Agonist-Induced Phosphorylation of the Endogenous AT1 Angiotensin Receptor in Bovine Adrenal Glomerulosa Cells
Roger D. Smith,
Albert J. Baukal,
Annamaria Zolyomi,
Zsuzsanna Gaborik,
Laszlo Hunyady,
Lu Sun,
Meng Zhang,
Hao-Chia Chen and
Kevin J. Catt
Endocrinology and Reproduction Research Branch (R.D.S., A.J.B.,
A.Z., L.S., M.Z., H.-C.C., K.J.C.) National Institute of Child
Health and Human Development National Institutes of Health
Bethesda, Maryland 20892-4510
Department of Physiology (Z.G.,
L.H.) Semmelweis University School of Medicine H-1444 Budapest,
Hungary
 |
ABSTRACT
|
---|
A polyclonal antibody was raised in rabbits
against a fusion protein immunogen consisting of bacterial
maltose-binding protein coupled to a 92-amino acid C-terminal fragment
of the rat AT1b angiotensin II (Ang II)
receptor. The antibody immunoprecipitated the photoaffinity-labeled
bovine AT1 receptor
(AT1-R), but not the rat
AT2 receptor, and specifically stained bovine
adrenal glomerulosa cells and AT1a
receptor-expressing Cos-7 cells, as well as the rat adrenal zona
glomerulosa and renal glomeruli. The antibody was employed to analyze
Ang II-induced phosphorylation of the endogenous
AT1-R immunoprecipitated from cultured bovine
adrenal glomerulosa cells. Receptor phosphorylation was rapid,
sustained for up to 60 min, and enhanced by pretreatment of the cells
with okadaic acid. Its magnitude was correlated with the degree of
ligand occupancy of the receptor. Activation of protein kinase A and
protein kinase C (PKC) also caused phosphorylation of the receptor, but
to a lesser extent than Ang II. Inhibition of PKC by staurosporine
augmented Ang II-stimulated AT1-R
phosphorylation, suggesting a negative regulatory role of PKC on the
putative G protein-coupled receptor kinase(s) that mediates the
majority of AT1-R phosphorylation. The antibody
should permit further analysis of endogenous
AT1-R phosphorylation in Ang II target cells.
 |
INTRODUCTION
|
---|
Angiotensin II (Ang II), the biologically active component
of the renin-angiotensin system, plays a major role in the physiology
of the cardiovascular system. The octapeptide hormone maintains blood
pressure and promotes salt and water retention by acting on a wide
range of target tissues, including vascular smooth muscle, the adrenal
cortex, pituitary, kidney, and several neuronal cell types (reviewed in
Ref.1). The actions of Ang II in target cells are mediated by specific
plasma membrane receptors, of which two distinct classes
(AT1 and AT2) have been identified and cloned
(2, 3, 4, 5). Both receptor subtypes are members of the seven-transmembrane
domain superfamily of G protein-coupled receptors (GPCRs), but share
only 32% amino acid sequence homology. The AT2 receptor is
widely distributed in the fetus but has a limited tissue distribution
in adults. Although its physiological functions remain obscure, recent
evidence suggests that the AT2 receptor mediates
anti-proliferative and/or apoptotic events in certain cell types (6).
In contrast, the AT1 receptor (AT1-R) is widely
distributed in adult tissues and mediates the major physiological
actions of Ang II. The classic signaling pathway activated by the
AT1-R is Gq/11-mediated activation of
phospholipase C-ß, with hydrolysis of the integral membrane lipid,
phosphatidyinositol 4,5-bisphosphate. The consequent generation of
water-soluble inositol 1,4,5-trisphosphate (which elicits the release
of Ca2+ from intracellular stores) and lipid-soluble
diacylglycerol (which activates protein kinase C) activates numerous
intracellular signaling pathways (reviewed in Ref.7). Although this
aspect of Ang II action is well characterized, several other functional
aspects of the AT1-R are poorly understood.
In recent years, agonist-induced GPCR phosphorylation, and its
relationship to receptor desensitization, have received much attention.
GPCRs can be phosphorylated by at least two types of protein kinases,
the GPCR kinases (GRKs) and second messenger-activated kinases such as
protein kinases A and C (8, 9). Although the role of GPCR
phosphorylation by second messenger-activated kinases is not yet clear,
GRK-mediated phosphorylation has been shown to favor the binding of
arrestin proteins that uncouple receptors from their cognate G
protein(s) (10, 11). This mechanism is responsible for the
desensitization of GPCR signaling that is commonly observed in cells
after initial stimulation by agonists. Agonist-induced phosphorylation
of several GPCRs including the ß1-adrenergic (12),
-opioid (13),
ETA and ETB endothelin (14), A3
adenosine (15), V2 vasopressin (16), and sst2A somatostatin
(17) receptors have been reported. However, since many of these studies
employed epitope-tagged receptors in transient expression systems,
their findings do not neccessarily reflect the behavior of native
receptors in normal target cells.
Although Ang II-induced phosphorylation of a transiently expressed,
epitope-tagged AT1-R has been observed in HEK 293 cells
(18), Ang II-induced phosphorylation of the native AT1-R
has not been reported in any normal cell type. This is due, in part, to
the lack of specific antibodies directed against the native
AT1-R. To address this problem, we employed a variety of
immunogens (based on several regions of the rat AT1b-R) to
raise anti-AT1-R antibodies in rabbits. One of these
antibodies, raised against a fusion protein immunogen consisting of
bacterial maltose-binding protein (MBP) coupled to the C-terminal
92-amino acid fragment of the rat AT1b-R,
immunoprecipitates the AT1-R and recognizes the receptor on
immunochemistry. With this antibody, it was possible to demonstrate Ang
II-induced phosphorylation of the native AT1-R in primary
cultures of bovine adrenal glomerulosa cells. This development should
allow for a more detailed analysis of the mechanisms of
AT1-R phosphorylation and its role in receptor
desensitization, internalization, and down-regulation in Ang II target
cells.
 |
RESULTS
|
---|
Antisera from rabbits immunized with the fusion protein (FP)
consisting of MBP coupled to the C-terminal 92 amino acids of the rat
AT1b angiotensin receptor were subjected to caprylic acid
precipitation and dialysis. The resulting preparation (anti-FP
antibody) was used either unpurified for immunoprecipitation or after
affinity purification for immunochemistry. Affinity purification was
achieved by sequential passage over MBP-Sepharose (to deplete anti-MBP
antibodies) and elution from FP-Sepharose (to enrich for
anti-AT1-R antibodies).
