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Falk Cardiovascular Research Center and Department of Medicine
(L.H., R.E.P., V.J.D., B.K.K.) Howard Hughes Medical Institute
(B.K.K.) Stanford University School of Medicine Stanford,
California 94305
Department of Pharmacology (L.H., L.M.)
University of Wuerzburg Wuerzburg, Germany
Department of
Medicine (R.E.P, V.J.D.) Brigham and Women’s Hospital Harvard
Medical School Boston, Massachusetts 02115
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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The cardiovascular effects of Ang II are frequently subject to rapid desensitization. For other types of G protein-coupled receptors, different mechanisms of desensitization have been characterized (9). These processes include 1) phosphorylation of specific intracellular receptor sites resulting in uncoupling from the G protein, 2) sequestration of receptors into endosomal vesicles, and 3) down-regulation of the total receptor number of a cell. The contribution of receptor sequestration to the desensitization process is still unclear. Tachyphylaxis of vascular contractile responses to Ang II has routinely been attributed to loss of cell surface receptors by sequestration (10, 11). Previously, indirect approaches have been used to study the internalization of angiotensin receptors. Sequestration of AT1 receptors has been observed as a rapid loss of cell surface ligand-binding sites accessible to radiolabeled derivatives of Ang II (11, 12, 13, 14). In addition, various cell types have been shown to internalize [125I]Ang II (12) or an Ang II-colloidal gold conjugate (15, 16). From the observation that Ang II was taken up by endocytosis, and angiotensin receptors were sequestered intracellularly, it was concluded that Ang II and its receptor were internalized as a receptor-ligand complex (14). However, due to the technical limitations of this approach, it was not possible to distinguish between the intracellular trafficking routes of Ang II and its receptors.
In the present study, we combined immunofluorescence staining of angiotensin receptors and fluorescein labeling of Ang II to follow the intracellular trafficking of Ang II and its AT1a and AT2 receptor subtypes by immunofluorescence microscopy. These experiments provide evidence for the internalization of Ang II and its AT1a receptor as a receptor-ligand complex, and they suggest that the receptor-ligand complex dissociates after endocytosis, with the receptors recycling to the plasma membrane and Ang II being transported to lysosomes. Sequestration of AT1a receptors into endosomes is in dynamic equilibrium with receptor recycling to the plasma membrane and continues after desensitization mechanisms have effectively attenuated the Ca2+ and inositol 1,4,5-trisphosphate (IP3) signaling pathways. The redistribution of receptor after agonist exposure is subtype specific, as the AT2 receptor does not undergo endocytosis.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and fluorescein-labeled Ang II (Fig. 1B). The flag epitope has been previously used to study the
intracellular trafficking pathway of the thrombin receptor (18, 19),
gastrin-releasing peptide receptor (20), and TRH receptor (21). Wild
type and flag epitope-tagged receptors were stably expressed in human
embryonal kidney 293 cells, and their ligand binding and signal
transduction properties were determined. The ligand binding properties
of the epitope-tagged receptor were indistinguishable from those of the
wild type AT1a receptor (Fig. 1A). Similarly, stimulation
of the flag-tagged receptor with Ang II induced an increase in
IP3 (Fig. 1C) and intracellular Ca2+ levels
(Fig. 1D). Under these conditions the cells were refractory to further
stimulation by Ang II (Fig. 1D), indicating desensitization of the
flag-AT1a receptor. The time course and magnitude of Ang
II-induced changes in intracellular Ca2+ and
IP3 were identical for flag-tagged and wild type
AT1a receptors (data not shown). Similar to the
AT1a receptor, flag epitope tagging of the AT2
receptor subtype at the amino-terminus did not change expression levels
or ligand binding characteristics of flag-AT2 compared with
those of the wild type AT2 receptor after transfection into
COS-7 or 293 cells (data not shown).
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). In
addition, fluorescein isothiocyanate-labeled angiotensin II (FITC-Ang
II) retained its agonist properties, as it was able to stimulate the
production of IP3 (data not shown) and induce
agonist-dependent endocytosis of AT1a receptors (see below)
to a similar extent as unlabeled Ang II.
