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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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), {delta}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 60,000–65,000 in SDS-PAGE (Fig. 1AGo). 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. 1AGo).



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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 85,000–140,000 in SDS-PAGE (Fig. 1BGo). 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. 2Go). 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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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. A–C, anti-HA antibody; D–H, 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 5–10% of cells in HA-AT1a-R-transfected cultures (Fig. 2Go). 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. 3Go). 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. 3Go). 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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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. A–C, Adrenal cortex; D–F, 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. 1Go), 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. 4Go). 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. 4Go). 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. 4Go) 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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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. 5Go). 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. 6Go). 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. 7Go). 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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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.

 


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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.

 


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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. 8Go). 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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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. 9Go). In contrast, the more prominent AT1-R phosphorylation induced by Ang II was augmented in the presence of staurosporine (Fig. 9Go). 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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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 60,000–65,000) 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. 10Go). 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. 10Go). 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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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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Initial attempts to raise an anti-AT1-R antibody employed synthetic peptide immunogens corresponding to sequences contained within the amino terminus (residues 1–26), intracellular loops (residues 18–70, 121–146, and 215–238), and extracellular loops (86–105, 172–194, and 269–278) 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{alpha} 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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.


    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 Scholar’s 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Peach MJ 1977 Renin-angiotensin system: biochemistry and mechanisms of action. Physiol Rev 57:313–370[Free Full Text]
  2. 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 351:230–233[CrossRef][Medline]
  3. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE 1991 Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351:233–236[CrossRef][Medline]
  4. 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 268:24539–24542[Abstract/Free Full Text]
  5. 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 268:24543–24546[Abstract/Free Full Text]
  6. 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 272:19022–19026[Abstract/Free Full Text]
  7. 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 307–356
  8. Lefkowitz RJ 1993 G protein-coupled receptor kinases. Cell 74:409–412[Medline]
  9. Inglese J, Freedman NJ, Koch WJ, Lefkowitz RJ 1993 Structure and mechanism of the G protein-coupled receptor kinases. J Biol Chem 268:23735–23738[Free Full Text]
  10. 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 24:953–959[Medline]
  11. 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 74:1095–1110[CrossRef][Medline]
  12. 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 270:17953–17961[Abstract/Free Full Text]
  13. Pei G, Kieffer BL, Lefkowitz RJ, Freedman NJ 1995 Agonist-dependent phosphorylation of the mouse {delta}-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. Mol Pharmacol 48:173–177[Abstract]
  14. 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 272:17734–17743[Abstract/Free Full Text]
  15. Palmer TM, Benovic JL, Stiles GL 1995 Agonist-dependent phosphorylation and desensitization of the rat A3 adenosine receptor. J Biol Chem 270:29607–29613[Abstract/Free Full Text]
  16. Innamorati G, Sadeghi H, Eberle AN, Birnbaumer M 1997 Phosphorylation of the V2 vasopressin receptor. J Biol Chem 272:2486–2492[Abstract/Free Full Text]
  17. 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 272:13869–13876[Abstract/Free Full Text]
  18. 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 271:13266–13272[Abstract/Free Full Text]
  19. 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 165:196–203[Medline]
  20. Mendelsohn FA, Dunbar M, Allen A, Chou ST, Millan MA, Aguilera G, Catt KJ 1986 Angiotensin II receptors in the kidney. Fed Proc 45:1420–1425[Medline]
  21. Lemp D, Haselbeck A, Klebl F 1990 Molecular cloning and heterologous expression of N-glycosidase F from Flavobacterium meningosepticum. J Biol Chem 265:15606–15610[Abstract/Free Full Text]
  22. Hausdorff WP, Caron MG, Lefkowitz RJ 1990 Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J 4:2881–2889[Abstract]
  23. Pronin AN, Benovic JL 1997 Regulation of the G protein-coupled receptor kinase GRK5 by protein kinase C. J Biol Chem 272:3806–3812[Abstract/Free Full Text]
  24. Tobin AB, Totty NF, Sterlin AE, Nahorski SR 1997 Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1{alpha}. J Biol Chem 272:20844–20849[Abstract/Free Full Text]
  25. 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 142:15–22[Medline]
  26. 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 269:31378–31382[Abstract/Free Full Text]
  27. Sandberg K, Ji H, Clark AJL, Shapira H, Catt KJ 1992 Cloning and expression of a novel angiotensin II receptor subtype. J Biol Chem 267:9455–9458[Abstract/Free Full Text]
  28. Harlow E, Lane D 1988 Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, pp 300–301
  29. Carson MC, Leach Harper CM, Baukal AJ, Aguilera G, Catt KJ 1987 Physicochemical characterization of photoaffinity-labeled angiotensin II receptors. Mol Endocrinol 1:147–153[Abstract]
  30. Laemmli UK 1970 Cleavage of structural head proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[Medline]