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Endocrinology Vol. 139, No. 1 365-375
Copyright © 1998 by The Endocrine Society

ARTICLES

Angiotensin II-Induced Nuclear Targeting of the Angiotensin Type 1 (AT1) Receptor in Brain Neurons1

Di Lu, Hong Yang, Gerry Shaw and Mohan K. Raizada

Departments of Physiology (D.L., H.Y., M.K.R.) and Neuroscience (G.S.), University of Florida, College of Medicine, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Mohan K. Raizada, Ph.D., Department of Physiology, P.O. Box 100274, Univer-sity of Florida, Gainesville, Florida 32610. E-mail: mraizada{at}phys.med.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (Ang II) interaction with the neuronal AT1 receptor results in a chronic stimulation of neuromodulation that involves the expression of norepinephrine transporter (NET) and tyrosine hydroxylase (TH). In view of this unique property and the presence of putative nuclear localization signal (NLS) consensus sequence in the AT1 receptor, this study was conducted to investigate the hypothesis that Ang II would induce nuclear sequestration of this G protein-coupled receptor and that the sequestration may have implications on Ang II-induced expression of NET and TH genes. Incubation of neuronal cultures with Ang II caused a time- and dose-dependent increase in the levels of AT1 receptor immunoreactivity in the nucleus. A 6.7-fold increase was observed with 100 nM Ang II, in 15 min, that was blocked by losartan, an AT1 receptor-specific antagonist. Ang II-induced nuclear sequestration was specific for AT1 receptor, because Ang II failed to produce a similar effect on neuronal AT2 receptors. The presence of the putative NLS sequence in the cytoplasmic tail of the AT1 receptor seems to be the key in nuclear targeting because: 1) nuclear targeting was attenuated by a peptide of the AT1 receptor that contained the putative NLS sequence; and 2) Ang II failed to cause nuclear translocation of the AT2 receptor, which does not contain the putative NLS.

Ang II also caused a time- and dose-dependent stimulation of P62 phosphorylation, a glycoprotein of the nuclear pore complex. A 6-fold stimulation of phosphorylation was observed with 100 nM Ang II, in 15 min, that was completely blocked by losartan and not by PD123,319, an AT2 receptor specific antagonist. Preloading of neurons with p62-pep (a peptide containing consenses of mitogen-activated protein kinase in p62) resulted in a loss of Ang II-induced p62 phosphorylation and stimulation of NET and TH messenger RNA levels.

In conclusion, these data demonstrate that Ang II induces nuclear sequestration of AT1 receptor involving NLS in the AT1 receptor and p62 of the nuclear pore complex in brain neurons. A possible role of such a nuclear targeting of the AT1 receptor on chronic neuromodulatory actions of Ang II has been discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (Ang II) interacts with the neuronal AT1 receptor and stimulates its chronic neuromodulatory actions involving the expression of norepinephrine transporter (NET), tyrosine hydroxylase (TH), and dopamine ß hydroxylase genes (1, 2). These actions of the AT1 receptor have been linked to the central control of blood pressure by Ang II (3, 4). AT1 receptor belongs to the G-protein-coupled receptor (GPCR) superfamily that contains seven transmembrane regions (5, 6). The receptor is linked to the Gq subtype of G protein and is coupled to inositol phosphate and protein kinase C signal transduction pathway (7, 8). Other studies have indicated that the AT1 receptor is also coupled to many other signaling kinases, including Ras, Raf-1, mitogen-activated protein kinase (MAP kinase), phospholipase A, Ca2+-dependent calmodulin protein kinase, Jak-Stat, and jun kinase (9, 10, 11). In spite of the observations, the precise involvement of these signaling molecules in diverse cellular and physiological actions of Ang II is not well understood.

We have focused our efforts to elucidate the signal transduction pathways involved in the chronic Ang II-induced neuromodulation. Our studies have established that the interaction of Ang II with the neuronal AT1 receptor initiates a cascade of signaling events involving the activation of Ras, Raf-1, and MAP kinase (9). Activation of MAP kinase leads to increased activities of serum response element and AP1-binding elements that result in the increased transcription of NET, dopamine ß hydroxylase, and TH genes (12). Thus, it is evident, from the above discussion, that the neuronal AT1 receptor is a unique GPCR: 1) it is linked to Ras, Raf-1, and MAP kinase signal transduction pathways involved in the chronic neuromodulatory actions of Ang II; and 2) desensitization and down-regulation of AT1 receptor, induced by Ang II, does not seem to have an effect on chronic stimulation of neuromodulation (13). This is, in contrast to other GPCRs, where agonist-induced cellular responses are immediately followed by desensitization and down-regulation of the receptor (14). Thus, we hypothesized that a distinct Ang II-induced intracellular targeting of the neuronal AT1 receptor could account for the uniqueness. This view is supported by the sequence analysis of the AT1 receptor, which indicates the presence of a sequence of basic amino acids in the cytoplasmic tail (KKFKK, amino acids307–311), which could possibly form a nuclear localization signal (NLS) sequence. NLS sequences are involved in the nuclear transport of various signaling proteins that exert transcriptional control by their sequestration into the nucleus (15, 16). Thus, our objective in this investigation was to test the hypothesis that Ang II induces translocation of AT1 receptor into the nuclear compartment in the neurons and to determine the involvement of NLS in this targeting. The data demonstrate that, indeed, the AT1 receptor is targeted to the nucleus, and we present evidence that the putative NLS sequence and phosphorylation of p62, a protein of the nuclear pore complex (NPC), are important in this targeting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
One-day-old Wistar Kyoto rats were obtained from our breeding colony, which originated from Harlan Sprague-Dawley (Indianapolis, IN). DMEM, plasma-derived horse serum (PDHS), and trypsin (150 U/mg) were from Central Biomedia (Irwin, MD). [32P]-Orthophosphate (1 mCi = 37 MBq) and chemiluminescence assay reagents were from Dupont/NEN (Boston, MA). Nitrocellulose membrane was from Micron Separations, Inc. (Westboro, MA). Ang II was purchased from Sigma Chemical Co. (St. Louis, MO). Losartan potassium (Dup 753) was a gift from DuPont/Merck (Wilmington, DE). PD123,319 was from RBI (Natick, MA). Polyclonal antirabbit AT1 receptor antibody (306, catalog no. SC579) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). It was prepared by using a peptide corresponding to amino acids306–359 of the AT1 receptor. The antibody was specific for AT1 receptor and was mouse, rat, and human reactive. No cross-reaction of the AT1 receptor antibody with the AT2 receptor was observed. Monoclonal antibody to AT1 receptor was obtained from Dr. G. Vinson, Department of Biochemistry, Queen Mary and Westfield College, London, England. The antibody has been characterized by many investigators (17, 18, 19, 20, 21, 22, 23). AT2 receptor-specific antibody was a gift from Dr. Steven Fluharty, University of Pennsylvania, Philadelphia, PA. Specificity of this antibody has been established previously (24). Rabbit anti-p62 antibody was obtained from Dr. John Hanover, NIH, Bethesda, MD. Its ability to specifically recognize p62 of the NPC has been discussed elsewhere (25). Protein A/G PLUS-agarose was purchased from Santa Cruz Biotechnology. Genemed Biotechnologies, Inc. (San Francisco, CA) provided synthetic peptides used in this study. All other reagents were purchased from Fisher Scientific (Pittsburgh, PA) and were of the highest quality available.

