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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 60,000–65,000
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).
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). 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 5–10% 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.
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). 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.
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), 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
60,000–65,000, 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 60,000–65,000 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 60,000–65,000 band as
the phosphorylated AT1-R.
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).
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 5–10 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.
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). 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.
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). 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.
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 268–359) 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 Freund’s complete adjuvant and boosted intradermally after 2 weeks (and subsequently every 4 weeks) with 50 µg FP in incomplete Freund’s 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 (8–16% 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
(8–16% 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 (8–16% 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 4, 1997. Revision received January 28, 1998. Accepted for publication January 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-opioid receptor:
involvement of G protein-coupled receptor kinases but not protein
kinase C. Mol Pharmacol 48:173–177[Abstract]
. J Biol Chem 272:20844–20849This article has been cited by other articles:
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  L. Szidonya, M. Cserzo, and L. Hunyady Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions J. Endocrinol., March 1, 2008; 196(3): 435 - 453. [Abstract] [Full Text] [PDF]
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 S. Shacham, M. N. Cheifetz, M. Fridkin, A. J. Pawson, R. P. Millar, and Z. Naor Identification of Ser153 in ICL2 of the Gonadotropin-releasing Hormone (GnRH) Receptor as a Phosphorylation-independent Site for Inhibition of Gq Coupling J. Biol. Chem., August 12, 2005; 280(32): 28981 - 28988. [Abstract] [Full Text] [PDF]
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 A. SPAT and L. HUNYADY Control of Aldosterone Secretion: A Model for Convergence in Cellular Signaling Pathways Physiol Rev, April 1, 2004; 84(2): 489 - 539. [Abstract] [Full Text] [PDF]
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 B. H. Shah, J. Alberto Olivares-Reyes, A. Yesilkaya, and K. J. Catt Independence of Angiotensin II-Induced MAP Kinase Activation from Angiotensin Type 1 Receptor Internalization in Clone 9 Hepatocytes Mol. Endocrinol., March 1, 2002; 16(3): 610 - 620. [Abstract] [Full Text] [PDF]
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 A. Garcia-Caballero, J. A. Olivares-Reyes, K. J. Catt, and J. A. Garcia-Sainz Angiotensin AT1 Receptor Phosphorylation and Desensitization in a Hepatic Cell Line. Roles of Protein Kinase C and Phosphoinositide 3-Kinase Mol. Pharmacol., March 1, 2001; 59(3): 576 - 585. [Abstract] [Full Text]
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J. A. Olivares-Reyes, S. Jayadev, L. Hunyady, K. J. Catt, and R. D. Smith Homologous and Heterologous Phosphorylation of the AT2 Angiotensin Receptor by Protein Kinase C Mol. Pharmacol., November 1, 2000; 58(5): 1156 - 1161. [Abstract] [Full Text]
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 S. Jayadev, R. D. Smith, G. Jagadeesh, A. J. Baukal, L. Hunyady, and K. J. Catt N-Linked Glycosylation Is Required for Optimal AT1a Angiotensin Receptor Expression in COS-7 Cells Endocrinology, May 1, 1999; 140(5): 2010 - 2017. [Abstract] [Full Text]
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R. D. Smith, L. Hunyady, J. A. Olivares-Reyes, B. Mihalik, S. Jayadev, and K. J. Catt Agonist-Induced Phosphorylation of the Angiotensin AT1a Receptor Is Localized to a Serine/Threonine-Rich Region of Its Cytoplasmic Tail Mol. Pharmacol., December 1, 1998; 54(6): 935 - 941. [Abstract] [Full Text]
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M. Zhang, X. Zhao, H.-C. Chen, K. J. Catt, and L. Hunyady Activation of the AT1 Angiotensin Receptor Is Dependent on Adjacent Apolar Residues in the Carboxyl Terminus of the Third Cytoplasmic Loop J. Biol. Chem., May 19, 2000; 275(21): 15782 - 15788. [Abstract] [Full Text] [PDF]
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Q. Liu and A. Schonbrunn Agonist-induced Phosphorylation of Somatostatin Receptor Subtype 1 (Sst1). RELATIONSHIP TO DESENSITIZATION AND INTERNALIZATION J. Biol. Chem., January 26, 2001; 276(5): 3709 - 3717. [Abstract] [Full Text] [PDF]
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J. A. Olivares-Reyes, R. D. Smith, L. Hunyady, B. H. Shah, and K. J. Catt Agonist-induced Signaling, Desensitization, and Internalization of a Phosphorylation-deficient AT1A Angiotensin Receptor J. Biol. Chem., October 5, 2001; 276(41): 37761 - 37768. [Abstract] [Full Text] [PDF]
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