ISO-stimulated adenylyl cyclase response (Fig. 1). Under assay
conditions favoring the detection of
ARK1-initiated
desensitization (29) , the maximum adenylyl cyclase response in
membranes from ISO-pretreated cells was 69 ± 8% and 72 ±
10% of control values for the
AR and
AR, respectively; EC
remained essentially
unchanged for the
AR and increased 2.7 ±
0.7-fold for the
AR. With a 30-min ISO pretreatment,
desensitization was even more pronounced; the maximum adenylyl cyclase
response in membranes from ISO-pretreated cells was 57 ± 5 and
60 ± 10% of control values for the
AR and
AR, respectively. EC
increased
insignificantly for the
AR and 3.2 ± 0.8-fold
for the
AR.
AR resembles that of the
AR. CHW
cells stably transfected with either the
AR (top) or the
AR (bottom) were
exposed to control or 1 µM ISO-containing medium for 5 or
30 min at 37 °C. Cells were then washed, and membranes were
prepared for ISO-stimulated adenylyl cyclase assay. Shown are the mean
values from six separate experiments performed in triplicate. For each
experiment, the difference between the ISO-induced maximal cyclase
activity and the basal cyclase activity seen in control cells was
designated 100%, and all other cyclase activities in that experiment
were normalized to this value. Basal cyclase activities were 3.5
± 0.8 and 4.9 ± 1 pmol cAMP/min/mg of protein; maximal
ISO-stimulated values were 12.2 ± 1.8 and 17.2 ± 2.4 pmol
cAMP/min/mg of protein for the
AR and the
AR membranes,
respectively.
AR and
AR desensitization observed in
CHW cells derives from a GRK-initiated mechanism, we would expect the
desensitization to be homologous or receptor-specific. To assess the
homologous nature of short term
AR desensitization, we
used 293 cells transiently co-transfected with the
AR
and the D
-dopamine receptor plasmids. When these cells
were prestimulated with a receptor-saturating dose of ISO for 3 min,
their accumulation of cAMP in response to a subsequent 4-min, maximal
stimulation with ISO or dopamine differed significantly from that
observed in nonprestimulated control cells. The response to ISO was
reduced by 51 ± 12%, and the response to dopamine was reduced by
only 12 ± 3% (Fig. 2). The disparity between reductions
in responses to the two agonists may derive from homologous
desensitization of the
AR on the one hand and
heterologous desensitization of the D
-dopamine receptor on
the other hand. Alternatively, the disparity between ISO-induced
reductions in agonist response may derive from other receptor-specific
differences in regulation. To test these possibilities, we
prestimulated the cells with dibutyryl cAMP in order to activate
exclusively PKA and engender only heterologous desensitization of both
the
AR and the D
-receptor (Fig. 2). In contrast to the ISO-prestimulated cells, the
dibutyryl cAMP-prestimulated cells showed equivalent desensitization of
ISO- and dopamine-induced responses; a 17 ± 1% reduction in
response to ISO and a 16 ± 6% reduction in response to dopamine
was seen. PKA-induced desensitization thus appears equivalent for the
AR and D
-dopamine receptor. Moreover, for
the D
-dopamine receptor, dibutyryl cAMP-induced
desensitization is indistinguishable from ISO-induced desensitization.
Together, these observations suggest that, with receptor-saturating
concentrations, agonist-induced desensitization of the
AR in transfected 293 cells is largely homologous. To
estimate the contribution of PKA to this homologous desensitization, we
used H-89 (32) to inhibit PKA in these intact cells.
Prestimulation with ISO under these conditions failed to affect the
response to subsequent stimulation with dopamine, but it still resulted
in
AR desensitization, manifested by a 23 ± 6% (p < 0.025) reduction in the response to subsequent
stimulation with ISO (Fig. 2).
AR. 293 cells transiently co-transfected with the
12
AR and human D
-dopamine receptor were
metabolically labeled with [
H]adenine and
challenged with control medium (control cells) or stimulus-containing
medium at 37 °C for varying times: 3 min with ISO (10
µM ISO); 10 min with dBcAMP (2 mM dibutyryl cAMP); 3 min with 10 µM ISO preceded by a
40-min incubation with 20 µM H-89 (ISO/H-89).