Immunoprecipitation of AT1-Rs
Anti-FP antibodies were assayed for their ability to
immunoprecipitate [125I]azido-Ang II
photoaffinity-labeled AT1-Rs from bovine adrenal
glomerulosa cells. In the absence of antibody, no photoaffinity-labeled
AT1-R was precipitated. However, both the unpurified and
affinity-purified anti-FP antibodies were able to immunoprecipitate the
receptor, which ran as a diffuse band of Mr 60,00065,000
in SDS-PAGE (Fig. 1A
).
Immunoprecipitation was specific since preincubation of the anti-FP
antibody with the FP immunogen completely abolished receptor
precipitation. In contrast, neither of two commercially available
anti-AT1-R antibodies (sc1173 and sc579) was able to
immunoprecipitate the receptor (Fig. 1A
).

View larger version (59K):
[in this window]
[in a new window]
|
Figure 1. Immunoprecipitation of Photoaffinity-Labeled
AT1-Rs
Solubilized
125I-[Sar1,(4-N3)Phe8]Ang
II photoaffinity-labeled membranes from (A) bovine adrenal glomerulosa
cells or (B) rat AT1a- or AT2-expressing Cos-7
cells were subjected to immunoprecipitation with (A) the indicated
antibodies [preincubated without (-) or with (+) FP], or (B) the
anti-FP antibody (Ab) or wheat-germ agglutinin (W) as indicated, and
resolved by SDS-PAGE.
|
|
To determine whether the anti-FP antibody was specific for the
AT1-R, and did not recognize the AT2-R,
solubilized membranes prepared from photoaffinity-labeled Cos-7 cells
transiently expressing either the rat AT1a-R or the rat
AT2-R were subjected to immunoprecipitation. Using the
lectin, wheat-germ agglutinin (which binds the carbohydrate moieties of
glycoproteins) as a positive control, the photoaffinity-labeled rat
AT1a-R and AT2-R each ran as diffuse bands of
Mr 85,000140,000 in SDS-PAGE (Fig. 1B
). However, only the
photoaffinity-labeled rat AT1a-R was immunoprecipitated by
the anti-FP antibody.
Immunochemistry of AT1-Rs
The ability of the anti-FP antibody to recognize the
AT1-R on immunocytochemistry was evaluated by comparing its
staining patterns to those obtained using an anti-hemagglutinin (HA)
antibody in Cos-7 cells transiently expressing an HA epitope-tagged rat
AT1a-R (HA-AT1a-R). The anti-HA antibody failed
to stain the untransfected cells, but 510% of the cells in cultures
transiently transfected with the HA-AT1a-R were
immunoreactive, consistent with the expected transfection efficiency
(Fig. 2
). The specificity of the staining
was indicated by its abolition after preincubation of the anti-HA
antibody with an excess of HA peptide.

View larger version (46K):
[in this window]
[in a new window]
|
Figure 2. Immunocytochemical Analysis of
HA-AT1-Rs
Anti-HA and anti-FP antibodies were preincubated with or without their
immunogens (HA peptide and FP, respectively) before immunostaining as
described in the text. A and D, Untransfected Cos-7 cells; B, C, E and
F, HA-AT1a-R-expressing Cos-7 cells; G and H, bovine
adrenal glomerulosa cells. AC, anti-HA antibody; DH, anti-FP
antibody. C, Anti-HA antibody preabsorbed with HA peptide: F and H,
Anti-FP antibody preabsorbed with FP. The antibody appears
red, and the nuclear counterstain appears
blue. Magnification x300.
|
|
When this experiment was repeated using the anti-FP antibody in
place of the anti-HA antibody, similar results were obtained. Whereas
no staining was observed in untransfected cells, the anti-FP antibody
specifically stained 510% of cells in
HA-AT1a-R-transfected cultures (Fig. 2
). Furthermore, the
individual cell staining pattern observed with the anti-FP antibody was
similar to that obtained with the anti-HA antibody, with diffuse
cytoplasmic staining and prominent perinuclear haloes. These findings
demonstrate that the anti-FP antibody specifically recognizes the
AT1-R in transfected cells and that (at least in Cos-7
cells) it does not cross-react with other cellular antigens. When the
anti-FP antibody was used for immunocytochemical analysis of primary
cultures of bovine adrenal glomerulosa cells, more than 95% of the
cells exhibited specific staining, with a similar distribution to that
seen in HA-AT1a-R-transfected Cos-7 cells.
Immunohistochemical studies with the anti-FP antibody were performed on
two rat tissues known to express high levels of the AT1-R,
the adrenal zona glomerulosa (19) and the renal glomerulus (20). The
anti-FP antibody heavily stained the zona glomerulosa, and the signal
was abolished by preincubation of the antibody with the FP immunogen
(Fig. 3
). Appropriately, the antibody did
not specifically stain the fasciculata/reticularis zones, which are
virtually devoid of AT1-Rs in the rat adrenal gland (19).
In contrast, a commercially available anti-AT1-R antibody
(sc1173) did not stain the zona glomerulosa. In the rat kidney, the
anti-FP antibody heavily stained the glomeruli, and this was again
inhibited by preincubation of the antibody with the FP immunogen (Fig. 3
). No staining was observed in the renal tubules. In contrast to the
anti-FP antibody, the sc1173 antibody failed to stain the renal
glomeruli.

View larger version (43K):
[in this window]
[in a new window]
|
Figure 3. Immunohistochemical Analysis of AT1-Rs
in Rat Adrenal Cortex and Kidney
Anti-FP antibody was incubated with or without FP before incubation
with tissue sections as described in the text. AC, Adrenal cortex;
DF, kidney. A and D, Not preabsorbed; B and E, preabsorbed with FP.
Staining patterns in adrenal cortex (C) and kidney (F) obtained using
the sc1173 antibody are also shown. Orientation: zg, zona glomerulosa;
zf, zona fasciculata; g, glomerulus; t, tubule. Antibody staining
appears red. The nuclear counterstain appears
blue, and the vimentin counterstain appears
green. Magnification x100.
|
|
Phosphorylation of AT1-Rs
Since the anti-FP antibody was shown to immunoprecipitate the
AT1-R (Fig. 1
), we used it to evaluate agonist-induced
phosphorylation of AT1-Rs in membranes prepared from
32Pi metabolically-labeled bovine adrenal
glomerulosa cells. Initial studies were hampered by a low
signal-to-noise ratio and the presence in SDS-PAGE of additional
phosphoproteins that obscured the receptor (data not shown). However,
these problems were overcome by employing overnight salt/urea
extraction of the membranes at 4 C, followed by overnight incubation of
the solubilized membranes at 37 C before immunoprecipitation at 4 C.
The use of this protocol enabled the 32P-labeled
phospho-AT1-R to be clearly visualized in SDS-PAGE analysis
after immunoprecipitation of the solubilized membrane fraction.