Preparation of neuronal cells in primary culture
Neuronal cells in primary culture were established essentially as described previously (26, 27). Briefly, areas containing hypothalamus and brain stem of 1-day-old Wistar Kyoto normotensive rat brains were dissected, brain cells dissociated by trypsin, and plated in poly L-lysine precoated tissue culture dishes in DMEM containing 10% PDHS (26, 27). Culture dishes of 35-mm diameter (3 x 106 cells) or 100-mm diameter (2 x 107 cells) were prepared for experiments. After 48 h, cultures were treated with 1% cytosine arabinoside for 3 days, followed by establishment of culture for an additional 10 days, before their use in experiments. These cultures contain 90–95% neuronal cells and 5–10% astroglial cells (4, 26, 27).

Immunofluorescent staining of neurons for AT1 and AT2 receptors
Neuronal cells were fixed in 1% buffered-formalin. In certain experiments, nuclei from neuronal cells were isolated (see below), fixed onto glass slides in 1% buffered-formalin. Cells or nuclei were treated with methanol for 5 min at -10 C and preincubated with 10% FBS for 1 h at 37 C. This was followed by incubation with mouse monoclonal anti-AT1 receptor antibody at 1 µg/ml in PBS containing 0.5% BSA for 16 h at 4 C. Rhodamine-conjugated antimouse IgG was used as the second antibody, followed by counterstaining with 4'-6-diamidine-2-phenylindole-dihydrochloride (DAPI) to identify nuclear DNA and nuclei, as described previously (12, 13). Appropriate controls, in which either primary antibody was replaced by growth medium, without AT1 receptor antibody or without secondary antibody, were run in parallel to determine nonspecific staining. Use of polyclonal AT1 receptor antibody provided a similar staining pattern, although the monoclonal antibody provided a picture with lower background. The cells were processed for confocal microscopy, as described previously (1). Distribution of AT2 receptor immunoreactivity was determined as described above for the AT1 receptor, except that a polyclonal AT2 receptor-specific antibody and rhodamine-conjugated antirabbit second antibody were used. The specificity of this antibody has previously been established (25).

Isolation of nuclei and immunoblotting of AT1 receptors from neuronal cells
Neuronal cells, grown in 100-mm-diameter tissue culture dishes, were used to separate nuclear fraction from the rest of the cell fraction, as described by us elsewhere (12, 13). This protocol yields highly pure nuclear fraction that was minimally contaminated by cytoplasmic marker proteins (12, 13). Purified nuclei were lysed in the nuclear lysis buffer (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 1% Triton x 100, 1% deoxycholic acid, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM phenylemethylsulfonyl fluoride, 10 µg/ml aprotinin, and 0.8 µg/ml leupeptin). Lysate was centrifuged at 12,000 x g for 5 min at 4 C, and the supernatant was saved as nuclear extract for AT1 receptor immunoblotting.

Extract containing 400 µg protein was mixed with 1 µg rabbit anti-AT1 receptor polyclonal antibody overnight at 4 C. Immunoprecipitates were collected on protein A/G agarose and subjected to SDS-PAGE, followed by immunoblotting with the use of AT1 receptor monoclonal antibody, as described elsewhere (13). Although both antibodies, on their own, provided similar results, this combination use of polyclonal (for immunoprecipitation) and monoclonal (for immunoblotting) AT1 receptor antibodies provided highly specific AT1 receptor bands with very little background. Protein-bound antibody was detected by HRP-labeled second antibody and enhanced by a chemiluminescence assay reagent. Bands corresponding to the AT1 receptor (~49 kDa protein) were visualized by exposure to x-ray film. X-ray films were scanned by using a UVP Imagestore 5000 System (UV Product, San Gabriel, CA). Data were analyzed using a SW 5000 Gel Analysis program and presented as observed density in the AT1 receptor band that was normalized for equal loading by total protein, as described previously (9, 12). In certain experiments, neuronal cells and nuclear extracts were subjected to SDS-PAGE, proteins transferred to membrane that was subjected to Western blotting with the use of either polyclonal or monoclonal AT1 receptor antibody, essentially as described previously (28).