Cells were then washed and rechallenged for 4 min at 37 °C with
control medium (for basal activity) or medium containing 10 µM of either ISO or dopamine. Plotted is the difference between the
stimulated and cognate basal values for cAMP production, as a percent
of the appropriate control cell value. Control cell values for percent
conversion of
H into cAMP were 0.20 ± 0.08 (basal),
2.4 ± 0.9 (ISO-stimulated), and 2.4 ± 0.1
(dopamine-stimulated). For ISO-prestimulated cells, these values were
2.5 ± 0.6 (basal), 3.6 ± 0.6 (ISO-stimulated), and 4.3
± 0.2 (dopamine-stimulated). Untransfected cells demonstrated
<3% of transfected cell responses. The means ± S.D. of two
independent experiments performed in triplicate are shown. Compared
with naive cells, *, p < 0.05;**, p <
0.025;***, p < 0.005. Compared with cells prestimulated and
restimulated with ISO,
, p < 0.05;
, p < 0.025. Responses elicited after H-89 pretreatment differed
from those elicited without such pretreatment (p <
0.05).
AR. By
flow cytometry, the desensitized cells appear to lose only 13 ±
1% of their plasma membrane receptors by the time of restimulation with
agonist. Over the range of receptor expression used in these
experiments, such a small change in receptor number has no effect on
ISO-stimulated cAMP production (data not shown).
AR in CHW or 293 cells is to be
attributed to a GRK-initiated mechanism, one should be able to
demonstrate the expression of both GRK(s) and the associated inhibitory
-arrestin(s) in these cells, as well as agonist-induced
phosphorylation of the
AR. Fig. 3shows that
both CHW and 293 cells express
ARK1 and
-arrestin-1 (M
50,000). Additionally, the CHW membrane
fraction (data not shown) and 293 cells (Fig. 3) express
-arrestin-2 (M
45,000). Similar
immunoblots (data not shown) demonstrate the expression of GRK5 by 293
cells.
ARK and
-arrestin isoform expression in 293 and CHW cells. Top
panel, 30 µg of 293 cell homogenate, 50 µg of CHW cell
cytosolic fraction, and 25 ng of purified
ARK1 were loaded
separately onto a 10% SDS-polyacrylamide gel and subjected to
electrophoresis and immunoblotting for
ARK isoforms as described
under ``Experimental Procedures.'' Bottom panel, 50
µg of 293 cell homogenate, 50 µg of CHW cell cytosolic
fraction, and 5 µg of homogenate from 293 cells transfected with
expression plasmids for either
-arrestin-1 (
arr1) or
-arrestin-2 (
arr2) were loaded separately onto a 12%
SDS-polyacrylamide gel and treated as above, but with antiserum
specific for
-arrestin isoforms. The positions of biotinylated
molecular weight markers (Amersham Corp.) are indicated with arrows.
AR in
intact 293 cells is demonstrated in Fig. 4. In unstimulated
cells (lane1), the
AR exists as a
phosphoprotein migrating with a M
of
69-79
10
. Upon stimulation with a
receptor-saturating dose of ISO (10 µM) for 3 min or with
3 µM TPA for 10 min, the 293 cells increase
AR phosphorylation approximately 2-fold above basal
levels. This phosphorylation corresponds to 2.4 ± 0.9 mol of
phosphate incorporated/mol of receptor (n = 3
experiments).
AR phosphorylation increases only
approximately 50% above basal levels when 293 cells are stimulated for
10 min with 3 mM dibutyryl cAMP or 50 µM forskolin, 0.5 mM IBMX, even though such forskolin
stimulation is sufficient to increase cellular cAMP at least 2.2-fold
more than the 3-min ISO stimulation (assuming a 35% transfection
efficiency, data not shown). Used at a concentration that inhibited
heterologous desensitization (Fig. 2), H-89 pretreatment of
cells abolished forskolin- and dibutyryl cAMP-induced
AR phosphorylation but inhibited ISO- and TPA-induced
AR phosphorylation by only approximately 60 and 40%,
respectively (Fig. 4). When 1 µM staurosporine is
used to abolish forskolin-, dibutyryl cAMP-, and TPA-stimulated
AR phosphorylation, ISO-stimulated
AR
phosphorylation is reduced by 56 ± 16% (data not shown, n = 3). However, these cell-permeant kinase inhibitors may
overestimate the relative activity of PKA in agonist-stimulated
AR phosphorylation. At the concentrations used in
these experiments, H-89 or staurosporine inhibits purified
ARK1
activity by about 15% (data not shown), and H-89 appears to inhibit
partially the protein kinase C-mediated
AR
phosphorylation demonstrated above. The results from these experiments,
in aggregate, indicate that a kinase or kinases other than PKA or
protein kinase C must be involved in agonist-induced phosphorylation of
the
AR.