Whereas little or no phosphorylated AT1-R was found in
control cells, treatment of bovine adrenal glomerulosa cells with Ang
II for 5 min caused the appearance of a broad band of Mr
60,00065,000, which comigrated with the [125I]azido-Ang
II photoaffinity-labeled AT1-R (Fig. 4
). This band was not present in the
absence of antibody (data not shown) and was abolished by preincubation
of the antibody with an excess of the FP immunogen (Fig. 4
). When
solubilized adrenal glomerulosa cell membranes were treated with the
deglycosylating enzyme, peptide-N-glycosidase F (PNGase F) (which
cleaves N-linked oligosaccharides from glycoproteins) (21), migration
of the Ang II-induced phosphoprotein in SDS-PAGE shifted from
Mr 60,00065,000 to Mr 40,000. This
corresponds to the location of the deglycosylated photoaffinity-labeled
receptor (Fig. 4
) and is consistent with the predicted size (41 kDa) of
the nonglycosylated AT1-R protein (2, 3). In addition,
boiling of the immune complexes before SDS-PAGE caused identical
degrees of aggregation and comigration (with lower electrophoretic
mobility) of the Ang II-induced phosphoprotein and the
photoaffinity-labeled receptor (data not shown). Taken together, these
data confirm the identity of the Mr 60,00065,000 band as
the phosphorylated AT1-R.

View larger version (55K):
[in this window]
[in a new window]
|
Figure 4. Angiotensin II-Stimulated Phosphorylation of
AT1-Rs in Bovine Adrenal Glomerulosa Cells
Cells were labeled with 32Pi for 4 h
before the addition of vehicle (C) or 100 nM Ang II (A) for
5 min as indicated. Membranes were then prepared and solubilized as
described in the text. After overnight incubation at 37 C in the
presence (PNG) or absence (Con, FP) of 10 U/ml PNGase F,
AT1-Rs were immunoprecipitated by the addition of anti-FP
antibody [preincubated with (FP) or without (Con, PNG) FP as
indicated] and resolved by SDS-PAGE. The migration of untreated (C)
and PNGase F-treated (PNG) 125I-azido-Ang II
photoaffinity-labeled bovine adrenal glomerulosa cell
AT1-Rs immunoprecipitated with the anti-FP antibody are
also shown (Azido).
|
|
Analysis of the concentration- and time-dependence of Ang II-stimulated
AT1-R phosphorylation in bovine adrenal glomerulosa cells
revealed that receptor phosphorylation was increased by 100
pM Ang II and reached a maximum at agonist concentrations
of 10 nM or higher (Fig. 5
).
In general, the extent of AT1-R occupancy by Ang II was
correlated with the degree of AT1-R phosphorylation.
Receptor phosphorylation reached a peak at 510 min and was still
elevated above the basal level 60 min after addition of the ligand
(Fig. 6
). In other experiments, Ang
II-induced AT1-R phosphorylation was apparent as early as 1
min after Ang II addition (data not shown). Pretreatment of the cells
with the protein phosphatase inhibitor, okadaic acid (1
µM), enhanced the AT1-R phosphorylation
observed after 5 min stimulation with Ang II (Fig. 7
). This finding indicates that the
phosphorylated AT1-R is subject to dephosphorylation by
cellular protein phosphatases during the early stages of Ang II
stimulation. Preincubation of cells with the tyrosine kinase inhibitor,
genistein (100 µM), did not inhibit Ang II-induced
AT1-R phosphorylation (data not shown), consistent with
phosphorylation of the receptor on serine and/or threonine residues and
not on tyrosine residues.

View larger version (82K):
[in this window]
[in a new window]
|
Figure 5. Concentration Dependence of Ang II-Stimulated
AT1-R Phosphorylation
Bovine adrenal glomerulosa cells were labeled with
32Pi for 4 h before addition of the
indicated concentrations of Ang II for 5 min. Membranes were then
prepared and solubilized as described in the text. AT1-Rs
were immunoprecipitated by the addition of anti-FP antibody and
resolved by SDS-PAGE. A representative example is shown from three
independent experiments.
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Figure 6. Time Dependence of Ang II-Stimulated
AT1-R Phosphorylation
Bovine adrenal glomerulosa cells were labeled with
32Pi for 4 h before the addition of 100
nM Ang II for the indicated times. Membranes were then
prepared and solubilized as described in the text. AT1-Rs
were immunoprecipitated by the addition of anti-FP antibody and
resolved by SDS-PAGE. A representative example is shown from three
independent experiments.
|
|

View larger version (48K):
[in this window]
[in a new window]
|
Figure 7. Enhancement of AT1-R Phosphorylation by
Okadaic Acid
Bovine adrenal glomerulosa cells were labeled with
32Pi for 4 h before the addition of
vehicle (Con) or 1 µM okadaic acid (OA) for 10 min.
Vehicle (C) or 100 nM Ang II (A) were added for a further 5
min as indicated. Membranes were then prepared and solubilized as
described in the text. AT1-Rs were immunoprecipitated by
the addition of anti-FP antibody and resolved by SDS-PAGE. A
representative example is shown from two independent experiments.
|
|
GPCRs have been reported to be phosphorylated by second
messenger-activated kinases as well as GRKs (8, 9, 22). The effects of
protein kinase C (PKC) activation by tetradecanoylphorbol-13-acetate
(TPA), Ca2+/calmodulin-dependent kinase activation by the
Ca2+ ionophore, ionomycin, and protein kinase A (PKA)
activation by a combination of forskolin and isobutylmethylxanthine
(IBMX) on AT1-R phosphorylation were analyzed in bovine
adrenal glomerulosa cells. Ionomycin had only a minor effect, but both
TPA and forskolin/IBMX stimulated significant increases in
AT1-R phosphorylation, although to a lesser degree than
that elicited by Ang II (Fig. 8
). Thus,
both PKC and PKA, but not Ca2+/calmodulin-dependent
kinases, have the capacity to phosphorylate the unliganded
AT1-R in bovine adrenal glomerulosa cells.