Osmotic loading of synthetic peptides into neurons
A peptide corresponding to the AT1 receptor tail [amino acids295–315, LNPLFYGFLGKKFKKYFLALL (AT1-pep)] and its mutant, in which lysine residues at amino acids305,306,308,309 were replaced by alanine (AT1-mut), were synthesized. The rationale for selecting this sequence was based on the identification of a putative NLS sequence (amino acids307–311) in this region of the receptor. Another peptide corresponding to amino acids189–198 of p62 [GSPFTPATLA (p62-pep)] and its mutant, in which Thr193 was replaced by Ala (p62-mut), were synthesized. All peptides were synthesized by Genemed Biotechnologies Inc. This region contained the consensus recognition sequence for MAP kinase phosphorylation (29). The reason for selecting this region of the p62 was based on our previous observation that Ang II stimulates MAP kinase and that MAP kinase is involved in chronic neuromodulatory actions of Ang II (9, 12). Osmotic loading of these peptides was carried out essentially as described previously (30). In brief, neuronal cells were rinsed in PBS, pH 7.4, incubated for 10 min with loading solution (0.5 M sucrose, 10% polyethylene glycol 1000, 10% FBS, and 200 µg/ml AT1-pep or p62pep or their mutants in DMEM, buffered with 25 mM HEPES, pH 6.8), followed by rapidly rinsing the cells with a hypotonic solution [6.5 vol H2O: 3.5 vol DMEM, buffered with 25 mM HEPES, pH 6.8 (30)]. After this treatment, cells were incubated with DMEM, containing 10% PDHS, and immediately used for experiments.

Effect of Ang II on phosphorylation of neuronal p62 protein
Neuronal cells were grown in 100-mm-diameter tissue culture dishes. They were rinsed in phosphate-free DMEM and were incubated with 20 mCi [32P]-orthophosphate in phosphate-free DMEM for 2 h at 37 C. After incubation with 100 nM Ang II at 37 C, cells were rinsed six times with ice-cold PBS, pH 7.4, and lysed for 20 min on ice in the lysis buffer (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 1% Triton X-100, 1% deoxycholic acid, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 0.8 µg/ml leupeptin). Samples were centrifuged at 12,000 g for 5 min at 4 C, and supernatants containing 200 µg protein were used for immunoprecipitation by anti-p62 (5 µg) specific antibody. Immune complexes were collected on agarose beads, conjugated with protein A+G (Santa Cruz Biotechnology), suspended in Laemmli’s sample buffer, boiled for 3 min, and subjected to SDS/PAGE, followed by autoradiography (9, 12). The radiolabeled band corresponding to a 62,000 MW protein was quantitated essentially as described previously (12, 13).

Quantitation of NET and TH messenger RNAs (mRNAs) by RT-PCR
Neuronal cells were osmotically loaded with p62-pep or p62-mut and stimulated with Ang II for 4 h. Total RNA was isolated, and NET and TH mRNA levels were quantitated by a semiquantitative RT-PCR, essentially as described previously (1, 2).

Experimental groups and data analysis
Each data point for the measurement of nuclear AT1 receptor immunoreactivity, p62 phosphorylation, and NET and TH mRNAs was collected from three culture dishes. Each culture dish contained cells generated by pooling of dissociated brain cells from 8–10 rats. Each data point was repeated at least 3 times. Comparisons between data points were made by using one-way ANOVA and Dunnett’s test, using Statistica Software (Tulsa, OK). All immunofluorescence experiments were repeated at least 6 times. One hundred to 250 cells were examined, and representative images were collected.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of polyclonal and monoclonal AT1 receptor antibodies
Western blot analysis of neuronal proteins, with the use of rabbit anti-AT1 receptor polyclonal antibody, provided a predominant protein band corresponding to approximately 49 kDa, similar to that observed previously (13). A minor protein band of approximately 60 kDa was also observed. Nuclear fraction prepared from 100 nM Ang II-treated neurons also showed a predominant (~49 kDa) and a minor (~60 kDa) protein bands (Fig. 1Go). Western blot analysis of control neurons and nuclear extracts of Ang II-treated neurons, with the use of monoclonal antibody, provided identical results (Fig. 1Go). This indicated that both monoclonal and polyclonal antibodies, raised against AT1 receptor, recognize protein of approximately 49 kDa as a major AT1 receptor protein in neuronal cells. Specificity of these antibodies was further confirmed in control Western blotting, in which PBS or normal rabbit serum were substituted for primary antibodies. No protein band of approximately 49 kDa was observed in these controls. Neuronal cell extract or nuclear extract from Ang II-treated cells was subjected to immunoprecipitation with polyclonal AT1 receptor antibody, followed by immunoblotting with monoclonal antibody, essentially as described in Materials and Methods. This protocol also provided a predominant protein band of approximately 49 kDa, with a minor band of approximately 60 kDa, similar to that seen with the individual antibodies on Western blots (Fig. 1CGo). These data showed that both antibodies recognize the same AT1 receptor protein in neuronal cells and in the nuclear fraction of Ang II-treated neurons. These data are consistent with, at least, the observations reported with the monoclonal AT1 receptor antibody. They show that this antibody recognizes AT1 receptor protein of varying size (44–70 kDa), indicating that the SDS-PAGE mobility of the receptor may be tissue specific (17, 18, 19, 20, 21, 22, 23).



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  		Figure 1. Western blot analysis of neuronal AT1 receptor by polyclonal and monoclonal AT1 receptor-specific antibodies. Neuronal cultures were treated with 100 nM Ang II for 15 min at 37 C. Whole-cell (W) extracts and nuclear (N) extracts, containing 100 µg protein from Ang II-treated neurons, were subjected to Western blot analysis, essentially as described elsewhere (28). A, Polyclonal antibody; B, monoclonal antibody; C, immmunoprecipitation with the polyclonal antibody, followed by immunoblotting by monoclonal antibody (from Dr. Vinson).

 
Further characterization of both AT1 receptor antibodies was carried out by immunofluorescence, followed by confocal microscopy. Figure 2Go shows that the distribution of AT1 receptor immunoreactivity in neuronal cells was comparable with both polyclonal (Fig. 2BGo) and monoclonal (Fig. 2DGo) antibodies. AT1 receptor immunoreactivity was predominantly localized in the plasma membrane; however, some staining was detected in the intracellular compartment. Although the staining pattern was comparable with both antibodies, the monoclonal antibody provided significantly lower background. No significant staining was observed when primary antibodies were replaced with normal rabbit serum for polyclonal (Fig. 2AGo), or PBS for monoclonal, antibody (Fig. 2CGo). In addition, no staining was seen when fluorescently-labeled secondary antibodies were deleted from the staining protocol. Finally, both antibodies provided little descrete staining for AT1 receptor immunoreactivity in PC12 cells (Fig. 2Go, E and F). This is consistent with other observations indicating that these cells predominantly express AT2, and not AT1, receptor subtypes (31, 32). Thus, monoclonal antibody was used for all subsequent immunofluorescent experiments, and a combination of polyclonal for immunoprecipitation, followed by immunoblotting by monoclonal antibodies, was used in quantitation of an AT1 receptor band of approximately 49 kDa.



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  		Figure 2. Immunofluorescent localization of AT1 receptor immunoreactivity in neuronal cells by polyclonal and monoclonal AT1 receptor-specific antibodies. Neuronal cells were fixed and subjected to immunofluorescent staining with the use of 2.5 µg/ml polyclonal antibody (C) or 1 µg/ml monoclonal antibody (D), essentially as described in Materials and Methods. Controls in which primary antibodies were replaced with normal rabbit serum (A) or PBS (B) also were prepared. Cells were processed for confocal microscopy. Data are representative of examination of approximately 200 cells. PC12 cells were established to predominantly express AT2 receptors, essentially as described elsewhere (31–32). They were incubated with polyclonal (E) or monoclonal (F) antibodies and analyzed by confocal microscopy similar to that described for neuronal cells.

 
Characterization of the effects of Ang II on AT1 receptor targeting
AT1 receptor immunoreactivity was predominantly localized on the plasma membrane of neuronal cell soma (Fig. 3AGo). Confocal imaging also indicated that a significant AT1 receptor immunoreactivity was present in the cytoplasmic compartment of the neuronal cells (Fig. 3AGo). In contrast, no detectable level of the receptor immunoreactivity was observed in the nuclear compartment. A dramatic redistribution of AT1 receptors was observed when neurons were incubated with 100 nM Ang II for 15 min. Ang II-treated neurons revealed receptor immunoreactivity, apparently in the nuclei (Fig. 3AGo). The nuclear sequestration was time- and dose-dependent and reached maximal levels in 15–30 min with 100 nM Ang II. It was blocked by coincubation of neurons with Ang II and 10 µM losartan, an AT1 receptor subtype-specific antagonist and not by 10 µM PD123319, an AT2 receptor-specific antagonist (Fig. 3AGo). Nuclear fractions from the control and Ang II-treated neurons were prepared according to previously established protocol (12, 13). They were analyzed by immunoblotting to confirm the nuclear sequestration of AT1 receptor immunoreactivity detected by immunofluorescence technique. Nuclear fractions from control, untreated neurons showed low, but significant, levels of AT1 receptor immunoreactivity (Fig. 3BGo). Ang II caused a 6.7-fold increase in the levels of AT1 receptor immunoreactivity in the nuclear fraction that was blocked by losartan and not by PD123319 (Fig. 3BGo). This indicated that the occupancy of AT1 receptor by Ang II is important in its nuclear targeting. Ang II caused a time-dependent increase in the nuclear sequestration of this immunoreactivity. It was 3-fold in 5 min and reached optimal levels (6-fold) in 15 min, followed by a 15% decrease in 30 min (Fig. 4Go). Increases in AT1 receptor immunoreactivity in the nuclear fraction were associated with its parallel decrease in the rest of the cell fractions (Fig. 4Go). Nuclear sequestration of AT1 receptor immunoreactivity also was Ang II concentration-dependent. Ang II as low as 10 nM showed a 3-fold increase, whereas 100 nM caused a maximal (4-fold) increase in nuclear immunoreactivity (Fig. 5Go).



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  		Figure 3. Effects of Ang II receptor antagonists on Ang II-induced nuclear targeting of the AT1 receptor. A, Immunofluorescence by confocal microscopy: Neuronal cultures, established in 35-mm dishes, were treated without (control) or with 100 nM Ang II, in the absence or presence of 10 µM losartan (LOS) or 10 µM PD123319 (PD), for 15 min. Cells were stained with DAPI (blue), after treatments with monoclonal antibody to the AT1 receptor and rhodamine-labeled antimouse IgG, as described in Materials and Methods; 150–200 cells were examined, and the data are representative of these cells. B, Immunoblotting of nuclear fractions of Ang II-treated neurons: Experimental conditions are essentially as described in Fig. 1AGo and in Materials and Methods. Top, A representative autoradiogram; bottom: quantitation of bands corresponding to the AT1 receptor. Data are mean ± SE (n = 3) and are normalized for equal loading, as described in Materials and Methods. *, Significantly different (P < 0.05) from control and Ang II + Los-treated cells.

 


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  		Figure 4. Time course of Ang II-induced nuclear targeting of the AT1 receptor. Neuronal cultures were incubated with 100 nM Ang II for indicated time periods, subjected to an fractionation protocol for the isolation of nuclear and the rest of the cell fractions (12–13). Fractions were used to analyze AT1 receptor immunoreactivity, as described in Materials and Methods. Top, A representative autoradiogram; bottom, data are mean ± SE (n = 3); *, significantly different (P < 0.05) from control.

 


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  		Figure 5. Dose-response of Ang II-induced nuclear targeting of the AT1 receptor. Neuronal cultures were incubated with indicated concentrations of Ang II for 15 min at 37 C, and immunoblotting or nuclear fractions were essentially as described in Materials and Methods and the legend to Fig. 4Go. Top, Representative autoradiogram; bottom, data are mean ± SE (n = 4).

 
Neuronal cultures express functional AT2 subtype of Ang II receptors. It, like AT1 receptors, belongs to the G protein-coupled receptor (GPCR) superfamily and possess limited homology with the AT1 receptor (33, 34). The effect of Ang II on the neuronal AT2 receptor distribution was studied to determine the specificity of AT1 receptor nuclear translocation. Figure 6Go shows that AT2 receptor immunoreactivity was primarily localized on neuronal plasma membrane. No significant amount of immunoreactivity was visible in either the cytoplasmic or the nuclear compartments. Incubation with 100 nM Ang II for 30 min caused no significant redistribution of AT2 receptor immunoreactivity on the plasma membrane. In addition, no nuclear translocation and sequestration of the AT2 receptor immunoreactivity was observed.



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  		Figure 6. Effect of Ang II on neuronal AT2 receptor distribution. Neuronal cultures were incubated without (control) or with 100 nM Ang II for 15 min at 37 C, essentially as described in Fig. 1Go. This was followed by immunofluorescence analysis with AT2 receptor antibodies and confocal microscopy, as described in Materials and Methods.

 
Role of putative NLS and p62 in Ang II-induced nuclear sequestration of the AT1 receptor
Our next objective was to determine the role of the putative NLS sequence, present in the AT1 receptor, on nuclear translocation of AT1 receptor. Neuronal cultures were preloaded with the AT1-pep or AT1-mut, followed by stimulation with Ang II and immunoblotting of nuclear fractions for AT1 receptor immunoreactivity. Figure 7AGo shows that preincubation of neurons with the AT1-pep caused an 85% decrease in the AT1 receptor immunoreactivity in the nuclear fraction. AT1-mut had no effect on this nuclear targeting. Figure 7BGo confirms these observations with the use of confocal microscopy of nuclear preparations. Ang II-induced nuclear sequestration (Fig. 7BGo) was attenuated by AT1-pep and not by AT1-mut. Nuclei from control neurons or neurons treated with AT1-pep or with AT1-mut showed very little AT1 receptor immunoreactivity.



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  		Figure 7. The effect of AT1-pep on Ang II-induced nuclear targeting of the AT1 receptor in neurons. A, Immunoblotting: Neuronal cells were loaded without or with AT1-pep or its mutant analog, AT1-mut. This was followed by incubation with 100 nM Ang II for 15 min at 37 C. Nuclei were isolated, and AT1 receptor immunoblotting was carried out as described in Materials and Methods. Top, A representative autoradiogram; bottom, quantitation of bands corresponding to the AT1 receptor, as described in Materials and Methods. Data are mean ± SE (n = 3). *, Significantly different (P < 0.05) from control; **, significantly different (P < 0.05) from Ang II treatment. B, Immunofluorescence by confocal microscopy: Experimental conditions are identical to those described in 4A. Immunofluorescence for the AT1 receptor in nuclear fraction was carried out as described in Materials and Methods. Blue represents DAPI staining of nuclear DNA, whereas red represents AT1 receptor immunostaining.

 
It is well established that the NPC plays an important role in the transport of cytoplasmic proteins across the nuclear membrane and their sequestration into the nucleus (35, 36). NPC is an intricate organization of specific proteins and macromolecules whose activity may be regulated by phosphorylation events (35). p62 is a protein of the NPC which contains many potential phosphorylation sites for kinases, including MAP kinase (29). Thus, it could be an important target for phosphorylation-mediated regulation of the NPC activity. Based on this, and our observations that Ang II stimulates MAP kinase (9, 12), we hypothesized that p62 phosphorylation may be important in Ang II-induced nuclear targeting of the AT1 receptor. Thus, we studied the effects of Ang II on p62 phosphorylation and involvement of this phosphorylation in the nuclear sequestrating of the AT1 receptor.

Figure 8Go shows that Ang II caused a time-dependent increase in the incorporation of [32P]-orthophosphate into p62. A 2-fold increase in the phosphorylation, observed as early as 5 min, reached a maximal level of 6-fold in 15 min, followed by a slight decline in 30 min. Interestingly, the time course of p62 phosphorylation was similar to that observed for nuclear sequestration of the AT1 receptor. Figure 9Go shows that Ang II-induced p62 phosphorylation was blocked by losartan and not by PD123319, indicating the involvement of the AT1 receptor in this process.



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  		Figure 8. Ang II stimulation of p62 phosphorylation, as a function of time. Neuronal cultures, prelabeled with [32P]orthophosphate, were incubated with 100 nM Ang II, for indicated time periods. Cells were lysed, [32P]-labeled p62 immunoprecipitated, and subjected to SDS-PAGE and autoradiography, essentially as described in Materials and Methods. Top: A representative autoradiogram; bottom, mean ± SE (n = 3); *, significantly different (P < 0.05) from control.

 


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  		Figure 9. Ang II stimulation of p62 phosphorylation in neurons. Neuronal cultures, prelabeled with [32P]-orthophosphate, were incubated with 100 nM Ang II,in the presence of 10 µM losartan (Los) or PD123319 (PD) for 30 min. Cells were lysed, [32P]-labeled p62 immunoprecipitated, and subjected to SDS-PAGE and autoradiography, as described in Materials and Methods. Top, A representative autoradiogram; bottom, quantitation of the radioactive band corresponding to the mean 62-kDa protein. Data are mean ± SE (n = 3). *, Significantly different (P < 0.05) from control; **, significantly different (P < 0.05) from Ang II-treated cells.

 
Neuronal cultures were preloaded with p62-pep to determine its effects on Ang II-induced nuclear sequestration of the AT1 receptor. We have established that p62-pep completely blocks Ang II-induced phosphorylation of p62, whereas it has no effect on Ang II-induced phosphorylation of AT1 receptor or Stat3. Figure 10AGo shows that the nuclei isolated from neurons preloaded with p62-pep before Ang II treatment, had little AT1 receptor immunoreactivity. In contrast, nuclei from neurons preloaded with p62-mut showed levels of AT1 receptor immunoreactivity comparable with the Ang II-treated neurons. This indicated that Ang II stimulates phosphorylation of p62 and that inhibition of p62 phosphorylation causes attenuation of Ang II-induced nuclear translocation of AT1 receptors. Figure 10BGo provides confocal microscopic evidence of this observation. Preloading of p62-pep resulted in a significant decrease in AT1 receptor immunofluorescence in the nuclei of Ang II-treated neurons. p62-mut showed no such inhibitory action.



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  		Figure 10. Effect of p62-pep on Ang II-induced nuclear translocation of neuronal AT1 receptor. A, Immunoblotting: p62-pep or p62-mut were osmotically loaded in neuronal cells, as described elsewhere (23). After 100 nM Ang II treatment for 15 min at 37 C, nuclei were isolated and subjected to AT1 receptor immunoblotting analysis, essentially as described in Materials and Methods. Top, A representative autoradiogram; bottom, a quantitation of 62-kDa bands. Data are mean ± SE (n = 3). *, Significantly different (P < 0.05) from control; **, significantly different (P < 0.05) from Ang II-treated cells. B, Immunofluorescence by confocal microscopy: Experimental conditions were identical to those described in the legend to Fig. 7AGo. Blue represents DAPI staining of nuclear DNA, and red represents AT1 receptor immunoreactivity.

 
Finally, we studied the role of p62 phosphorylation and nuclear sequestration of the AT1 receptor in effecting Ang II’s cellular actions. The effects of Ang II on NET and TH mRNA levels were studied. We hypothesized that if p62 phosphorylation is important in chronic neuromodulation, then Ang II stimulation of NET and TH mRNA levels would be inhibited by blocking the phosphorylation of p62. The data in Fig. 11Go, indeed, illustrate this point. Preloading of p62-pep, after Ang II stimulation, resulted in a 70% decrease in Ang II stimulation of NET and TH mRNA levels. p62-mut had no such inhibitory effect. It is pertinent to point out that the experimental conditions also inhibit Ang II-induced nuclear sequestration of Stat3 and AT1 receptors.



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  		Figure 11. Effects of p62-pep on Ang II stimulation of NET and TH mRNAs in neurons. Neuronal cells were osmotically loaded with p62-pep or p62-mut. Cells subjected to the same treatment without the peptide were used as control. This was followed by incubation, without or with 100 nM Ang II, for 4 h at 37 C. NET and TH mRNA levels were measured as described in Materials and Methods. Top, A representative autoradiogram; bottom, quantitation of bands corresponding to NET and TH cDNAs, as described elsewhere (1, 2). Data are mean ± SE (n = 3). *, Significantly different (P < 0.05) from control; **, significantly different (P < 0.05) from Ang II-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have established that the neuronal AT1 receptor, a GPCR, is targeted to the nucleus when it is stimulated by Ang II. The molecular basis for this nuclear targeting is consistent with the presence of the NLS in its cytoplasmic tail and involves phosphorylation of p62, an NPC protein. The observations are in accordance with earlier reports in which nuclear Ang II receptors have been demonstrated in hepatocytes (37, 38). The evidence for nuclear targeting includes the following: 1) Neuronal cells express the AT2 receptor subtype, which also belongs to the GPCR superfamily, shares a limited homology, and binds Ang II with a comparable affinity with the AT1 receptor (33, 34). In spite of these similarities, the AT2 receptor is distinct from the AT1 receptor, both physiologically and functionally (7, 39, 40). In addition, Ang II does not cause nuclear translocation of this receptor under the same experimental conditions that cause nuclear targeting of the AT1 receptor. 2) AT1 receptor on astroglial cells is not translocated into the nucleus under the influence of Ang II. This is true, in spite of the presence of high levels of functional AT1 receptors in astroglial cells that are coupled to stimulation of glucose transporter and plasminogen-activator inhibitor-1 (41, 42). In addition, Ang II stimulation also does not cause nuclear sequestration of the AT1 receptor in vascular smooth-muscle cells (Lu et al., unpublished observation), in spite of its involvement in chronic hypertrophic and hyperplasic actions of Ang II in these cells (43). 3) Observations derived from two different methodologies (confocal microscopy and immunoprecipitation) provide complementary results in demonstrating the Ang II-induced nuclear sequestration of the AT1 receptor. Two distinct antibodies, generated in two separate laboratories, against two different regions of the AT1 receptor, demonstrate essentially the same observation. The data also show that Ang II-induced nuclear targeting does not seem to involve a major modification in the size of the AT1 receptor, because the nuclear receptor is comparable with the plasma membrane receptor. 4) We provide evidence that the putative NLS sequence, present in the cytoplasmic tail region of the receptor, plays an important role in its nuclear targeting. This view is based on the observation that the AT1-pep competitively blocks Ang II-induced nuclear targeting of the AT1 receptor. These data could also explain the failure of nuclear translocation of the neuronal AT2 receptor, which does not contain putative NLS sequence (33, 34). These data clearly show that the AT1 receptor becomes associated with the nuclear fraction of appropriately treated neurons. Although the confocal images suggest that the receptor is actually within the nucleus, the resolution of this technique is not high enough to firmly make this conclusion. Therefore, although the data presented, using NLS and p62 peptides, is consistent with the view that the receptor translocates into the nucleus, it is also possible that the receptor associates with the exterior of the nucleus (for example, by binding to the nuclear membrane). Experiments are currently underway to definitively answer this question. Another significant finding in this study is the involvement of p62 in Ang II-induced nuclear targeting of this GPCR. p62 is an important nucleoporin that participates in the functioning of the NPC during the transport of proteins across the nuclear membrane (35). In spite of a clear involvement of p62 phosphorylation in this targeting, the precise mechanism remains to be elucidated.

This study raises a number of issues concerning the functional relevance of the nuclear targeting of the AT1 receptor in the neurons. These issues are worthy of discussion that will help provide future direction in this field of investigation. First, what are the cellular and physiological consequences of the nuclear targeting of the AT1 receptor in neurons? We suggest that it may be involved in the chronic neuromodulatory actions of Ang II. This idea is supported by the observation that both nuclear targeting for the AT1 receptor and the Ang II stimulation of mRNAs for NET and TH are blocked by the inhibition of p62 phosphorylation. Thus, nuclear AT1 receptors may interact with the DNA, or a specific nuclear protein, to regulate the transcription of NET, TH, or other genes relevant to the neuromodulatory actions of Ang II. There is no evidence in support of this view, because this report is the first example of nuclear targeting of a GPCR. However, analogous situations are well established for steroid hormone receptors. Steroid hormone receptors contain NLS sequences that play an important role in their nuclear translocation. Nuclear targeting is accompanied by specific DNA binding and transcriptional control of various steroid-hormone-responsive genes (44, 45). Second, AT1 receptors are present, both on the neuronal plasma membrane and in the cytoplasmic compartment, and presently, which compartment contributes to the nuclear AT1 receptors remains an open question. Binding of Ang II to the plasma membrane AT1 receptor activates the Ras-MAP kinase signal transduction pathway (9). Activation of MAP kinase results not only in the stimulation of downstream signaling leading to neuromodulation (12) but also in the phosphorylation of the AT1 receptor (13). The phosphorylated receptors are internalized (13). This would suggest that the plasma membrane receptors are translocated into the nucleus. This view is further supported by the observation that both the phosphorylated AT1 receptor and the nuclear AT1 receptor lack Ang II binding activity (13). However, this does not preclude other possibilities of nuclear targeting of cytosolic receptors. Finally, it remains to be determined whether nuclear targeting is specific for the AT1 receptor or it could be a general phenomenon for other GPCR. It would be surprising that AT1 receptor was unique in exhibiting agonist-induced nuclear translocation. Indeed, examination of amino acid sequences of other G protein-coupled receptors reveals that several others have sequences in the C-terminal cytoplasmic loop that conform to the consensus expected for the NLS sequence. For example, human M1, M3, and M5A muscarinic acetylcholine receptors contain sequences KRRWRK, KKKRRK, and RWKKKV, respectively (46, 47, 48). Similarly, the human platelet activating factor receptor contains the sequence KKFRKH, which also might have NLS activity (49). It remains to be seen whether these receptors, like AT1 receptor, also translocate to the nucleus after agonist stimulation.


   Acknowledgments
 
The authors wish to thank Drs. John Hanover, the NIH; Gavin Vinson, Queen Mary and Westfield College; and Steven Fluharty, University of Pennsylvania; for their generosity in providing antibodies to p62, AT1 receptor, and AT2 receptor, respectively. The Confocal Microscopic Facility of the Center for Structural Biology, at the University of Florida, was valuable in the completion of this study. Excellent assistance from Jennifer Brock, in the preparation of the manuscript, is acknowledged.


   Footnotes
 
1 This work was supported by NIH Grants HL-33610 and NS-22695. Back

Received February 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lu D, Yu K, Paddy MR, Rowland NE, Raizada MK 1996 Regulation of norepinephrine transport system by angiotensin II in neuronal cultures of normotensive and spontaneously hypertensive rat brains. Endocrinology 137:763–772[Abstract]
  2. Yu K, Lu D, Rowland NE, Raizada MK 1996 Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats. Endocrinology 137:2503–2513[Abstract]
  3. Wright JN, Harding JN 1994 Brain angiotensin receptor subtypes in the control of physiological and behavioral response. Neurosci Biobehav Rev 18:21–53[CrossRef][Medline]
  4. Raizada MK, Lu D, Sumners C 1994 AT1 receptors and angiotensin actions in the brain and neuronal cultures of normotensive and hypertensive rats. In: Mukhopadhyay A, Raizada MK (eds) Current Concepts: Tissue Renin-Angiotensin System as Local Regulators in Reproductive and Endocrine Organs. Plenum Press, New York, pp 331–348
  5. 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]
  6. Inagami T, Yamano Y, Bardhan S, Chaki S, Guo DF, Ohyama K, Kambayashi Y, Takahashi K, Ichiki T, Tsuzuki S 1995 Cloning, expression and regulation of angiotensin II receptors. Adv Exp Med Biol 377:311–317[Medline]
  7. Sumners C, Raizada MK, Kang J, Lu D, Posner P 1994 Receptor-mediated effects of angiotensin II on neurons. Front Neuroendocrinol 15:203–230[CrossRef][Medline]
  8. Catt KJ, Sandberg K, Balla T 1993 Angiotensin II receptor and signal transduction mechanisms. In: Raizada MK, Phillips MI, Sumners C (eds) Cellular and Molecular Biology of Renin Angiotensin System. CRC Press, Boca Raton, 307–356
  9. Yang H, Lu D, Yu K, Raizada MK 1996 Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci 16:4047–4058[Abstract/Free Full Text]
  10. Marrero MB, Paxton WG, Schieffer B, Ling BN, Bernstein KE 1996 Angiotensin II signaling events mediated by tyrosine phosphorylation. Cell Signal 8:21–26[CrossRef][Medline]
  11. Dostal DE, Booz GW, Baker KM 1996 Angiotensin II signaling pathways in cardiac fibroblasts: conventional vs. novel mechanisms in mediating cardiac growth and function. Mol Cell Biochem 157:15–21[Medline]
  12. Lu D, Yang H, Raizada MK 1996 Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase. J Cell Biol 135:1609–1617[Abstract/Free Full Text]
  13. Yang H, Lu D, Vinson GP, Raizada MK 1997 Involvement of MAP kinase in angiotensin II-induced phosphorylation and intracellular targeting of neuronal AT1 receptors. J Neurosci 17:1660–1669[Abstract/Free Full Text]
  14. Sasamura H, Dzau VJ, Pratt RE 1994 Desensitization of angiotensin receptor function. Kidney Int 46:1499–1501[Medline]
  15. Jans DA, Hubner S 1996 Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol Rev 76:651–685[Abstract/Free Full Text]
  16. Boulikas T 1993 Nuclear localization signals (NLS). Crit Rev Eukaryot Gene Expr 3:193–227[Medline]
  17. Barker S, Marchant W, Ho MM, Puddefoot JR, Hinson JP, Clark AJ, Vinson GP 1993 A monoclonal antibody to a conserved sequence in the extracellular domain recognizes the angiotensin II AT1 receptor in mammalian target tissues. J Mol Endocrinol 11:241–245[Abstract/Free Full Text]
  18. Kapas S, Hinson JP, Puddefoot JR, Teja R, Barker S, Kapas S, Hinson JP 1994 Internalization of the type 1 angiotensin II receptor (AT1 is required for protein kinase C activation but not for inositol trisphosphate release in the angiotensin II stimulated rat adrenal zona glomerulosa cell. Biochem Biophys Res Commun 204:1292–1298[CrossRef][Medline]
  19. Vinson GP, Ho MM, Puddefoot JR, Teja R, Barker S, Kapas S, Hinson JP The relationship between the adrenal tissue renin-angiotensin system, internalization of the type 1 angiotensin II receptor (AT1) and angiotensin II function in the rat adrenal zona glomerulosa cell. Adv Exp Med Biol 377:319–329
  20. Vinson GP, Ho MM, Puddefoot JR, Teja R, Barker S, Kapas S, Hinson JP 1995 Internalization of the type 1 angiotensin II receptor (AT1) and angiotensin II function in the rat adrenal zona glomerulosa cell. Endocr Res 21:211–217[Medline]
  21. Montiel M, Quesada J, Jimenez E 1995 Charge heterogeneity of the AT1 angiotensin II receptor subtype in the rat lung. J Endocrinol 147:153–159[Abstract/Free Full Text]
  22. Marisgliante S, Resta L, Muscella A, Vinson GP, Marzullo A, Storelli C 1996 AT1 angiotensin II receptor subtype in the human larynx and squamous laryngeal carcinoma. Cancer Lett 110:19–27[CrossRef][Medline]