AR, we sought
to inhibit this agonist-induced phosphorylation by overexpression of a
dominant-negative mutant (K220R) of
ARK1. Kong et al.(30) have shown this mutant to be a competitive inhibitor
of wild-type
ARK1 with regard to phosphorylation of the purified
AR. As shown in Fig. 5A,
agonist-induced
AR phosphorylation is increased
40% in
ARK1-overexpressing cells and decreased
50% in
ARK1 K220R-overexpressing cells, compared with control cells. By
immunoblotting, we found overexpression of both
ARK1 and
ARK1
K220R in these 293 cells to be
20-fold over endogenous levels (data
not shown). Fig. 5B shows that whereas overexpression
of
ARK1 K220R reduces agonist-induced
AR
phosphorylation by 45 ± 8%, it has essentially no effect on TPA-
or forskolin-induced
AR phosphorylation. Thus, a GRK
in 293 cells seems to effect about 50% of the rapid, agonist-induced
phosphorylation of the
AR.
AR,
we should expect that overexpression of one or more GRKs would not only
increase agonist-induced
AR phosphorylation but also
decrease agonist-induced cAMP signaling by virtue of more rapid or
enhanced desensitization. When cells were co-transfected with the
AR and either
ARK1,
ARK2, GRK5, or rhodopsin
kinase,
AR phosphorylation increased
3.2-4.5-fold over that seen in the absence of GRK overexpression (Fig. 6, A and B). When 293 cells from the
same transfected populations used in phosphorylation assays were used
in intact cell cyclase assays, we found that this augmented
AR phosphorylation correlated with a significant
27-41% reduction in ISO-stimulated cAMP production (Fig. 6C). By contrast, 50 µM
forskolin-stimulated cAMP production was not affected by GRK
overexpression; GRK-overexpressing cells showed 98 ± 3% of
control cell responses.
AR phosphorylation, we used purified
GRKs to phosphorylate the
AR in purified plasma
membrane fragments. Fig. 7shows that phosphorylation of the
AR by
ARK1 was agonist-dependent. The
phosphorylated
AR (indicated by the arrow)
co-migrates with the
AR from photoaffinity-labeled
Ac
AR-infected Sf9 cell membranes (Fig. 7) and
is absent from samples of uninfected Sf9 cell membranes subjected to
ISO-stimulated phosphorylation by
ARK1 (data not shown). Similar
results were obtained when phosphorylation reactions were conducted
with
ARK2 or GRK5 (data not shown).
AR
in Sf9 cell membranes. Ac
AR-infected Sf9 cell
membranes were incubated with [
-
P]ATP with
or without 100 µM ISO and in the absence or presence of
purified
ARK1, as described under ``Experimental
Procedures.'' Samples were then subjected to SDS-polyacrylamide
gel electrophoresis in 10% gels. Shown (at left) is an
autoradiogram of one such gel, representative of three performed. The
position of the phosphorylated
AR is indicated by the arrow, and it co-migrates with the photoaffinity-labeled
AR (at right). For photoaffinity labeling,
membranes from Sf9 cells were resuspended in 75 mM Tris
Cl, pH 8 (25 °C), 12.5 mM MgCl
,
2 mM EDTA. Approximately 2 pmol of
AR in
these membranes were incubated with 2 nM [
I]iodocyanopindololdiazirine (
I-CYP)(Amersham Corp.), with or without 20
µM of the
AR antagonist(-)-alprenolol, as
described previously(46) . Photolysis was performed at 0 °C
for 10 min with a 254-nm ultraviolet lamp suspended 15 cm above the
samples. Samples were then processed as described previously (46) and
0.1 pmol of receptor was
loaded/lane.
-arrestin-1 or
-arrestin-2 along with the
AR in 293 cells and assessed the effect of this
overexpression on ISO-stimulated cellular cAMP accumulation (Fig. 8). Each
-arrestin isoform was expressed to levels
20-fold above endogenous levels ( Fig. 3and data not shown).
Under these conditions,
-arrestin isoforms reduce ISO-stimulated
cAMP production by 20% (p < 0.01). Similar levels of
ARK1 overexpression reduce ISO-stimulated cAMP production by 33%,
significantly more (p < 0.05) than the
-arrestin
isoform effect (Fig. 8). ISO-stimulated cAMP production was
diminished even further, by 49%, when we co-expressed the
AR with
ARK1 and
-arrestin-2 (p < 0.05 compared with the
ARK1 effect). Thus, the effects
of
ARK1 and
-arrestin isoform overexpression on
AR-mediated signaling appear to be additive in this
system.
ARK1 and
-arrestin isoforms
diminish ISO-induced cAMP responses by interacting specifically with
the
AR, and not with some downstream component of the
signaling pathway, we took two approaches. First, we stimulated the
cells used in Fig. 8with 25-50 µM forskolin
and found cellular cAMP responses all to be within 5% of control cell
values. Second, we overexpressed either
ARK1 or
-arrestin-2
with the
AR and replicated the
AR
experiments described above. Although coupled to G
like the
AR, the
AR fails to undergo short
term desensitization(14) . In contrast to experiments in
AR-expressing cells, overexpression of neither
ARK1 nor
-arrestin-2 affects ISO-stimulated cAMP production
in
AR-expressing cells (Fig. 8).
AR in
intact cells involves agonist-induced phosphorylation of the receptor
itself. Our cell culture model for
AR phosphorylation
suggests that any of four GRKs, when overexpressed, can phosphorylate and initiate desensitization of the human
AR. Furthermore, because we can immunologically
identify at least
ARK1 and GRK5 in untransfected 293 cells, our
ARK1 dominant negative experiments suggest that either
ARK1,
GRK5, or both kinases actually do phosphorylate the
AR in intact cells. Whereas the ability of GRK5 to
phosphorylate activated receptors has been previously characterized,
our work is the first to correlate this phosphorylation with functional
receptor desensitization.
AR is
well established(20, 29, 33) . For the
AR, the actions of PKA and
ARK1 appear to be
independent events that contribute roughly equally to receptor
phosphorylation and desensitization induced by receptor-saturating
concentrations of agonist. Our results in transfected 293 cells suggest
a similar scheme for the
AR. Assessed with either
inhibitors of PKA and GRKs or with specific activators of PKA,
agonist-promoted phosphorylation and desensitization of the
AR appear to be due to approximately equal
contributions from GRKs and PKA.
AR appears to be remarkably similar to the
AR. Although certain intracellular domains of the
AR may differentiate it from the
AR
at the level of G
coupling (34, 35) and
agonist-promoted receptor sequestration(36) , the primary
structures relevant to rapid desensitization are indeed similar between
the receptor subtypes. Both receptors possess PKA sites in their third
intracellular loops(6) . While the
AR
cytoplasmic tail contains 11 serine and threonine residues thought to
be candidates for GRK-mediated
phosphorylation(10, 20) , the cognate
AR region contains 10. This minor difference is almost
certainly insignificant, however, since the actual stoichiometry of
agonist-induced
AR phosphorylation by intact cells
(like that of the
AR) approximates 1 mol of
phosphate/mol of receptor(37) . Furthermore, regions of the
activated
AR other than the cytoplasmic tail substrate
domain are known to be very important in
ARK1
activation(38) . The receptor's first intracellular loop
is one such region. Indeed, a peptide corresponding to amino acids
57-71 of the human
AR has been shown to inhibit
ARK1-mediated
AR phosphorylation and
ARK1-initiated
AR desensitization(39) .
The human
AR shares 73% homology and 80% similarity
with the
AR in this domain(6) .
AR
desensitization. Desensitization in their system, however, was evident
only by an increase in the EC
for ISO-stimulated adenylyl
cyclase activity in membranes from ISO-pretreated cells. Unlike our
studies in CHW and 293 cells, their studies showed no desensitization
of the ISO-stimulated adenylyl cyclase maximal response. Their membrane
cyclase assays were probably confounded, though, by the presence of
ARs in the SK-N-MC cells(40) . The
AR can mediate virtually all of the ISO-stimulated
SK-N-MC cell adenylyl cyclase response when concentrations of ISO reach
the high (
1 µM) levels required to elicit the maximal
response of adenylyl cyclase(40) . Since the
AR fails to desensitize(14) , it is therefore
not surprising that the maximal adenylyl cyclase response to
ISO also fails to desensitize in SK-N-MC cells. This
AR contribution to ISO-stimulated SK-N-MC adenylyl
cyclase activity also complicates the interpretation of other
observations made by Zhou and Fishman(5) . For example, when
SK-N-MC cells are permeabilized and loaded with heparin, a
ARK1
inhibitor, no effect on ISO-induced desensitization is seen in (5) . From these data, Zhou and Fishman (5) infer that
GRK-initiated desensitization is unimportant for the
AR. If
AR-mediated signaling in
SK-N-MC cell membranes obscures GRK-initiated
AR
desensitization, however, one might expect no effect of heparin on the
residual apparent
AR desensitization. Similarly,
AR-mediated signaling may underlie the failure of PKA
activators to engender desensitization of ISO-stimulated adenylyl
cyclase in SK-N-MC cells(5) . In light of this failure to
desensitize the
AR with PKA activation, the mechanism
by which the peptide inhibitor of PKA attenuates ISO-induced
desensitization in permeabilized SK-N-MC cells (5) remains
unclear. In our
AR-transfected 293 cell model system,
we were able to study the ISO-stimulated cAMP signal of specifically
the
AR by fully antagonizing the endogenous 293 cell
AR with ICI-118,551.
AR is
linked to GRK5 and
ARK1 circumstantially. The relative expression
of GRK5 mRNA is highest in myocardium(10) , where the human
AR is also highly expressed(1) . A coordinate
50% down-regulation of the
AR and 2-3-fold
up-regulation of
ARK1 has been observed in human chronic heart
failure(11) , in which decreased cardiac responsiveness to
-adrenergic agonists is thought to exacerbate the disease (for
review, see (1) ). Nonetheless, uncoupling of the diseased
myocardial human
AR from effectors remains to be
demonstrated. Previous human studies may have had limited sensitivity
to detect
ARK1-mediated
AR uncoupling, however,
since they utilized adenylyl cyclase assays of washed (i.e.
ARK1-depleted) membranes prepared without phosphatase
inhibitors (41) . Porcine models of heart failure, by contrast,
have succeeded in demonstrating
AR
uncoupling(42) . Recently,
AR uncoupling
(preceding down-regulation) in porcine heart failure has been
correlated with significant increases in myocardial GRK
activity(47) . Also linking
ARK1 with the
AR in the heart are recent data from transgenic mice,
showing that
AR-mediated events are attenuated by
myocardial overexpression of
ARK1 and potentiated by myocardial
overexpression of a
ARK1 antagonist(43) . By directly
demonstrating
ARK1-mediated phosphorylation of the human
AR, our data considerably strengthen the credibility
of
ARK1 as a potential therapeutic target in chronic heart
failure.
-muscarinic
acetylcholine receptor(44) , and the
-adrenergic receptor(26) , the
AR, like the
AR (26) ,
appears to be a relatively promiscuous GRK substrate. What factors,
then, determine the relative importance of any particular GRK to
desensitization of the
AR expressed in any particular
tissue or cell type? The anatomic distributions of GRK expression are
distinct, but overlapping(7) , and few data exist comparing
expression levels among individual GRKs (besides
ARK1 and
ARK2(16) ). Nonetheless, it seems reasonable to speculate
that cell-specific differences in both GRK subtype and quantity
primarily determine which GRK, if any, significantly affects the
signaling through the
ARs expressed in that cell.
Correlating GRK-mediated receptor phosphorylation with receptor
desensitization, as we have done with the
AR, seems
critical to the evaluation of receptor-GRK interaction. Because
distinct peptide substrate specificities have been demonstrated for
ARK1 and GRK5 (44, 45) , it might be that various
GRKs phosphorylate the same receptor at distinct serine or threonine
residues, thereby engendering variable effects on receptor
desensitization. The transfected cell system we have developed for
studying the
AR should prove valuable in exploring
these possibilities.
AR,
-adrenergic receptor; PKA,
cAMPdependent protein kinase;
ARK,
-adrenergic receptor
kinase; G protein, guanine nucleotide-binding regulatory protein; GRK,
G protein-coupled receptor kinase; G
, stimulatory G
protein; ISO, (-)-isoproterenol; CHW, Chinese hamster fibroblast;
293 cells, human embryonic kidney cells; IBMX,
3-isobutyl-1-methylxanthine; TPA,
12-O-tetradecanoylphorbol-13-acetate; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfon-amide
2HCl;
PBS, phosphate-buffered saline.
We thank Dr. Madan Kwatra and Susan Trukawinski for
help in preparing the AR recombinant baculovirus, Dr.
Julie A. Pitcher for in vitro assays of H-89 and staurosporine
effects on GRK-mediated phosphorylation, and Humphrey Kendall and Grace
Irons for expert technical assistance.
AR in CHW
cells has recently been documented by Zhou et
al.(48) , and the ability of heparin to inhibit this
desensitization in permeabilized cells suggests a GRK-dependent
mechanism(48) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.