View larger version (65K):
[in this window]
[in a new window]
|
Figure 8. Effects of Second Messenger-Activated Protein
Kinases on AT1-R Phosphorylation
Bovine adrenal glomerulosa cells were labeled with
32Pi for 4 h before the addition of
vehicle (C), 100 nM Ang II (A), 200 nM TPA (T),
10 µM ionomycin (Ion), or a combination of 0.5
mM isobutylmethylxanthine and 50 µM forskolin
(For) as indicated for 5 min. Membranes were then prepared and
solubilized as described in the text. AT1-Rs were
immunoprecipitated by the addition of anti-FP antibody and resolved by
SDS-PAGE. A representative example is shown from three independent
experiments.
|
|
Since Ang II activates PKC in bovine adrenal glomerulosa cells (7), we
investigated the role of this kinase in Ang II-induced
AT1-R phosphorylation. Adrenal glomerulosa cells were
preincubated for 10 min with a concentration (500 nM) of
staurosporine that is sufficient to inhibit PKC [but has no effect on
GRKs (18)] before treatment with TPA or Ang II. TPA stimulated
AT1-R phosphorylation in control cells, and this was
abrogated by pretreatment of the cells with staurosporine (Fig. 9
). In contrast, the more prominent
AT1-R phosphorylation induced by Ang II was augmented in
the presence of staurosporine (Fig. 9
). Similar results were obtained
when the highly selective PKC inhibitor, bisindolylmaleimide (1
µM), was used in place of staurosporine (data not shown).
These findings suggest that although PKC may mediate a minor component
of Ang II-stimulated AT1-R phosphorylation, its inhibition
removes a negative regulatory influence on the major pathway to
AT1-R phosphorylation. It is probable that this major,
non-PKC, component of Ang II-induced receptor phosphorylation in
adrenal glomerulosa cells is mediated by one or more GRKs.

View larger version (40K):
[in this window]
[in a new window]
|
Figure 9. PKC Inhibition Enhances Ang II-Stimulated
AT1-R Phosphorylation
In panel A, bovine adrenal glomerulosa cells were labeled with
32Pi for 4 h before the addition of
vehicle (Con) or 500 nM staurosporine (SP) for 10 min.
Vehicle (C), 100 nM Ang II (A), or 200 nM TPA
(T) were then added for a further 5 min as indicated. Membranes were
prepared and solubilized as described in the text. AT1-Rs
were immunoprecipitated by the addition of anti-FP antibody and
resolved by SDS-PAGE. Quantification of mean (± SEM)
AT1-R phosphorylation from three independent experiments is
shown in panel B.
|
|
Immunoblotting of the AT1-R
Immunoblotting studies using membranes prepared from bovine
adrenal glomerulosa cells indicated that both the unpurified and
affinity-purified anti-FP antibodies specifically recognized a cluster
of two to four bands that comigrated in SDS-PAGE with the
photoaffinity-labeled receptor (data not shown). However, the
recognition of a band(s) with the same Mr as the
AT1-R (which runs as a diffuse band with Mr
60,00065,000) does not unequivocally identify such band(s) as the
receptor. We therefore determined whether the bands recognized by the
anti-FP antibody migrated with the same increased mobility as the
photoaffinity-labeled receptor after deglycosylation by PNGase F.
Whereas the photoaffinity-labeled receptor exhibited the expected
increase in migration (with Mr 40,000) after treatment with
PNGase F, the bands recognized by immunoblotting showed no change in
their mobility (data not shown). Furthermore, whereas the
photoaffinity-labeled AT1-R migrated as a high
Mr smear after boiling, the cluster of bands recognized by
the anti-FP antibody did not alter their mobility (data not shown).
Taken together, these findings indicate that the bands recognized in
immunoblotting by the anti-FP antibody are cross-reacting species and
not the AT1-R.
We compared the results obtained using the anti-FP antibody in
immunoblotting of bovine adrenal glomerulosa cell membranes with those
obtained using the commercially supplied sc579 and sc1173 antibodies.
Whereas sc1173 recognized two discrete bands, which comigrated with the
photoaffinity-labeled receptor, sc579 recognized only a single discrete
band with an Mr intermediate between those of the two
sc1173 bands (data not shown). However, none of these bands shifted to
a higher electrophoretic mobility after treatment with PNGase F (data
not shown). Hence, in addition to the inability of either antibody to
immunoprecipitate the photoaffinity-labeled receptor, and the inability
of the sc1173 antibody to stain the rat adrenal glomerulosa and renal
glomeruli, the sc579 and sc1173 antibodies also fail to recognize the
AT1-R in immunoblotting.
We therefore evaluated whether the HA.11 antibody was able to recognize
the HA-AT1a-R in immunoblotting. The antibody did not
recognize any bands in untransfected Cos-7 cells or in cells expressing
the wild-type AT1a-R. However, in
HA-AT1a-R-expressing cells, the HA.11 antibody recognized
multiple bands that comigrated with the photoaffinity-labeled receptor
(Fig. 10
). Recognition of these bands
was abolished by preincubation of the HA.11 antibody with the HA
peptide (data not shown). After boiling, the photoaffinity-labeled
HA-AT1a-R ran as a high Mr smear, which
correlated with the appearance of additional high Mr bands
in immunoblotting (Fig. 10
). In addition, both the
photoaffinity-labeled HA-AT1a-R, as well as a single band
recognized in immunoblotting, migrated with Mr 40,000 after
deglycosylation with PNGase F (data not shown). Taken together, these
data indicate that the HA.11 antibody specifically recognizes the
HA-AT1a-R in immunoblotting and does not cross-react with
other species.

View larger version (60K):
[in this window]
[in a new window]
|
Figure 10. Immunoblotting of HA-AT1-Rs
Solubilized
125I-[Sar1,(4-N3)Phe8]Ang
II photoaffinity-labeled membranes from untransfected (Con),
AT1a-R-expressing (AT1a), or
HA-AT1a-R-expressing (HA-AT1a) Cos-7 cells were
heated to 48 C for 1 h or 100 C for 10 min as indicated before
SDS-PAGE and transfer to PVDF. After detection of the
photoaffinity-labeled receptors in a PhosphorImager (Azido), the
membrane was probed with the HA.11 antibody (Blot).
|
|
 |
DISCUSSION
|
---|
Initial attempts to raise an anti-AT1-R antibody
employed synthetic peptide immunogens corresponding to sequences
contained within the amino terminus (residues 126), intracellular
loops (residues 1870, 121146, and 215238), and extracellular
loops (86105, 172194, and 269278) of the rat AT1b-R.
Although some of the antibodies generated using these peptides were
able to immunoprecipitate the receptor, they were of low titer. In
contrast, the use of a fusion protein (FP) immunogen containing the
C-terminal 92 amino acid cytoplasmic tail of the rat AT1b-R
made possible the generation of a high-titer antibody that specifically
immunoprecipitates the AT1-R and recognizes the receptor on
immunochemistry. However, this antibody did not recognize the
receptor in immunoblotting.
The ability of the anti-FP antibody to immunoprecipitate the
AT1-R, but its failure to recognize the receptor in
immunoblotting, may result from the antibody recognizing primarily a
conformational epitope(s) that is preserved during cell or tissue
preparation for immunochemistry, or when receptors are solubilized for
immunoprecipitation, but is destroyed during SDS-PAGE. Alternatively,
putative epitope(s) contained within the hydrophobic seventh
transmembrane domain of the receptor (which is contained in the FP) may
be masked on immunoblots as a result of hydrophobic interactions both
between AT1-Rs themselves and between AT1-Rs
and additional comigrating hydrophobic membrane proteins. Indeed, the
latter possibility may present a general problem for the detection of
GPCRs in immunoblots.
Although the anti-FP antibody specifically immunoprecipitated the
photoaffinity-labeled AT1-R, our initial attempts to
immunoprecipitate the putative phosphorylated receptor from Ang
II-stimulated bovine adrenal glomerulosa cells were hampered by a low
signal-to-noise ratio and the presence in SDS-PAGE of several
additional phosphoproteins, some of which obscured the
phospho-AT1-R. In principle, there are three possible
explanations for these additional phosphoproteins. First, since the
antibody cross-reacts with non-AT1-R proteins on
immunoblotting, it may also cross-react with other phosphoproteins in
immunoprecipitation (although on immunochemistry it did not cross-react
with any cellular antigens in untransfected Cos 7 cells or in the rat
adrenal zona reticularis/fasciculata). Second, the additional
phosphoproteins may represent species that associate with the receptor
physiologically and that, therefore, genuinely coimmunoprecipitate with
the receptor. Third, the additional phosphoproteins may represent
species that associate nonphysiologically (possibly via hydrophobic
interactions) with the solubilized receptor and therefore spuriously
coimmunoprecipitate with the receptor. Superimposed upon these
possibilities is the additional problem of low cellular
AT1-R abundance with resulting low signal-to-noise ratio of
phospho-AT1-R over nonspecific and/or cross-reacting
phosphoproteins (which may be more abundant than the
phospho-AT1-R). These technical problems are characteristic
not only of the anti-FP antibody, but were also encountered when the
anti-HA antibody was employed to immunoprecipitate the
phospho-HA-AT1-R from Ang II-stimulated Cos-7 cells (data
not shown).
Preextraction of cell membranes with salt/urea, followed by
preincubation of solubilized membranes at 37 C (before
immunoprecipitation at 4 C), was required to overcome this problem. The
mechanisms whereby these treatments unmasked the
phospho-AT1-R are unknown. However, salt/urea extraction of
membranes might be expected to dissociate species that are not integral
membrane proteins but which physiologically associate with the
AT1-R, or to remove similarly associated
cross-reacting species from membranes, whereas incubation of
solubilized membranes at 37 C might dissociate protein aggregates bound
by hydrophobic interactions. Since neither pretreatment alone was
sufficient to reveal the phospho-AT1-R (data not shown), it
is probable that more than one of these proposed mechanisms operates to
obscure the immunoprecipitated phospho-AT1-R in
SDS-PAGE.
Under the above experimental conditions, it was possible to demonstrate
phosphorylation of the native AT1-R in adrenal glomerulosa
cells. The degree of ligand occupancy of AT1-Rs in such
target cells correlated with the magnitude of receptor phosphorylation.
This finding is consistent with a receptor phosphorylation mechanism
that entails a conformational change of the agonist-liganded receptors
that allows phosphorylation on exposed intracellular sites by active
GRKs or second messenger-activated kinases. Whereas little
AT1-R phosphorylation was detected in quiescent cells,
receptor phosphorylation was apparent as early as 1 min after Ang II
addition. Thereafter, maximal receptor phosphorylation was sustained up
to 40 min, and appreciable phosphorylation was still apparent at 60
min. Despite this prolonged phosphorylation, the
phospho-AT1-R was subject to dephosphorylation by okadaic
acid-sensitive protein phosphatases even during the first 5 min of Ang
II stimulation. This suggests that phospho-AT1-Rs are
rapidly dephosphorylated but, after internalization and recycling to
the plasma membrane, bind fresh ligand and undergo a further round(s)
of phosphorylation. If such cycles of phosphorylation/dephosphorylation
are maintained in the continuous presence of ligand, the
phospho-AT1-R measured in our assay would represent the net
phosphorylation status of the cell receptor population at each time
point. In this paradigm, the AT1-R dephosphorylation
observed 1 h after Ang II stimulation may in fact result from
down-regulation of cell-surface AT1-R receptors induced by
the continuous presence of ligand.
Activation of the second messenger-activated kinases, PKA and PKC (but
not of Ca2+/calmodulin-dependent kinases), increased
phosphorylation of the (unliganded) AT1-R in bovine adrenal
glomerulosa cells, but the magnitude of receptor phosphorylation was
less than that stimulated by Ang II. This finding indicates that the
majority of Ang II-induced AT1-R phosphorylation is not
mediated by PKC [which is activated by Ang II in bovine adrenal
glomerulosa cells (7)], but most likely by GRKs. When
AT1-Rs were expressed in HEK293 cells, TPA stimulated about
50% of the receptor phosphorylation seen in response to Ang II, and
staurosporine inhibited about one third of Ang II-stimulated receptor
phosphorylation (18). In contrast, inhibition of PKC by staurosporine
in bovine adrenal glomerulosa cells augmented Ang II-stimulated
AT1-R phosphorylation. The latter finding suggests that
although PKC may make a contribution to Ang II-induced
AT1-R phosphorylation, it also negatively regulates the
activity of the putative GRK(s) that mediates the majority of this
phosphorylation. Consistent with this hypothesis, phosphorylation of
GRK5 by PKC has been reported to reduce its ability to phosphorylate
light-activated rhodopsin in vitro (23). Bovine adrenal
glomerulosa cells express GRKs 2, 3, and 5 (but not GRK 6) as
determined by immunoblotting with specific antibodies (data not shown),
and each of these kinases has been shown to phosphorylate the
AT1-R when over-expressed in HEK 293 cells (18). It remains
to be determined which GRK(s) mediates Ang II-induced AT1-R
phosphorylation in bovine adrenal glomerulosa cells. However, the
possibility that additional (non-GRK) kinases may also be able to
phosphorylate the AT1-R cannot be excluded, since casein
kinase 1
has recently been demonstrated to phosphorylate the
m3-muscarinic receptor in an agonist-dependent manner (24).
In conclusion, we have generated a polyclonal antibody that
specifically immunoprecipitates the AT1-R and recognizes
the receptor on immunochemistry. The use of this antibody has permitted
the demonstration of agonist-induced phosphorylation of the native
AT1-R in primary cultures of bovine adrenal glomerulosa
cells, an observation not previously reported. The opposite effects of
PKC inhibition on AT1-R phosphorylation observed in bovine
adrenal glomerulosa cells (this report) compared with
HA-AT1-R expressing HEK 293 cells (18) indicate the need
for further investigation of GPCR phosphorylation in normal cells. The
use of the anti-FP antibody in the protocol outlined here should
facilitate the analysis of endogenous AT1-R phosphorylation
during the actions of Ang II in its principal target cells in
cardiovascular, neuronal, and endocrine tissues.
 |
MATERIALS AND METHODS
|
---|
Materials
DMEM, Medium 199, donor horse serum, FBS, and
antibiotic/antimycotic solutions were from Biofluids (Rockville, MD).
Angiotensin II was from Peninsula Laboratories (Belmont, CA).
32Pi was from Amersham (Arlington Heights, IL),
and
125I-[Sar1,(4-N3)Phe8]Ang
II was from Covance Laboratories (Vienna, VA). Ionomycin and okadaic
acid were from Calbiochem (San Diego, CA). Protein G Plus Sepharose and
CN Sepharose 4B were from Pharmacia (Uppsala, Sweden).
Peptide-N-glycosidase F (E.C.3.5.1.52) was from Boehringer Mannheim
(Indianapolis, IN). Rabbit polyclonal anti-AT1-R antibodies
(sc579 and sc1173) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Sepharose-coupled wheat-germ agglutinin, protease inhibitors,
isobutylmethylxanthine, 12-O-tetradecanoylphorbol
13-acetate, staurosporine, and forskolin were from Sigma (St. Louis,
MO). All other fine chemicals, which were of analytical grade or
higher, were from Sigma.
Cell Culture
Primary cultures of glomerulosa cells were prepared from bovine
adrenal glands as previously described (25). For photoaffinity
labeling, 107 cells were plated in 10-cm plastic culture
dishes (Becton Dickinson, Lincoln, NJ) in DMEM containing 10%
(vol/vol) donor horse serum, 2% (vol/vol) FBS, 100 µg/ml
streptomycin, 100 IU/ml penicillin, 5 µg/ml fungizone, 25 µg/ml
gentamicin, 8 µg/ml trimethoprim, and 40 µg/ml sulfamethoxazole.
Cells were cultured in a humidified atmosphere of 5% CO2
in air at 37 C and formed confluent monolayers after 3 days. Cells for
immunocytochemical staining were seeded at 0.25 x 106
cells per 35-mm culture dish on polylysine-coated glass coverslips and
used after 3 days in culture.
Transient Expression of AT1-Rs
Cos-7 cells were maintained in DMEM containing 10% (vol/vol)
FBS, 100 µg/ml streptomycin, and 100 IU/ml penicillin (Cos-7 medium).
A HindII/NotI fragment of the rat
AT1a receptor cDNA and a HindIII/NsiI
fragment of the rat AT2 receptor were subcloned into the
eukaryotic expression vector, pcDNAI/Amp (Invitrogen, San Diego, CA),
as previously described (26).
The influenza HA-epitope (YPYDVPDYA) was inserted after the codons of
the amino-terminal first two amino acids (MA) into the cDNA of the rat
AT1a receptor using the Mutagene kit (Bio-Rad, Hercules,
CA). The sequence of the tag was verified by dideoxy sequencing using
Sequenase II (Amersham). The epitope-tagged receptor was detected using
the HA.11 monoclonal antibody (BAbCO, Richmond, CA). The presence of
the epitope tag had no effect on ligand binding or inositol phosphate
signaling and internalization properties of the receptor (data not
shown).
Sparsely seeded Cos-7 cells growing on polylysine-coated glass
coverslips were transfected with the required receptor cDNA for 6
h at 37 C in OptiMEM containing 10 µg/ml of LipofectAMINE (both from
GIBCO/BRL, Gaithersburg, MD). After changing to Cos-7 medium, the cells
were cultured for a further 48 h before use.
Preparation of Antiserum
An FP consisting of MBP linked to a 92-amino acid C-terminal
fragment (residues 268359) of the rat AT1b angiotensin
receptor was cloned into Escherichia coli using the Protein
Fusion and Purification System from New England Biolabs (Beverly, MA).
Briefly, a PvuII/HindIII fragment of the rat
AT1b angiotensin receptor (27) was purified by agarose
electrophoresis and ligated into the E. coli expression
vector, pMAL-c2, in-frame with the MBP gene. The ligated plasmid was
used to transform E. coli strain TB1, and expression of the
FP was induced with isopropyl-ß-D-thiogalactoside. After
20 h, bacteria were sonicated in 200 mM NaCl/1
mM EDTA/20 mM Tris, pH 7.4, centrifuged, and
the supernatant was loaded onto an amylose affinity column. After
washing, bound FP was eluted with 10 mM maltose, divided
into aliquots, and frozen at -20 C.
New Zealand white rabbits were immunized ip with 100 µg FP in
Freunds complete adjuvant and boosted intradermally after 2 weeks
(and subsequently every 4 weeks) with 50 µg FP in incomplete
Freunds adjuvant. Igs were purified from crude rabbit antisera by
caprylic acid precipitation as described (28) and dialyzed extensively
against PBS at 4 C. The antibody was depleted of anti-MBP Igs and
enriched for anti-AT1-R Igs by sequential immunoaffinity
chromatography over MBP- and FP-Sepharose columns, respectively. Igs
were eluted from the FP-Sepharose column with 50 mM glycine
(pH 3) directly into 100 mM Tris (pH 10.5), subjected to
ultrafiltration through a Centricon concentrator (Amicon, Beverly, MA),
and stored in aliquots at -20 C in a 1:1 solution of PBS and glycerol.
Depletion of anti-MBP Igs and enrichment of anti-AT1-R Igs
were confirmed by immunoblotting against MBP and FP, respectively (data
not shown).
Immunoprecipitation of Photoaffinity-Labeled
AT1-Rs
Confluent monolayers of bovine adrenal glomerulosa cells were
washed three times with ice-cold Medium 199, before overnight
incubation at 4 C in the same medium containing the photoaffinity
ligand,
125I-[Sar1,(4-N3)Phe8]Ang
II (125I-azido-Ang II) (29) (
107 cpm/dish).
Cells were then washed three times with ice-cold PBS and exposed to UV
light for 10 sec. Noncovalently bound 125I-azido-Ang II was
removed by incubating the cells for 10 min in ice-cold 150
mM NaCl containing 50 mM acetic acid. After
further washes in ice-cold PBS, dishes were drained and the cells were
scraped into lysis buffer (LB-: 50 mM Tris, pH 8.0, 100
mM NaCl, 20 mM NaF, 10 mM Na
pyrophosphate, 5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstatin,
10 µg/ml benzamidine, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1
mM Na3VO4, 1 µM
okadaic acid) and probe-sonicated (Sonifier Cell Disruptor: Heat
Systems Ultrasonics, Plainview, NY) for 2 x 20 sec. After removal
of nuclei by centrifugation for 10 min at 750 x g,
membranes were collected by centrifugation for 45 min at 200,000
x g. Membrane pellets were solubilized by Dounce
homogenization in ice-cold LB+ (LB- supplemented with 1% (vol/vol) NP
40, 1% (wt/vol) Na deoxycholate, and 0.1% (wt/vol) SDS). After
clarification for 10 min at 10,000 x g, solubilized
membranes were incubated for 4 h at 4 C with 2.5% (vol/vol)
Protein G Plus Sepharose. The precleared supernatant was then divided
into aliquots and stored at -20 C before use.
Solubilized membranes were subjected to immunoprecipitation by the
addition of 10 µl of antibody and 2% (vol/vol) Protein G Plus
Sepharose overnight at 4 C with tumbling. Immune complexes were
collected by centrifugation and washed three times with ice-cold LB+
lacking protease inhibitors. After the final wash, immune complexes
were eluted into Laemmli sample buffer (30) for 1 h at 48 C. After
resolution by SDS-PAGE (816% resolving gel) and transfer to
polyvinylidene fluoride (PVDF) membranes, photoaffinity-labeled
AT1-Rs were visualized using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Immunochemistry of AT1 Receptors
For immunocytochemistry, cells grown on glass coverslips were
fixed in 4% (wt/vol) paraformaldehyde in PBS at room temperature for
10 min, washed three times in PBS for 5 min, and then treated with 3%
(vol/vol) normal goat serum in PBS containing 0.2% (vol/vol) Triton
X-100, followed by three 5-min washes in PBS. Cells were then incubated
for 1 h with anti-FP antibody (1:90) or mouse anti-HA antibody
(1:1000) in PTB [PBS containing 0.3% (vol/vol) Triton X-100 and 0.1%
(wt/vol) BSA] followed by three 5-min washes in PBS before incubation
for 1 h with indocarbocyanine-conjugated goat anti-rabbit (or
mouse) F(ab')2 fragments (Jackson ImmunoResearch Labs, West
Grove, PA) at 1:750 dilution in PTB. After three final 5-min washes
with PBS, cells were rinsed with distilled water. Nuclei were stained
for 30 sec with the DNA-binding chromophore,
4'-6-diamidino-2'-phenylindole (0.13 µg/ml in water), followed by
three washes in water. Cells were then viewed in a Zeiss Chroma
fluorescence microscope (Carl Zeiss, Thornwood, NY) and photographs
were taken using Fuji Provia 1600 film.
For immunohistochemistry, tissues harvested from freshly killed rats
were frozen immediately in 2-methylbutane at -40 C and stored at -80
C. Twelve-micrometer sections were cut using a Frigocut-E 2800 cryostat
(Reichert, Heidelberg, Germany), dried in air at 37 C, mounted onto
silanized glass slides (Digene, Beltsville, MD), and stored at -80 C
before use. Sections were stained with the anti-FP antibody as
described above. Counterstaining was provided by
4'-6-diamidino-2'-phenylindole (adrenal), or by mouse anti-vimentin
(Sigma) at 1:1000 (kidney). Sections were secured under coverslips with
Cytoseal 60 (Stephens Scientific, Riverdale, NJ) before viewing in the
fluorescence microscope.
Phosphorylation of AT1-Rs
Confluent cultures of bovine adrenal glomerulosa cells in 10-cm
dishes were rendered quiescent by overnight incubation in serum-free
medium and then labeled for 4 h at 37 C in Pi-free
DMEM containing 0.1% (wt/vol) BSA and 150 µCi/ml
32Pi. After three washes in KRH [118
mM NaCl, 2.4 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgCl2, 10
mM glucose, 0.1% (wt/vol) BSA, 20 mM HEPES, pH
7.4], cells were incubated in the same medium for 10 min in a 37 C
water bath. Vehicle or Ang II was then added for the required time.
After three washes with ice-cold PBS, cells were drained before
scraping into LB- and probe-sonicated for 45 sec. After removal of
nuclei at 750 x g, membranes were extracted by the
addition of an equal volume of LB- containing 2 M NaCl and
8 M urea overnight with tumbling at 4 C. Membranes were
collected at 200,000 x g and solubilized in LB+ with
Dounce homogenization. After clarification at 14,000 x
g, solubilized membranes were incubated with 2.5% (vol/vol)
Protein G Plus Sepharose for 1 h at 4 C. The precleared
supernatant was incubated overnight at 37 C, before immunoprecipitation
of AT1-Rs by the addition of 10 µl of anti-FP antibody
and 2% (vol/vol) Protein G Plus Sepharose overnight at 4 C. After
washing of immune complexes in LB+ lacking protease inhibitors,
32P-labeled phospho-AT1-Rs were eluted in
Laemmli sample buffer (30) for 1 h at 48 C, resolved by SDS-PAGE
(816% gradient resolving gel), and visualized in the
PhosphorImager.
Immunoblotting of HA-Tagged AT1-Rs
Photoaffinity-labeled membranes prepared from
HA-AT1a-R-expressing Cos-7 cells were solubilized and
resolved by SDS-PAGE (816% resolving gel) before transfer to PVDF.
Membranes were blocked for 1 h at room temperature in TBS
containing 0.05% (vol/vol) Tween 20 and 5% (wt/vol) dried milk
proteins (TBST/5% milk) before incubation for 1 h in TBST/5%
milk containing HA.11 mouse monoclonal antibody (1:1000). After washing
for 30 min in TBST, membranes were incubated for 30 min in TBST/5%
milk containing 1:5000 horseradish peroxidase-conjugated goat
anti-mouse antibody (Kirkegaard & Perry, Gaithersburg, MD). After a
further 30-min wash in TBST, immune complexes were developed using
enhanced chemiluminescence (ECL) reagents (Kirkegaard & Perry) and
exposed to Kodak Biomax (Eastman Kodak, Rochester, NY) x-ray film.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Tamas Balla for many fruitful discussions and Xue
Zhao for preparing bovine adrenal glomerulosa cells. R.D.S. is the
recipient of an International Fellowship (FS/95018) from the British
Heart Foundation. L.H. was supported by an International Research
Scholars award from the Howard Hughes Medical Institute.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. K. J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 6A-36, 9000 Rockville Pike, Bethesda, Maryland 20892-4510.
Received for publication December 4, 1997.
Revision received January 28, 1998.
Accepted for publication January 30, 1998.
 |
REFERENCES
|
---|
-
Peach MJ 1977 Renin-angiotensin system: biochemistry and
mechanisms of action. Physiol Rev 57:313370[Free Full Text]
-
Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ et al 1991 Cloning and expression of a complementary DNA encoding a bovine adrenal
angiotensin II type-1 receptor. Nature 351:230233[CrossRef][Medline]
-
Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein
KE 1991 Isolation of a cDNA encoding the vascular type-1 angiotensin II
receptor. Nature 351:233236[CrossRef][Medline]
-
Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE,
Dzau VJ 1993 Expression cloning of type 2 angiotensin II receptor
reveals a unique class of seven-transmembrane receptors. J
Biol Chem 268:2453924542[Abstract/Free Full Text]
-
Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H,
Hamakoubo T, Inagami T 1993 Molecular cloning of a novel angiotensin II
receptor isoform involved in phosphotyrosine phosphatase inhibition.
J Biol Chem 268:2454324546[Abstract/Free Full Text]
-
Horiuchi M, Hayashida W, Kambe T, Yamada T, Dzau, VJ 1997 Angiotensin type 2 receptor dephosphorylates Bcl-2 by activating
mitogen-activated protein kinase phosphatase-1 and induces apoptosis.
J Biol Chem 272:1902219026[Abstract/Free Full Text]
-
Catt KJ, Sandberg K, Balla T 1993 Angiotensin II receptors
and signal transduction mechanisms. In: Raizada MK, Phillips MI,
Sumners C (eds) Cellular and Molecular Biology of the Renin-Angiotensin
System. CRC Press, Boca Raton, FL, pp 307356
-
Lefkowitz RJ 1993 G protein-coupled receptor kinases. Cell 74:409412[Medline]
-
Inglese J, Freedman NJ, Koch WJ, Lefkowitz RJ 1993 Structure
and mechanism of the G protein-coupled receptor kinases. J Biol
Chem 268:2373523738[Free Full Text]
-
Ferguson SSG, Zhang J, Barak LS, Caron MG 1996 G-protein-coupled receptors kinases and arrestins: regulators of
G-protein-coupled receptor sequestration. Biochem Soc Trans 24:953959[Medline]
-
Ferguson SSG, Barak LS, Zhang J, Caron MG 1996 G-protein-coupled receptor regulation: role of G-protein-coupled
receptor kinases and arrestins. Can J Physiol Pharmacol 74:10951110[CrossRef][Medline]
-
Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG,
Lefkowitz RJ 1995 Phosphorylation and desensitization of the human
ß1-adrenergic receptor. J Biol Chem 270:1795317961[Abstract/Free Full Text]
-
Pei G, Kieffer BL, Lefkowitz RJ, Freedman NJ 1995 Agonist-dependent phosphorylation of the mouse
-opioid receptor:
involvement of G protein-coupled receptor kinases but not protein
kinase C. Mol Pharmacol 48:173177[Abstract]
-
Freedman NJ, Ament AS, Oppermann M, Stoffel RH, Exum ST,
Lefkowitz RJ 1997 Phosphorylation and desensitization of the human
endothelin A and B receptors. J Biol Chem 272:1773417743[Abstract/Free Full Text]
-
Palmer TM, Benovic JL, Stiles GL 1995 Agonist-dependent
phosphorylation and desensitization of the rat A3 adenosine
receptor. J Biol Chem 270:2960729613[Abstract/Free Full Text]
-
Innamorati G, Sadeghi H, Eberle AN, Birnbaumer M 1997 Phosphorylation of the V2 vasopressin receptor. J Biol Chem 272:24862492[Abstract/Free Full Text]
-
Hipkin RW, Friedman J, Clark RB, Eppler M, Schonbrunn A 1997 Agonist-induced desensitization, internalization, and phosphorylation
of the sst2A somatostatin receptor. J Biol Chem 272:1386913876[Abstract/Free Full Text]
-
Oppermann M, Freedman NJ, Alexander RW, Lefkowitz RJ 1996 Phosphorylation of the type 1A angiotensin II receptor by G
protein-coupled receptor kinases and protein kinase C. J Biol Chem 271:1326613272[Abstract/Free Full Text]
-
Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia
JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWM 1989 Identification of angiotensin II receptor subtypes. Biochem Biophys Res
Commun 165:196203[Medline]
-
Mendelsohn FA, Dunbar M, Allen A, Chou ST, Millan MA, Aguilera
G, Catt KJ 1986 Angiotensin II receptors in the kidney. Fed Proc 45:14201425[Medline]
-
Lemp D, Haselbeck A, Klebl F 1990 Molecular cloning
and heterologous expression of N-glycosidase F from
Flavobacterium meningosepticum. J Biol Chem 265:1560615610[Abstract/Free Full Text]
-
Hausdorff WP, Caron MG, Lefkowitz RJ 1990 Turning off the
signal: desensitization of beta-adrenergic receptor function. FASEB J 4:28812889[Abstract]
-
Pronin AN, Benovic JL 1997 Regulation of the G protein-coupled
receptor kinase GRK5 by protein kinase C. J Biol Chem 272:38063812[Abstract/Free Full Text]
-
Tobin AB, Totty NF, Sterlin AE, Nahorski SR 1997 Stimulus-dependent phosphorylation of G-protein-coupled receptors
by casein kinase 1
. J Biol Chem 272:2084420849[Abstract/Free Full Text]
-
Guillemette G, Baukal AJ, Balla T, Catt KJ 1987 Angiotensin-induced formation and metabolism of inositol polyphosphates
in bovine adrenal glomerulosa cells. Biochem Biophys Res Commun 142:1522[Medline]
-
Hunyady L, Bor M, Balla T, Catt KJ 1994 Identification of a
cytoplasmic Ser-Thr-Leu motif that determines agonist-induced
internalization of the AT1 angiotensin receptor. J
Biol Chem 269:3137831382[Abstract/Free Full Text]
-
Sandberg K, Ji H, Clark AJL, Shapira H, Catt KJ 1992 Cloning
and expression of a novel angiotensin II receptor subtype. J Biol
Chem 267:94559458[Abstract/Free Full Text]
-
Harlow E, Lane D 1988 Antibodies: A Laboratory Manual. Cold
Spring Harbor Laboratory, New York, pp 300301
-
Carson MC, Leach Harper CM, Baukal AJ, Aguilera G, Catt KJ 1987 Physicochemical characterization of photoaffinity-labeled
angiotensin II receptors. Mol Endocrinol 1:147153[Abstract]
-
Laemmli UK 1970 Cleavage of structural head proteins during
the assembly of the head of bacteriophage T4. Nature 227:680685[Medline]