  23. Harrison-Bernard LM, Navar LG, Ho MM, Vinson GP, El-Dahr SS 1997 Immunohistochemical localization of Ang II AT1 receptor in adult kidney using a monoclonal antibody. Am J Physiol 273:F170–F177
  24. Reagan LP, Theveniau M, Yang XD, Siemens IR, Yee DK, Reisine T, Fluharty SJ 1993 Development of polyclonal antibodies against angiotensin type 2 receptors. Proc Natl Acad Sci USA 90:7956–7960[Abstract/Free Full Text]
  25. Davis LI, Blobel G 1987 Nuclear pore complex contains a family of glycoproteins that includes p62:glycosylation through a previously unidentified cellular pathway. Proc Natl Acad Sci USA 84:7552–7556[Abstract/Free Full Text]
  26. Raizada MK, Muther TF, Sumners C 1984 Increased angiotensin II specific receptors in neuronal cultures of spontaneously hypertensive rat brain. Am J Physiol 247:C364–C372
  27. Raizada MK, Lu D, Tang W, Kurian P, Sumners C 1993 Increased angiotensin II type 1 receptor gene expression in neuronal cultures from spontaneously hypertensive rats. Endocrinology 132:1715–1722[Abstract]
  28. Winston SE, Fuller SA, Huwel JGR 1992 Western Blotting. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K K (eds) 1992 Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology. John Wiley & Sons, ed. 2, New York, pp 10–33
  29. Cordes V, Waizenerger I, Krohne G 1991 Nuclear pore complex glycoprotein p62 of xenopus laevis and mouse cDNA cloning and identification of its glycosylation region. Eur J Cell Biol 55:31–47[Medline]
  30. Ahmad F, Li PM, Meyerovitch J, Goldstein BJ 1995 Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway. J Biol Chem 270:20503–20508[Abstract/Free Full Text]
  31. Kijima K, Matsubara H, Murasawa S, Maruyama K, Mori Y, Inada M 1995 Gene transcription of angiotensin II type 2 receptor is repressed by growth factors and glucocorticoids in PC12 cells. Biochem Biophys Res Commun 216:359–366[CrossRef][Medline]
  32. Brechler V, Jones PW, Levens NR, de Gasparo M, Bottari SP 1993 Agonistic and antagonistic properties of angiotensin analogs at the AT2 receptor in PC12W cells. Regul Pept 44:207–213[CrossRef][Medline]
  33. Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo 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]
  34. Nakajima M, Mukoyama M, Pratt RE, Horiuchi M, Dzau VJ 1993 Cloning of cDNA and analysis of the gene for mouse angiotensin II type 2 receptor. Biochem Biophys Res Commun 197:393–399[CrossRef][Medline]
  35. Schlenstedt G 1996 Protein import into the nucleus. FEBS Lett 389:75–79[CrossRef][Medline]
  36. David LI 1995 The nuclear pore complex. Annu Rev Biochem 64:865–896[CrossRef][Medline]
  37. Booz GW, Conrad KM, Hess AL, Singer HA, Baker KM 1992 Angiotensin II binding sites on hepatocyte nuclei. Endocrinology 130:3641–3649[Abstract]
  38. Tang SS, Rogg H, Schumacher R, Dzau VJ 1992 Characterization of nuclear angiotensin II binding sites in rat liver and comparison with plasma membrane receptor. Endocrinology 131:374–380[Abstract]
  39. Nahmias C, Strosberg AD 1995 The angiotensin AT2 receptor: searching for signal-transduction pathways and physiological function. Trends Pharmacol Sci 16:223–225[CrossRef][Medline]
  40. Clauser E, Curnow KM, Davies E, Conchon S, Teutsch B, Vianello B, Monnot C, Corvol P 1996 Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements. Eur J Endocrinol 134:403–411[Abstract/Free Full Text]
  41. Tang W, Richards EM, Raizada MK, Sumners C 1995 Angiotensin II increases glucose uptake and glucose transporter-1 mRNA levels in astroglia. Am J Physiol 268:E384–E390
  42. Rydzewski B, Zelezna B, Tang W, Sumners C, Raizada MK 1992 Angiotensin II stimulation of plasminogen activator inhibitor-1 gene expression in astroglial cells from the brain. Endocrinology 130:1255–1262[Abstract]
  43. Hsueh WA, Do YS, Anderson PW, Law RE 1995 Angiotensin II in cell growth and matrix production. Adv Exp Med Biol 377:217–223[Medline]
  44. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P 1995 The nuclear receptor superfamily: the ed. 2ecade. Cell 83:835–839[CrossRef][Medline]
  45. Lopes da Silva S, Burbach JP 1995 The nuclear hormone receptor family in the brain: classics and orphans. Trends Neurosci 18:542–548[CrossRef][Medline]
  46. Bonner TI, Young AC, Brann MR, Buckley NJ 1988 Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1:403–410[CrossRef][Medline]
  47. Peralta EG, Ashkenazi A, Winslow JW, Smith DH, Ramachandran J, Capon DJ 1987 Distinct primary structures, ligand binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J 6:3923–3929[Medline]
  48. Allard WJ, Sigal IS, Dixon RA 1987 Sequence of the gene encoding the human M1 muscarinic acetylcholine receptor. Nucleic Acids Res 15:10604[Free Full Text]
  49. Chase PB, Halonen M, Regan JW 1993 Cloning of a human platelet-activating factor receptor gene: evidence for an intron in the 5'-untranslated region. Am J Respir Cell Mol Biol 8:240–244



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