Ang II Induces Internalization of AT1a
Receptors but not of AT2 Receptors
Confocal laser scanning microscopy was used to visualize the
cellular localization of AT1a and AT2 receptors
after immunostaining. In unstimulated human 293 cells stably expressing
flag-tagged AT1a receptors, the AT1a subtype
was detected in the plasma membrane by M1 antibody staining of
nonpermeabilized cells (Fig. 2A). No
antibody binding was detectable in untransfected 293 cells (data not
shown). In addition to its surface localization, a smaller amount of
AT1a receptor was found in intracellular vesicles in
permeabilized cells (Fig. 2B). Flag-tagged AT2 receptors
were localized on the cell surface, and no intracellular
AT2 receptors could be detected (Fig. 2C). The presence of
an intracellular pool of receptor has been observed for other G
protein-coupled receptors, including the 
2c-adrenergic
receptor (24) and the thrombin receptor (19). In the case of the
thrombin receptor, there is evidence that the intracellular pool of
receptor serves as a reservoir of receptor protected from thrombin
cleavage and activation (19). The functional role of intracellular
AT1a and 2c-adrenergic receptor remains to
be determined.
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). Using
this approach, it was possible to label the plasma membrane
AT1a receptors with antibody (Fig. 3A) without labeling the
intracellular pool of AT1a receptors, and the fate of
plasma membrane receptors could be determined. AT1a
receptors were internalized into small intracellular vesicles upon
stimulation with 1 µM Ang II (Fig. 3B) or 1
µM FITC-Ang II for 10 min (Fig. 3C). Addition of the
AT1-specific antagonist PD 134756 alone did not change the
cellular distribution of receptors (Fig. 3D). Internalization of
receptors induced by Ang II or FITC-Ang II was completely blocked in
the presence of the antagonist PD 134756 (Fig. 3, E and F). The
AT2 receptor subtype was localized on the cell surface in
unstimulated cells (Fig. 3G), but it did not undergo endocytosis upon
stimulation with Ang II (Fig. 3H) or FITC-Ang II (Fig. 3I). Even
prolonged exposure of AT2 receptor-expressing cells to Ang
II for up to 2 h did not result in a detectable redistribution of
the cell surface receptors (not shown). This observation is consistent
with radioligand binding experiments showing no sequestration of
AT2 receptors after exposure of cells to Ang II (25, 26).
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, A and B). The presence of internalized
AT1a receptors in endosomes was further confirmed by
colocalization with the transferrin receptor using a monoclonal
antibody against the human transferrin receptor (data not shown).
Internalization of the AT1a receptor was reversible upon
removal of the agonist. If cells were stimulated with Ang II for 10 min
and then incubated in fresh medium for 50 min to remove Ang II,
AT1a receptors recycled to the plasma membrane (Fig. 4C, arrow), whereas TR-transferrin remained in intracellular
vesicles after removal of Ang II (Fig. 4D).
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, E and F).
The endocytosis of FITC-Ang II was AT1a receptor mediated,
as it could not be detected in the presence of the receptor antagonist
PD 134756, in cells transfected with AT2 receptor, or in untransfected
cells (data not shown). However, upon removal of the agonist for 50 min
following the initial 10-min exposure to FITC-Ang II, the fluorescent
angiotensin remained in intracellular vesicles (Fig. 4G). Only part of
these FITC-Ang II-containing vesicles were transferrin positive (Fig. 4, G and H, arrowhead), suggesting that the intracellular
trafficking pathways of FITC-Ang II and transferrin beyond the endosome
are distinct.
Previous studies have demonstrated that an Ang II-colloidal gold
conjugate can be internalized into lysosomal structures in vascular
smooth muscle cells (15, 16). To test, whether the intracellular
vesicles, which contain FITC-Ang II after prolonged labeling periods,
are lysosomes, TR-ovalbumin was used as a marker for lysosomal
targeting of endocytosed ligands. When 293 cells expressing
AT1a receptors were incubated with TR-ovalbumin for 30–60
min, the fluorescent ovalbumin was detected in intracellular vesicles,
which could be stained with an antiserum against the lysosomal membrane
protein lgp-120 (28) (results not shown). After 60 min of exposure of
cells to Ang II and TR-ovalbumin, AT1a receptors and
TR-ovalbumin were partially separated in the cells, with
AT1a receptor-positive vesicles appearing in the periphery
of the cells (Fig. 5, A and C,
arrowhead) and ovalbumin-containing vesicles in the
periphery (Fig. 5B, arrowhead) and the center of the cells
(Fig. 5, B and C, arrow). After removal of the agonist,
AT1a receptors returned to the plasma membrane (Fig. 5, A
and F, arrow), whereas the TR-ovalbumin remained in
intracellular vesicles (Fig. 5, E and F, arrowhead).
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, G–I). After removal of FITC-Ang II
from the media and washout for 50 min, both labels showed significant
colocalization in intracellular vesicles (Fig. 5, K–M,
arrowhead), indicating that FITC-Ang II was
diverted to the lysosomal system. Taken together, these results suggest
that AT1a receptors can recycle to the plasma membrane
after endocytosis, and that Ang II itself is transported to
lysosomes.
Continuous Endocytosis and Recycling of
AT1a Receptors
Internalization of AT1 receptors can be
detected by radioligand binding assay in a variety of cell types as a
decrease in cell surface receptor number by 50–75% (10, 11, 29). This
sequestration of AT1a receptor reaches an equilibrium after
20–30 min of agonist stimulation. The observation that
AT1a receptors recycle to the plasma membrane after agonist
removal suggests that this equilibrium might be a dynamic one, with
continuous internalization and recycling of the receptor to the cell
surface rather than a static equilibrium, where receptors do not
recycle in the presence of agonist. To test this hypothesis, maximum
internalization of AT1a receptors was induced by exposure
of 293 cells to 1 µM Ang II for 30 min. Under this
condition, no further decrease in cell surface receptor number could be
observed (Fig. 6A). Receptor signaling
through intracellular IP3 accumulation was already
desensitized after 10 min of exposure to agonist (see Fig. 1C).
Surprisingly, cells were able to internalize FITC-Ang II (Fig. 6B) as
well as AT1a receptors labeled with M1 antiflag antibody
(Fig. 6C) into intracellular vesicles, even after the maximum receptor
internalization was reached. This phenomenon could be blocked by
addition of the receptor antagonist PD 134756 (data not shown). This
result demonstrates that endocytosis and recycling of receptors to the
plasma membrane occur continuously even in the presence of receptor
agonist. The internalization and recycling process of the
AT1a receptor seems to be independent of receptor
signaling, as receptor internalization and recycling could be detected
even in the presence of agonist for up to 50 min. At this time, signal
transduction by the AT1a receptor via IP3 (Fig. 1C) or Ca2+ pathways (Fig. 1D) was completely desensitized
(29, 30). However, Ang II-stimulated diacylglycerol levels in vascular
smooth muscle cells remain elevated even after 30- to 60-min exposure
to Ang II, and it has been suggested that the sustained diacylglycerol
accumulation is linked to the internalization of receptor-ligand
complexes (10). In 293 cells stably expressing AT1a
receptors, inhibition of protein kinase C by staurosporin or
down-regulation of protein kinase C by prolonged treatment with the
phorbol ester phorbol 12-myristate 13-acetate did not change the
internalization of angiotensin receptors (29).
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, A and B). Upon removal of Ang II and
incubation of cells in normal medium, AT1a receptors
recycled to the cell surface (Fig. 7A, open squares, and
Fig. 7C). When quantitated by enzyme-linked immunosorbent assay,
approximately 65% of the previously internalized AT1a
receptors returned to the cell surface (Fig. 7A). Ammonium chloride and
chloroquine significantly inhibited the recycling of AT1a
receptors (Fig. 7, A, D, and E).
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In summary, we have provided experimental evidence that 1) Ang II causes a subtype-specific endocytosis of AT1a receptors but not AT2 receptors; 2) internalization and recycling of the AT1a receptor are dynamic processes and continue even after IP3 and Ca2+ signaling pathways have been desensitized; and 3) dissociation of the internalized receptor-ligand complex in endosomes is necessary for recycling of the AT1a receptor to the plasma membrane and further trafficking of Ang II.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fluorescent Labeling of Ang II
Ang II was labeled at the amino-terminus with FITC as described
previously (22). Briefly, 1 µmol Ang II was incubated with 1 µmol
FITC in 100 mM NaHCO3, pH 8.5, including 25%
acetone. After 2 h, the coupling reaction was stopped by adding 10
µmol glycine in 100 mM NaHCO3, pH 8.5.
FITC-Ang II was separated from unbound FITC by chromatography on a
Sephadex G-10 column. Experimental results obtained using Ang II that
was FITC labeled with this method were identical to those obtained with
commercially available FITC-Ang II (RBI Research Biochemicals
International, Natick, MA).
Radioligand Binding
Receptor binding assays were performed as described previously
(4). Briefly, transiently transfected COS-7 cells or stable 293 cell
transfectants were lysed in 10 mM Tris-HCl (pH 7.4) and 1
mM EDTA buffer and scraped off plates. Membrane homogenates
were prepared as previously described (4). Binding assays were
performed in 500 µl buffer (75 mM Tris-HCl, 1
mM EDTA, 12.5 mM MgCl2, and 0.1%
BSA, pH 7.4) for 1 h at 22 C. Saturation isotherms were obtained
by incubating membranes with varying concentrations of
[125I]Sar-Ile-Ang II (2200 Ci/mmol; DuPont-New England
Nuclear, Boston, MA; Amersham, Arlington Heights, IL). Nonspecific
binding was determined by the addition of 10 µM unlabeled
Ang II. Competition experiments were performed in the presence of
varying concentrations of Ang II (Sigma Chemical Co., St. Louis, MO) or
the AT1 and AT2 receptor-specific antagonists
PD 134756 (identical with DuP 753, losartan) and PD 123319 (kindly
provided by Parke Davis, Detroit, MI). Radioactivity bound to membranes
was separated from free ligand by filtration through GF/C filters
(Whatman, Clifton, NJ). Binding data were analyzed by nonlinear
regression using InPlot software (GraphPad Software, San Diego,
CA).
Measurement of Intracellular IP3
Levels
293 cells expressing AT1a receptors were grown on
six-well culture dishes (Falcon) and stimulated with 100 nM
Ang II in DMEM for 30 sec to 50 min. Reactions were stopped by adding 1
ml ice-cold 20% trichloroacetic acid (wt/vol) and extracted with
water-saturated diethyl ether. The concentration of IP3 in
these samples was determined using a competitive radioligand receptor
binding assay (Amersham) (36). Results shown are the mean ±
SEM from triplicate determinations of four independent
experiments.
Intracellular Calcium
To measure changes in intracellular calcium levels under
conditions identical to those used for immunofluorescence staining,
cells on coverslips were loaded with the long wavelength calcium
indicator fluo-3 as described previously (19). Briefly, fluo-3
acetoxymethylester (fluo-3/AM) was dissolved in 20% pluronic F-127 in
dimethylsulfoxide and was diluted 1:100 in DMEM before use. Cells on
coverslips were loaded with 5 µM fluo-3/AM for 30 min at
37 C, rinsed twice in fresh medium, and incubated for 15 min to allow
deesterification. Coverslips with attached cells were mounted in a
perfusion chamber in PBS for inspection in a Sarastro Phoibos 1000
confocal laser scanning microscope (Molecular Dynamics, Sunnyvale, CA).
Images were recorded at 8-bit resolution using the fluorescein channel
in time lapse mode in 5-sec intervals (objective, x20; 256 x 256
pixels). Cells were stimulated with 100 nM Ang II or 15
µM ionomycin. Fluorescence intensity was measured by
placing circular spots over the widest part of any given cell, and
average intensity was determined for a minimum of 20 cells/coverslip.
For each experiment, six coverslips were measured under identical
conditions, and experiments were repeated at least three times. The
data displayed indicate the mean ± SE.
Immunofluorescence Microscopy
Two days before the experiments, cells were split on glass
coverslips. After various treatments, cells were fixed in 4%
paraformaldehyde as described previously (18, 19). For experiments
using permeabilized cells, fixed specimens were incubated for 30 min in
blocking buffer containing 0.2% Nonidet P-40, 5% nonfat dry milk, and
50 mM HEPES (pH 7.6). Subsequently, primary antibodies
(final concentrations: M1 antiflag antibody, 10 µg/ml; 12CA5
antibody, 5 µg/ml) were applied in blocking buffer for 1 h.
Secondary antibodies [goat antirabbit F(ab')2 fragment of
IgG conjugated to Texas Red; Jackson ImmunoResearch, West Grove, PA;
goat antimouse IgG FITC conjugate, Amersham) were diluted 1:500 and
applied in blocking buffer. For selective detection of receptor antigen
localized on the cell surface, nonpermeabilized cells were labeled with
primary antisera in DMEM containing 10% FBS and 30 mM
HEPES, pH 7.6, at 37 C. For labeling of the endocytic pathways, cells
were incubated with TR-transferrin and TR-ovalbumin (Molecular Probes,
Eugene, OR). Specimens were inspected by confocal laser scanning
microscopy using a Sarastro Phoibos 1000 instrument or a Leica DM
microscope equipped with Leica TCS confocal scanner (Leica, Deerfield,
IL). Optical sections were scanned through cells 2 µm above the
surface of the coverslip. Images were stored on optical laser disk and
processed using Image Space (Molecular Dynamics, Mountain View, CA) and
Adobe Photoshop 2.5.1 software (Adobe Systems, Mountainview, CA). For
each experiment, three to five coverslips were treated and inspected
under identical conditions, and experiments were repeated at least
three times with identical results.
Enzyme-Linked Immunosorbent Assay of Cell Surface Receptor
Antigen
To quantitate the amount of cell surface angiotensin
AT1 receptors, stably transfected 293 cells were split onto
24-well plates (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ)
at 5 x 104 cells/well. The next day, cells were
washed with DMEM with 20 mM HEPES (pH 7.4) and 1 mg/ml BSA.
Cells were incubated with M1 anti-flag antibody for 30 min at 37 C to
label the epitope-tagged AT1 receptors on the cell surface.
After a brief rinsing step to remove unbound M1 antibody, cells were
incubated with 1 µM Ang II for 10 min to induce receptor
internalization. To monitor recycling of internalized receptors
previously decorated with the M1 antibody, cells were kept in fresh
medium without Ang II for 50 min at 37 C. At the end of the experiment,
cells were fixed for 5 min in freshly prepared 4% paraformaldehyde in
PBS. Plates were washed with PBS and incubated with alkaline
phosphatase-conjugated secondary antibody (Bio-Rad, Richmond, CA; 1:300
dilution in PBS-1% BSA). Plates were developed with alkaline
phosphatase chromogenic substrate p-nitrophenylphosphate.
OD405 was read after 20 min. Antibody binding data are
expressed as specific binding (total minus nonspecific, with
nonspecific being defined as the level of binding seen in untransfected
293 cells). Data shown are the mean ± SEM
(n = 3) for a representative experiment of three
performed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by the Howard Hughes Medical Institute (to B.K.K.), NIH grants (to R.E.P. and V.J.D.), and a fellowship (to L.H.) from the German Research Foundation (Deutsche Forschungsgemeinschaft, Bonn).
Received for publication April 30, 1996. Revision received April 2, 1997. Accepted for publication May 9, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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 D. Fessart, M. Simaan, and S. A. Laporte c-Src Regulates Clathrin Adapter Protein 2 Interaction with {beta}-Arrestin and the Angiotensin II Type 1 Receptor during Clathrin- Mediated Internalization Mol. Endocrinol., February 1, 2005; 19(2): 491 - 503. [Abstract] [Full Text] [PDF]
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C. J. Wruck, H. Funke-Kaiser, T. Pufe, H. Kusserow, M. Menk, J. H. Schefe, M. L. Kruse, M. Stoll, and T. Unger Regulation of Transport of the Angiotensin AT2 Receptor by a Novel Membrane-Associated Golgi Protein Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 57 - 64. [Abstract] [Full Text] [PDF]
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A. Heydorn, B. P. Sondergaard, B. Ersboll, B. Holst, F. C. Nielsen, C. R. Haft, J. Whistler, and T. W. Schwartz A Library of 7TM Receptor C-terminal Tails: INTERACTIONS WITH THE PROPOSED POST-ENDOCYTIC SORTING PROTEINS ERM-BINDING PHOSPHOPROTEIN 50 (EBP50), N-ETHYLMALEIMIDE-SENSITIVE FACTOR (NSF), SORTING NEXIN 1 (SNX1), AND G PROTEIN-COUPLED RECEPTOR-ASSOCIATED SORTING PROTEIN (GASP) J. Biol. Chem., December 24, 2004; 279(52): 54291 - 54303. [Abstract] [Full Text] [PDF]
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M. A. F. de Godoy, A. M. de Oliveira, and S. Rattan Angiotensin II-Induced Relaxation of Anococcygeus Smooth Muscle via Desensitization of AT1 Receptor, and Activation of AT2 Receptor Associated with Nitric-Oxide Synthase Pathway J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 394 - 401. [Abstract] [Full Text] [PDF]
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C. M. Filipeanu, F. Zhou, W. C. Claycomb, and G. Wu Regulation of the Cell Surface Expression and Function of Angiotensin II Type 1 Receptor by Rab1-mediated Endoplasmic Reticulum-to-Golgi Transport in Cardiac Myocytes J. Biol. Chem., September 24, 2004; 279(39): 41077 - 41084. [Abstract] [Full Text] [PDF]
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 C. Suarez, G. Diaz-Torga, A. Gonzalez-Iglesias, C. Cristina, and D. Becu-Villalobos Upregulation of angiotensin II type 2 receptor expression in estrogen-induced pituitary hyperplasia Am J Physiol Endocrinol Metab, May 1, 2004; 286(5): E786 - E794. [Abstract] [Full Text] [PDF]
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A. SPAT and L. HUNYADY Control of Aldosterone Secretion: A Model for Convergence in Cellular Signaling Pathways Physiol Rev, April 1, 2004; 84(2): 489 - 539. [Abstract] [Full Text] [PDF]
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 G. Du, P. Huang, B. T. Liang, and M. A. Frohman Phospholipase D2 Localizes to the Plasma Membrane and Regulates Angiotensin II Receptor Endocytosis Mol. Biol. Cell, March 1, 2004; 15(3): 1024 - 1030. [Abstract] [Full Text] [PDF]
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S. P. Singh, D. McDonald, T. J. Hope, and B. S. Prabhakar Upon Thyrotropin Binding the Thyrotropin Receptor Is Internalized and Localized to Endosome Endocrinology, February 1, 2004; 145(2): 1003 - 1010. [Abstract] [Full Text] [PDF]
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 D. Kumar, V. Menon, W. R. Ford, A. S. Clanachan, and B. I. Jugdutt Effect of Angiotensin II lype 2 Receptor Blockade on Activation of Mitogen-Activated Protein Kinases after Ischemia-Reperfusion in Isolated Working Rat Hearts Journal of Cardiovascular Pharmacology and Therapeutics, December 1, 2003; 8(4): 285 - 296. [Abstract] [PDF]
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M. Lopez-Ilasaca, X. Liu, K. Tamura, and V. J. Dzau The Angiotensin II Type I Receptor-associated Protein, ATRAP, Is a Transmembrane Protein and a Modulator of Angiotensin II Signaling Mol. Biol. Cell, December 1, 2003; 14(12): 5038 - 5050. [Abstract] [Full Text] [PDF]
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J.-H. Wu, K. Peppel, C. D. