(Received for publication, April 3, 1997, and in revised form, May 8, 1997)
From the Howard Hughes Medical Institute, Departments of Medicine
(Cardiology) and Biochemistry, Duke University Medical Center,
Box 3821, Durham, North Carolina 27710
Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, we tested the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-specific, desensitization occurred within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponded temporally with agonist-induced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32Pi-labeled cells. Agonist-induced receptor phosphorylation was not substantially affected by PKC inhibition but was reduced 40% (p < 0.03) by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogated agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augmented agonist-induced ET-R phosphorylation ~2-fold (p < 0.02), but each kinase reduced receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibited ET-R signaling by only ~25%, GRK2 inhibited ET-R signaling by 80% (p < 0.01). Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitated with the ET-Rs in an agonist-dependent manner. We conclude that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. We propose that because of its affinity for ET-Rs demonstrated by co-immunoprecipitation, GRK2 is the most likely of the GRKs to initiate ET-R desensitization.
A variety of vital cardiovascular and developmental functions are mediated through the G protein1-coupled, heptahelical endothelin A and endothelin B receptors (ETA-R and ETB-R) (1, 2). Like many G protein-coupled receptor signaling systems (3), the receptor/Gq/phospholipase C system(s) activated by the ETA-R and ETB-R have demonstrated diminishing responsiveness to prolonged stimulation (4-11). This desensitization may be particularly important in the regulation of ET-R-mediated signaling, since endothelins bind their receptors essentially irreversibly under physiological conditions (2). In several cell types or tissues, this desensitization has been characterized phenomenologically as homologous, or receptor-specific (5-11), but the molecular mechanisms effecting this desensitization remain to be elucidated.
For a growing number of G protein-coupled receptor signaling systems, phosphorylation of the receptor on serine and threonine residues appears to be an important mechanism of desensitization (3, 12). This phosphorylation can be accomplished by at least two classes of protein kinases: (i) second messenger-dependent kinases, such as cAMP-dependent protein kinase or PKC isoforms, and (ii) the family of G protein-coupled receptor kinases (GRKs), which are activated by and phosphorylate agonist-occupied receptors (13, 14). The GRK family comprises six cloned members, with highly homologous catalytic domains flanked by divergent N and C-terminal domains (14). In model receptor systems, GRK-mediated receptor phosphorylation appears to facilitate the binding of an inhibitory arrestin protein to the receptor, resulting in uncoupling of the receptor from its G protein in the process of homologous desensitization (12), within seconds to minutes of receptor activation (15, 16). This GRK-initiated process has been shown to attenuate severely (17, 18) or even to terminate (19) signaling through a variety of G protein-coupled receptors.
Like several receptors shown to be phosphorylated by GRKs (18, 20, 21), the human ETA-R and ETB-R each have cytoplasmic carboxyl-terminal tail domains that are rich in serine and threonine residues (22, 23). Only for the ETB-R, however, does the primary structure surrounding some of these residues resemble the receptor sequences previously demonstrated to be phosphorylated by GRKs (20). Indeed, the relatively low homology between the ETA-R and ETB-R in the cytoplasmic tail domain suggests the possibility that these receptors might be regulated differently by GRKs, or regulated by different GRKs. To date, no data directly address this issue. However, experiments with cytoplasmic tail truncation mutants of the ETA-R (24) and the ETB-R (25) suggest that large portions of each receptor's cytoplasmic tail may be removed without impairing agonist-induced desensitization of endothelin-1-stimulated chloride currents (24) or calcium transients (25). To test directly whether or not homologous desensitization of the human ETA-R and ETB-R proceeds via a GRK-initiated mechanism, we used a transfected, intact cell model for assessing both receptor phosphorylation and signaling through phospholipase C. In this model system, we also overexpressed individual GRKs with the ETA-R and ETB-R to test the hypothesis that different GRKs might phosphorylate and initiate desensitization of the ETA-R and ETB-R.
Embryonic rat thoracic aortic smooth muscle (A10)
cells and human embryonic kidney 293 cells were obtained from the
American Type Culture Collection, and all cell culture supplies were
obtained from Life Technologies, Inc.
125I-Tyr13-labeled ET-1,
myo-[2-3H]inositol,
L-[35S]methionine/L-[35S]cysteine
(EXPRE35S35S protein labeling mix),
[-32P]GTP, and [32P]orthophosphate were
procured from NEN Life Science Products. Biotinylated 12CA5 IgG and
staurosporine were from Boehringer Mannheim, 12CA5 ascites was from
BabCo, and the M2 monoclonal IgG1 (both biotinylated and
native) was from IBI. Horseradish peroxidase-conjugated streptavidin
and antispecies IgGs were from Jackson ImmunoResearch. Protein
A-Sepharose and protein G-Sepharose were obtained from Pharmacia
Biotech Inc. The AG1-X8 anion exchange resin was procured from Bio-Rad.
Peninsula Laboratories was the source of ET-1, and Sigma was the source
of angiotensin II. The carboxyl-terminal amide of SFLLRN was
synthesized on an Applied Biosystems 430A peptide synthesizer, using
FastMocTM chemistry, and the product was purified by high
pressure liquid chromatography.
The cDNAs for the human
ETA-R and ETB-R were the generous gifts of
Masachi Yanagisawa (Department of Molecular Genetics, University of
Texas Southwestern Medical Center, Dallas, TX) (23, 26). Cassette PCR
was employed to remove the endogenous signal sequence of each cDNA
and to add to each cDNA an influenza virus hemagglutinin signal
sequence (27) followed by either a hemagglutinin epitope recognized by
the 12CA5 monoclonal antibody (21) (for the ETA-R) or the
FLAGTM epitope (for the ETB-R) (27). The 5
(mutagenic) primers for the ETA-R and ETB-R,
respectively, were
5
-cgcgggaagcttacc(atgaagaccatcatcgccctgagctacatcttctgcctggtgttcgcc)gacgcc[tacccctacgacgtccccgactacgcc]gataatcctgagagatacagc-3
and
5
-cgcgggaagcttacc(atgaagaccatcatcgccctgagctacatcttctgcctggtgttcgcc)[gactacaaggacgatgatgacgcc]gaggagagaggcttcccgcct-3
. Parentheses surround the signal sequence, brackets surround the epitope
sequence, boldface type highlights the initiator methionine, and
underlined type denotes nucleotides 541-561 (ETA-R)
and 309-329 (ETB-R) of the native receptor sequences. For
the ETA-R, first the 1.1-kilobase pair
EcoRV/Xmn I cDNA fragment and then the
292-base pair HindIII/EcoRV-cut PCR product were
subcloned into pcDNA I (Invitrogen) to generate the
epitope-tagged ETA-R (t-ETA-R). For the
ETB-R, first the 1-kilobase pair
EcoRV/XbaI cDNA/polylinker fragment (23) and
then the 606-base pair HindIII/EcoRV-cut PCR product were subcloned into pcDNA I (Invitrogen) to generate
the epitope-tagged ETB-R (t-ETB-R). The
authenticity of the sequences amplified by PCR was verified by dideoxy
sequencing.
The bovine GRK2 N-terminal minigene construct was also made by cassette
PCR. The cDNA corresponding to amino acids 1-180 was amplified
with a 5-primer that also encoded the hemagglutinin epitope recognized
by 12CA5; the resulting fragment was ligated into pCMV5 at
BamHI and HindIII sites. Plasmid constructs for the 195-amino acid bovine GRK2 C-terminal domain polypeptide (28), human GRK6 (29), the hemagglutinin epitope-tagged AT1A-R
(18), bovine GRK2, the bovine GRK2 K220R dominant negative mutant,
bovine GRK3, and bovine GRK5 (21) have been described previously.
Rat A10 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum as well as penicillin (100 units/ml) and streptomycin (100 µg/ml) and incubated in 95% air, 5% CO2 at 37 °C in a humidified incubator. Human 293 cells were propagated and transfected by the calcium phosphate method as described (21).
For most t-ETA-R transfections, 10 µg of receptor plasmid was used, along with 5 µg of GRK, GRK2 polypeptide, or empty vector plasmid per 100-mm dish. For homologous desensitization experiments, 293 cells were transfected with 4 µg of t-ETA-R and 10 µg of AT1A-R per 100-mm dish, to ensure that phosphoinositide hydrolysis mediated by the AT1A-R exceeded that mediated by the t-ETA-R. For t-ETB-R transfections, 4 µg of receptor plasmid was used, along with either 4 µg of GRK, 2-6 µg of empty vector, or 6 µg of AT1A-R plasmid (for a total of 10 µg) per 100-mm dish. For AT1A-R experiments, cells were transfected as described (18). The day after transfection, cells from all identically transfected plates were pooled and aliquoted, at the same density, for functional or receptor expression assay (21). With transfection on day 1, cells were assayed on day 3 or 4 (with equivalent results).
Assays for Receptor Expressiont-ETA-R and t-ETB-R expression levels were assessed in pilot experiments by [125I]ET-1 binding assays, modified from Sakamoto et al. (23). Assays were performed in triplicate, and data were analyzed by least squares nonlinear regression with PrismTM software (Graphpad). These assays demonstrated receptor expression levels of 1-3 pmol/mg of total cell protein for both receptor subtypes.
The relative receptor expression among cell lines co-transfected with various plasmids was routinely assessed by immunofluorescence and flow cytometry, performed as described previously (21). Typical transfection efficiencies ranged from 35 to 50% and from 45 to 70% for the t-ETA-R and t-ETB-R, respectively. To obtain a surrogate for receptor expression in each transfected cell line, the mean fluorescence/cell was multiplied by the percentage of cells staining positive for receptor. Cells co-transfected with various plasmids typically demonstrated receptor expression levels within 30% of that measured in empty vector-co-transfected control cells. Cells with receptor expression levels outside this range were not used.
Cellular Phosphoinositide Hydrolysis and Desensitization AssaysA10 and 293 cells were metabolically labeled for 18-24 h with 2 µCi of [3H]inositol/ml of labeling medium, which differed from the cell-specific growth medium only in that it contained 5% fetal bovine serum. A10 cells were plated in labeling medium at 5.3 × 104/cm2 in 12-well dishes. After labeling, cells were washed with Dulbecco's phosphate-buffered saline and then exposed at 37 °C to 0.2% bovine serum albumin and 20 mM LiCl in Dulbecco's phosphate-buffered saline ("PI medium"), without (basal) or with (stimulated) 100 nM ET-1 for the indicated times. Each time point comprised a single 12-well plate containing triplicate wells for both basal and stimulated cells. Reactions were stopped by the addition of an equal volume of 0.8 M perchloric acid to the wells, and total inositol phosphates were assayed by anion exchange chromatography as described (18). To facilitate comparisons between independent experiments, cellular inositol phosphates obtained from each well were normalized to the amount of intracellular 3H counts in that well (determined by counting a 50-µl aliquot of neutralized cell extract before chromatography). The resulting value, multiplied by 100, is referred to as "percent conversion of 3H into inositol phosphates."
Signaling assays with 293 cells (see Figs. 7, 8, 9) were performed in an
analogous manner with the following modifications: cells were plated at
1.6 × 105/cm2 in labeling medium; each
12-well plate comprised triplicate wells of each transfected cell type;
and agonist stimulation transpired for 8 min. Homologous
desensitization assays with 293 cells were performed with three 12-well
plates manipulated in concert. During the "first period" of 3 min,
cells were exposed at 37 °C to PI medium without ("control
plate") or with ("desensitized plates") the indicated
concentrations of ET-1. PI medium was then removed from all wells.
Next, during the "signaling period," cells from both control and
desensitized plates were exposed for 5 min at 37 °C to PI medium
containing vehicle ("basal"), 100 nM ET-1, or 100 nM angiotensin II. Reactions were then terminated, as
above. For desensitized cells, basal inositol phosphate values for the signaling period were obtained by terminating reactions on one of the
desensitized plates at the outset of the signaling period.
Intact Cell Phosphorylation
These assays were performed as described previously (21). Briefly, cells metabolically labeled with 32Pi were stimulated with the indicated stimulus for 3 min (ET-1) or 10 min (TPA), washed, and solubilized in detergent buffer. Cells expressing the t-ETA-R were solubilized, and receptors were immunoprecipitated with 12CA5, as described (21). Cells expressing the t-ETB-R were solubilized in "M2 detergent buffer" (1% (w/v) Triton X-100TM, 0.05% SDS, 5 mM EDTA, 50 mM Tris-Cl, pH 8.0 (25 °C), 200 mM NaCl, with phosphatase and protease inhibitors as described (21)). The t-ETB-Rs were immunoprecipitated as described (21), but with the M2 monoclonal IgG1 and protein G-Sepharose. Immune complexes were dissociated and resolved on SDS-10% polyacrylamide gels, which were loaded with equivalent amounts of receptor protein per lane, as described (21). Autoradiography and Molecular Dynamics PhosphorImagerTM analysis of radioactive gels were also performed as described (21).
Stoichiometry of Receptor PhosphorylationEquilibrium biosynthetic labeling and immunoprecipitation were employed to determine the stoichiometry of ETB-R phosphorylation in intact 293 cells, as described previously (21).
Membrane GTPase Assays293 cells plated at 1 × 105/cm2 in 150-mm dishes were subsequently
exposed to 100 nM ET-1 ("desensitized") or vehicle
("naive") at 37 °C, in minimal essential medium containing 20 mM HEPES, pH 7.4, and 1 µM okadaic acid
(Calbiochem) (to inhibit possible phosphatase activity during the
subsequent acid washes (30)). After 3 min, the cells were washed three
times at 4 °C in 50 mM glycine, pH 2.5, in a protocol
shown by Chun et al. (31) to remove receptor-bound ET-1.
After a further wash in Dulbecco's phosphate-buffered saline, the
cells were scraped at 0 °C into 10 mM Tris-Cl, 2 mM EDTA, pH 7.4 (25 °C), supplemented with protease inhibitors (21). Cells were lysed with a motorized Teflon pestle, and
crude membranes were pelleted at 30,000 × g (4 °C)
for 15 min. Membranes were washed once at 4 °C in 10 mM
triethanolamine, pH 7.4 (30 °C), supplemented with protease
inhibitors (21), and finally resuspended at a protein concentration of
0.3 mg/ml in the same buffer. ET-1-stimulated hydrolysis of
[-32P]GTP was determined with triplicate 15-µg
aliquots of these membranes, as described (18). Reactions were stopped
at 10 min, a time at which reaction rates were still linear.
293 cells were
transfected with either an endothelin receptor or the
AT1A-R and the indicated plasmids (see Fig. 10); they were
plated at 7-9 × 104/cm2 in 90-mm dishes
and assayed the next day. As in the inositol phosphate signaling
assays, cells were washed and then exposed to PI medium for 8 min
(37 °C) in the absence (unstimulated) or presence (stimulated) of
100 nM ET-1 or 100 nM angiotensin II. (Albumin
was omitted from the PI medium for these experiments.) PI medium was
then replaced with 1 ml of cross-linking buffer (Dulbecco's
phosphate-buffered saline containing 10 mM HEPES, pH 7.4 (to increase buffering capacity), 10% (v/v) Me2SO, and 2.5 mM of the cell-permeant homobifunctional cross-linking
agent dithiobis(succinimidylpropionate) (Pierce). Cells were incubated at 25 °C for 30 min, with continuous rocking. (In AT-R experiments, the cross-linking buffer on stimulated plates contained 100 nM angiotensin II.) Cross-linking buffer was then replaced
with 1 ml/plate of the detergent buffer appropriate for the monoclonal IgG to be used for immunoprecipitating the receptor (see above), and
receptor immunoprecipitation proceeded as described previously (18,
21), but with 17 µg of IgG/sample. Before the addition of IgG to the
detergent-solubilized cells, aliquots were removed for protein assay
and for subsequent immunoblotting to determine the approximate fraction
of cellular GRK co-immunoprecipitated with the receptor.
Dithiobis(succinimidylpropionate)-mediated cross-links were reduced,
and immune complexes were dissociated by incubation in Laemmli buffer
(32) at 37 °C for 90 min. As described above for phosphorylation
assays, equivalent amounts of receptor protein were resolved on
SDS-10% polyacrylamide gels and then transferred to nitrocellulose by
semidry blotting (32). Immunoblotting was performed with biotinylated
monoclonal IgGs, either the GRK2/3-specific C5/7 or the
GRK4/5/6-specific A16/17 (33), and horseradish peroxidase-conjugated
streptavidin. Enhanced chemiluminescence (Amersham Corp.) was used to
develop the immunoblots, which were exposed to Biomax MRTM film
(Eastman Kodak Co.). Quantitation of band density was performed with a
laser densitometer. To confirm that equivalent amounts of receptor
protein had been loaded in each gel lane, immunoblots processed for
detecting GRK2 or -5 were stripped (50 °C for 3-4 h in 2% (w/v)
SDS, 100 mM 2-mercaptoethanol, 62.5 mM Tris-Cl,
pH 6.7 at 25 °C) and reprobed with either biotinylated M2 IgG (for
the t-ETB-R) or biotinylated 12CA5 (for the
t-ETA-R and AT1A-R).
Statistical Analysis
The t test for independent means was used to compare results for different cell types, and two-sided p values were calculated using ExcelTM software (Microsoft), assuming equal sample variances (34). Because of considerable variability in membrane GTPase activity among experiments, paired t testing was used to compare naive with desensitized cells and to compare GRK2 K220R cells with control cells in the membrane GTPase experiments. Concentration/response curves were fitted by least squares nonlinear regression with variable slope, using PrismTM software (Graphpad). In the text, numerical values are given as means ± S.D., whereas means ± S.E. are plotted in all figures.
To demonstrate rapid desensitization of the ETA-R, we
investigated the time course of ET-1-stimulated phosphoinositide
hydrolysis in intact A10 cells, which endogenously express the
ETA-R (31). Within 10 min, the A10 cell phospholipase C
response to ET-1 ceased, consistent with ETA-R
desensitization (Fig. 1). When these ET-1-desensitized cells were challenged with thrombin agonist peptide, however, their
phospholipase C response resumed. Thus, the ETA-R
desensitization observed in A10 cells was homologous, or
receptor-specific. Moreover, because the
ETA-R-dependent phosphoinositide hydrolysis in
A10 cells waned substantially by 6 min, this desensitization would seem
to involve mechanisms other than receptor sequestration, which has been
shown to follow a somewhat longer time course in these cells (31).
To examine the molecular mechanisms responsible for endothelin receptor
desensitization and to facilitate direct comparison between the cloned
human ETA-R and ETB-R, we employed transfected human 293 cells as a model system. To evaluate homologous
desensitization in this system, we assayed ET-1-stimulated
phosphoinositide hydrolysis in 293 cells co-transfected with the rat
AT1A-R and either the human t-ETA-R or
t-ETB-R. A 3-min challenge with ET-1 reduced the subsequent
inositol phosphate response to ET-1 in a dose-dependent manner for both the t-ETA-R and the t-ETB-R
(Fig. 2). However, the initial challenge with ET-1 had
no effect on the subsequent inositol phosphate response to angiotensin
II. Thus, rapid homologous desensitization characterizes the signaling
behavior of both the t-ETA-R and the t-ETB-R in
this model 293 cell system.
Receptor sequestration attendant to the first challenge with ET-1 could not explain the desensitization findings in transfected 293 cells. Separate aliquots of cells challenged with 100 nM ET-1 and then processed for flow cytometry demonstrated that only 12 ± 5% of the cell surface t-ETA-Rs and 9 ± 1% of the t-ETB-Rs were internalized within 3 min. These small decrements in cell surface receptor number were modeled by transfecting cells with varying amounts of receptor plasmid, and had no effect on ET-1-stimulated phosphoinositide hydrolysis (data not shown).
To determine whether or not receptor phosphorylation was involved in
the observed agonist-promoted desensitization of the t-ETA-R and t-ETB-R, we immunoprecipitated
these receptors from 293 cells metabolically labeled with
32Pi. Receptors were isolated by
SDS-polyacrylamide gel electrophoresis, and representative
autoradiograms are presented in Figs. 3 and 4. The t-ETB-R (migrating at
Mr ~48,000-54,000; Fig. 3) and the t-ETA-R (migrating at Mr
~61,000-79,000; Fig. 4) both underwent significant
agonist-dependent phosphorylation in intact cells within 3 min. Thus, agonist-dependent phosphorylation correlated temporally with the agonist-dependent desensitization seen
in Fig. 2. Attesting to the biochemical significance of this receptor phosphorylation, the stoichiometry of t-ETB-R
phosphorylation was 1 ± 0.2 mol of phosphate/mol of receptor
(n = 2).
Because endothelin receptor stimulation results in activation of PKC isoforms, we sought to determine the extent to which PKC might participate in the rapid agonist-dependent phosphorylation of the endothelin receptors. In experiments presented in Fig. 3, 293 cells were incubated with a PKC-selective concentration of staurosporine briefly and then challenged with either ET-1, to evoke agonist-induced receptor phosphorylation, or TPA, to activate PKC. Whereas staurosporine abolished the TPA-stimulated t-ETB-R phosphorylation by PKC, it had no significant effect on agonist-promoted t-ETB-R phosphorylation. Similar results were obtained with the ETA-R (data not shown). Thus, for both the t-ETA-R and the t-ETB-R, agonist-induced phosphorylation must be mediated by kinases other than PKC.
To test the possibility that agonist-promoted t-ETA-R
phosphorylation is mediated by one or more of the GRKs endogenously expressed by 293 cells (21), we performed phosphorylation experiments in cells transfected with a dominant-negative K220R mutant of GRK2 (21,
35). This K220R mutant of GRK2 cannot perform a phosphotransferase
reaction (35); therefore, for any receptor substrate of GRK2, the K220R
mutant can act as a competitive inhibitor of any GRK that could bind to
and subsequently phosphorylate the receptor (18, 21, 35-37).
Characteristically, however, the GRK2 K220R mutant inhibits GRK action
incompletely; even when present in 15-fold molar excess over GRK2, the
K220R mutant inhibits only 60% of agonist-stimulated
2-adrenergic receptor phosphorylation in
vitro (35). Co-expression of the GRK2 K220R with the
t-ETA-R in 293 cells resulted in a 40% reduction in
agonist-induced t-ETA-R phosphorylation (Fig. 4). In
contrast, GRK2 K220R expression had no effect on TPA-stimulated
t-ETA-R phosphorylation (Fig. 4). Thus, a substantial
fraction of the agonist-induced t-ETA-R phosphorylation appears to be mediated by one or more GRKs expressed endogenously in
293 cells.
The foregoing experiments demonstrate a temporal correlation between
agonist-induced phosphorylation and desensitization of the
t-ETA-R and t-ETB-R. To correlate the extent of
GRK-mediated receptor phosphorylation with the extent of
desensitization, we inhibited agonist-promoted ETA-R
phosphorylation in 293 cells with dominant-negative GRK2 and assessed
the effect of such inhibition on ET-1-stimulated GTPase activity in
membranes derived from these cells (Fig. 5A).
A 3-min exposure of cells to ET-1 reduced the subsequent
ET-1-stimulated membrane GTPase activity by 40%, compared with that
observed in membranes derived from control cells. With cells expressing
dominant-negative GRK2, however, exposure to ET-1 did not affect the
subsequent ET-1-stimulated membrane GTPase activity. Thus, abrogation
of desensitization appears to attend the inhibition of GRK-mediated
t-ETA-R phosphorylation by GRK2 K220R. In contrast to
dominant-negative GRK2-transfected cells, native GRK2-transfected cells
demonstrated desensitization indistinguishable from control cells (Fig.
5B), although GRK2 overexpression resulted in augmented
agonist-induced ETA-R phosphorylation (see below; Fig.
6). In 293 cells, agonist-induced ETA-R
phosphorylation mediated by endogenous GRK(s) therefore appears both
necessary and sufficient for ETA-R desensitization.
Which of the anatomically disseminated (12) mammalian GRKs can effect agonist-induced phosphorylation of the endothelin receptors? To address this question, we overexpressed GRK2, -5, and -6 along with each endothelin receptor in 293 cells and studied the effects of GRK overexpression on agonist-induced receptor phosphorylation. Overexpression of any of the three GRKs tested significantly increased the level of agonist-induced receptor phosphorylation (Fig. 6). For the t-ETA-R, co-overexpression of either GRK2, GRK5, or GRK6 augmented agonist-induced phosphorylation 1.6 ± 0.2-, 1.7 ± 0.3-, or 2.0 ± 0.6-fold, respectively, over that observed in control cells (p < 0.02) (Fig. 6A). For the t-ETB-R, co-overexpression of GRK2, -5, or -6 augmented agonist-induced phosphorylation 2.6 ± 0.4-, 2.1 ± 0.8-, or 2.4 ± 0.8-fold, respectively, over that observed in control cells (p < 0.02) (Fig. 6B). Under the conditions prevailing in these cellular assays, then, the t-ETA-R and t-ETB-R each appear susceptible to agonist-promoted phosphorylation mediated by each of the three widely expressed mammalian GRKs.
To correlate GRK-mediated receptor phosphorylation with effects on receptor signaling in the intact cell, we assayed ET-1-stimulated phosphoinositide hydrolysis in cells co-expressing the ET-Rs with GRK2 and GRK5 (Fig. 7, A and B). Cells overexpressing GRK2 with the t-ETA-R or t-ETB-R produce only 16 ± 4% or 22 ± 5%, respectively, of the maximal inositol phosphate response observed in control cells after an 8-min challenge with ET-1 (p < 0.002). By contrast, cells overexpressing GRK5 with the t-ETA-R or t-ETB-R produce 75 ± 8% or 73 ± 13%, respectively, of the maximal inositol phosphate response observed in control cells (p < 0.03) (Fig. 7, A and B). Despite these consistent and distinct effects on ET-R-promoted phosphoinositide hydrolysis, overexpression of GRK2 or GRK5 failed to affect phosphoinositide hydrolysis engendered by fluoroaluminate, which stimulates G proteins directly (38) (data not shown). Thus, overexpression of either GRK2 or GRK5 appears to diminish agonist-promoted phosphoinositide hydrolysis at the level of the endothelin receptor and not at the level of even the proximate signal-transducing protein.
To ascertain that the overexpressed GRKs were indeed inhibiting ET-1-promoted phosphoinositide hydrolysis at the level of the endothelin receptors, we tested the 293 cell GRK overexpression system with two additional Gq-coupled receptors. First, we transfected 293 cells with plasmids encoding the rat type IA angiotensin II receptor (AT1A-R), along with plasmids encoding GRK2 or GRK5. Using an identical transfection scheme, we previously demonstrated that overexpression of either GRK2 or GRK5 augments agonist-induced AT1A-R phosphorylation to the same degree (18). As can be seen in Fig. 7C, however, overexpression of these GRKs with the AT1A-R produces distinct yet similar effects on agonist-promoted phosphoinositide hydrolysis. Interestingly, the signaling suppression achieved by GRK5 overexpression is substantially greater with the AT1A-R (60%) than with either endothelin receptor (~25%), despite equivalent levels of GRK5 expression attained in each distinct cell line (assessed by immunoblotting; data not shown). Contrastingly, the profound signaling suppression achieved by GRK2 overexpression is similar for the AT1A-R, ETA-R, and ETB-R (Fig. 7).
To assess the receptor specificity of GRK-induced signal dampening further, we exploited the thrombin receptors expressed endogenously by 293 cells (Fig. 8). In phosphoinositide hydrolysis assays, cells like those in Fig. 7 were stimulated either with ET-1, to stimulate the transfected ET-Rs, or with the thrombin agonist peptide SFLLRN, to stimulate the 293 cell thrombin receptors. As before, overexpression of GRK2 suppressed ET-1-stimulated signaling profoundly, and overexpression of GRK5 suppressed ET-1-stimulated signaling modestly (Fig. 8). In these same cells, however, overexpression of either GRK2 or GRK5 suppressed thrombin receptor signaling to an equivalent, modest degree (Fig. 8). Thus, whether assessed with different Gq-coupled receptors in distinct cell lines or with different Gq-coupled receptors within the same cell, the signal-suppressing activity of overexpressed GRK5 appears receptor-specific.
Whereas overexpressing GRK2 or GRK5 achieved equivalent levels of augmented agonist-induced receptor phosphorylation, such overexpression achieved disparate levels of signaling suppression for the t-ETA-R, t-ETB-R, and AT1A-R. Thus, incremental receptor phosphorylation achieved by GRK overexpression failed to correlate with signaling suppression achieved by GRK overexpression. This apparent paradox prompted us to test further the mechanism by which GRK overexpression suppresses Gq-coupled receptor phosphoinositide signaling in 293 cells.
To determine whether or not receptor phosphorylation is required for receptor-specific signal dampening by overexpressed GRKs, we examined the effect of the dominant-negative GRK2 on receptor-mediated phosphoinositide hydrolysis in intact 293 cells. Surprisingly, the dominant-negative (K220R) GRK2 mutant suppressed receptor signaling as effectively as the wild type GRK2 for both ET-Rs (Fig. 9, and data not shown). Moreover, by transfecting graded amounts of GRK2 and GRK2 K220R plasmid DNA in parallel cell groups, we could find no significant disparity between the wild type and mutant GRK effect on signaling at any level of GRK protein expression (data not shown). Thus, GRK-mediated receptor phosphorylation appears unimportant to the suppression of receptor signaling observed in intact 293 cells overexpressing certain GRKs.
Because GRK2 had the most profound dampening effect on endothelin receptor signaling, we investigated the ability of the noncatalytic GRK2 polypeptide domains (14) to accomplish such signal dampening. Cellular expression of the 180-amino acid GRK2 N-terminal domain polypeptide diminished t-ETA-R-dependent phosphoinositide hydrolysis, by 27 ± 6% (p < 0.02). Expression of the 195-amino acid C-terminal domain polypeptide (28), however, had no effect on signaling (Fig. 9).
Both the phosphorylation-incompetent GRK2 mutant (GRK2 K220R) and the GRK2 N-terminal domain polypeptide suppressed endothelin receptor signaling. Could a simple physical association of GRK with the activated receptor, by itself, engender the observed suppression of signaling? To test this hypothesis, we immunoprecipitated the t-ETA-R, t-ETB-R, or AT1A-R from 293 cells co-transfected with either GRK2 or GRK5 and immunoblotted these immunoprecipitates to detect the presence of GRK co-immunoprecipitated with each receptor. Data obtained with the t-ETB-R and AT1A-R are depicted in Fig. 10.
When immunoprecipitated from unstimulated cells, both the ETB-R and the AT1A-R appeared to be associated with a small amount of either GRK2 or GRK5. For both receptors, there was a substantial increase in receptor-associated GRK2 attendant to activation by agonist (13 ± 8-fold for the ETB-R; 29 ± 3-fold for the AT1A-R). Only for the AT1A-R, however, did the amount of receptor-associated GRK5 increase substantially with agonist stimulation (8 ± 1-fold). Agonist-dependent GRK5 association with the receptor occurred exclusively for the AT1A-R, even in 293 cells co-transfected with the ETB-R, AT1A-R, and GRK5 (data not shown). Agonist-dependent association between an overexpressed GRK and the receptor thus appears to correlate with GRK-mediated suppression of signaling, a process that appears to be specific at the level of both the Gq-coupled receptor and the GRK.
Because of their exceptionally high affinity for their agonists, the ETA-R and ETB-R may remain stably associated with their agonists long after initial agonist binding (2, 39). The resulting persistence of the receptors' activated states makes intracellular mechanisms of receptor/effector desensitization exceedingly important for cellular homeostasis. In this investigation, we have shown for the first time that rapid, agonist-promoted desensitization of both the ETA-R and ETB-R involves phosphorylation of the receptors, and that this phosphorylation results from GRK activity in an intact, transfected cell model. Surprisingly, despite the dissimilarity of their cytoplasmic C-terminal tail domains, the ETA-R and ETB-R appear to interact with GRKs indistinguishably.
That the ETA-R and ETB-R are regulated by one or more GRKs in vivo is anatomically plausible, since the ET-Rs (40) as well as GRKs 2, 5, and 6 (12) are all expressed together, for example, in human myocardium. Moreover, the paradigm for GRK-initiated desensitization of only agonist-occupied receptors (12) is consonant with the homologous ETA-R and ETB-R desensitization observed in a multitude of experimental systems (5-11). The rapid rate of receptor/effector desensitization observed in some of these systems (4, 5, 7-9, 11) is also consistent with a GRK-initiated mechanism (15, 16). In this study, the use of dominant-negative GRK2 to inhibit both agonist-induced phosphorylation and desensitization of the ETA-R demonstrates that these events are indeed mediated by a GRK mechanism (18, 21, 35, 36).
This study describes three distinct aspects of the relationship between GRKs and the ET-Rs: (i) the dependence of ET-R desensitization upon GRK-mediated ET-R phosphorylation, demonstrated in experiments that use dominant-negative GRK2 to inhibit both processes; (ii) the ability of individual GRKs to phosphorylate the ET-Rs, demonstrated in experiments that show augmented agonist-induced ET-R phosphorylation in cells transfected with specific GRK expression plasmids; and (iii) the affinity of individual GRKs for the ET-Rs, suggested by GRK-specific inhibition of ET-R-dependent signaling in intact cells and demonstrated by agonist-dependent association of GRK with receptor. As we discuss below, this last point seems most informative regarding GRK substrate specificity.
The incremental ET-R phosphorylation seen in cells overexpressing a particular GRK demonstrates the ability of that GRK to phosphorylate the receptor under the conditions studied. However, this incremental phosphorylation is probably unimportant with regard to desensitization of the receptor/G protein signaling system. This latter inference is supported by our membrane GTPase data with the ETA-R. Desensitization of ET-1-stimulated GTPase was equivalent in cells that overexpressed GRK2 and in cognate control cells that expressed only endogenous levels of GRK2. We obtained similar results with the AT1A-R previously (18). In 293 cells expressing endogenous GRKs, both the ET-R and the AT1A-R undergo GRK-mediated phosphorylation to a stoichiometry of ~1 mol of phosphate/mol of receptor. For these receptors in 293 cells, then, it appears that this stoichiometry of ~1 suffices for maximal binding of arrestins to the receptor and hence for maximal desensitization assessed by receptor signaling in membrane preparations.
The most unexpected finding of this study emerged from examining the ability of overexpressed GRKs to inhibit ET-R-mediated phosphoinositide hydrolysis in intact 293 cells. We anticipated that cellular overexpression of GRKs would diminish ET-R-dependent phosphoinositide hydrolysis in intact cells by augmenting both agonist-promoted ET-R phosphorylation and desensitization. Surprisingly, however, GRK-mediated suppression of ET-R signaling in intact cells failed to correlate with the incremental, GRK-mediated receptor phosphorylation observed. Rather, the ability of an overexpressed GRK to suppress receptor-mediated signaling correlated with the GRK's ability to bind to the receptor in an agonist-dependent manner, as revealed by co-immunoprecipitation experiments. Bound to the GRK, the agonist-occupied receptor appears sterically hindered from stimulating its G protein and thus from signaling. As assessed by the parameters of agonist-dependent receptor binding and signal dampening, both the ETA-R and ETB-R appear to interact selectively with GRK2, as opposed to GRK5 or GRK6. Contrastingly, the AT1A-R appears relatively nonselective with regard to GRK2 and GRK5.
Although probably attributable to our level of GRK overexpression, GRK-dependent dampening of receptor-mediated signaling nonetheless reveals a novel perspective on GRK substrate specificity. We showed that GRK-dependent suppression of signaling was receptor-specific by controlling for GRK expression in distinct cell lines expressing different receptors and by stimulating different receptors within the same GRK-overexpressing cell. Since this GRK-dependent signal dampening correlates with agonist-promoted binding of the GRK to the receptor, it must reflect the affinity of a given GRK for the activated form of a particular receptor. This receptor affinity is likely to be very important for GRK-mediated receptor phosphorylation in untransfected cells, which express physiologic (i.e. very low (41)) levels of both receptor and GRK. Applying this reasoning to our results with the ET-Rs, we would expect GRK2 to be the most active of the GRKs tested in desensitizing the ET-Rs in vivo.
Why does a GRK's affinity for the ET-Rs, manifested by co-immunoprecipitation, fail to correlate with the observed degree of agonist-induced phosphorylation in our 293 cell overexpression system? Two explanations seem relevant. First, ET-R phosphorylation is facilitated by the high expression levels of both receptor and GRK in the transfected 293 cells (20-50-fold above those seen physiologically). By mass action, even GRKs with relatively low affinity for the agonist-occupied receptor may phosphorylate it in this system, although they may not do so when enzyme and substrate are present at physiological concentrations. Second, ET-R phosphorylation may be bridled by other intracellular proteins (particularly the arrestins), which may compete with even the high-affinity GRK(s) for receptor binding. In this regard, it is notable that phosphorylation of the receptor has been shown both to decrease the affinity of GRK binding (42) and to increase the affinity of arrestin isoform binding to the receptor (43).
GRK-mediated phosphorylation of the ET-Rs does not, of course, preclude a role for other kinases in the phenomenon of homologous ET-R desensitization. Since they are activated by the ET-Rs (2), PKC isoforms would seem likely candidates for regulating the ET-Rs. In support of this idea, both the ETA-R (five sites) (22) and the ETB-R (seven sites) (23) possess numerous serines and threonines that occur within consensus sequences for PKC-mediated phosphorylation (44), and we have shown that PKC can clearly phosphorylate the ET-Rs in our experimental system. Despite these data, PKC appears to play no role in the rapid, agonist-induced ET-R phosphorylation we have studied here. Indeed, second messenger-dependent kinases, probably because of the multitude of their potential substrates, have been shown to effect receptor phosphorylation or/and desensitization at rates considerably slower than that evinced for GRKs (16, 18). In other cell systems or at later time points after agonist stimulation, however, it would not be surprising to observe a more prominent role for PKC isoforms in ET-R desensitization.
The site or sites of GRK phosphorylation on the ET-Rs remain
speculative. Suggesting as yet unidentified diversity in GRK phosphorylation sequences, neither ET-R (22, 23) possesses serine or
threonine residues residing in sequences conforming to those defined
for the best characterized GRK substrates (20). By analogy with other
receptors shown to be GRK substrates, the molecular architecture of the
ET-Rs suggests that the ET-R cytoplasmic tail domains would most likely
serve as sites for GRK phosphorylation (20, 22, 23, 45). Previous
mutational studies have contended, though, that the C-terminal
cytoplasmic tail of neither the ETA-R (24) nor the
ETB-R (25) is important for agonist-promoted receptor
desensitization. Inferences drawn from these studies regarding ET-R
desensitization and its potential mechanisms should be cautious,
however. "ET-R desensitization" in both of these studies was
monitored by signals considerably downstream from the ET-Rs themselves
(calcium-activated chloride currents in Xenopus oocytes and
cytosolic calcium transients in Ltk cells,
respectively). Results from these downstream signaling mechanisms may
be confounded by rapid desensitization of the inositol trisphosphate
receptor (46) and perhaps the chloride channels, too. Furthermore,
complete ETB-R desensitization appeared to be achieved with
very low fractional receptor occupancy and in the setting of
considerable heterologous desensitization (25). Such an observation
conflicts strongly with the behavior of a GRK-initiated mechanism (47).
Even if these mutational studies are correct, however, GRK
phosphorylation sites on the ET-Rs may reside in the C tail remnants
proximal to the truncation sites or in other cytoplasmic receptor
domains (22, 23).
In light of the rapid desensitization of the ET-R/Gq/phospholipase C signaling system by GRK-initiated (and perhaps other) mechanisms, how can we explain the prolonged physiologic action of ET-1 administered pharmacologically (48)? Whereas ET-R-promoted phospholipase C activity desensitizes rapidly, ET-R-promoted phospholipase D activity (2) may desensitize much more slowly, as has been observed for the AT1A-R (18, 49). Furthermore, the attenuating effect of nitric oxide on ET-1-stimulated signaling (39), which may facilitate ET-1 signal termination in vivo, may be unphysiologically deficient in experimental systems demonstrating sustained ET-1-promoted activity (1, 48).
In investigating the role of GRKs in the homologous desensitization of the ETA-R and ETB-R, we have employed a co-transfected cell technique used by our laboratory (21) and subsequently others (37, 50-52) to show the effect of overexpressed GRKs on agonist-induced receptor phosphorylation and signaling. Our current study reveals how overexpressed GRKs, by binding to agonist-occupied receptors, can inhibit receptor signaling in a manner that may manifest GRK substrate specificity previously unappreciated. Using the approach employed with the ET-Rs and the AT1A-R, future studies should substantially expand our understanding of GRK substrate specificity.
We are grateful to Dr. Richard Premont for subcloning the human GRK6 cDNA into pcDNA I; to Humphrey Kendall and Grace Irons for expert technical assistance; and to Mary Holben and Donna Addison for secretarial assistance.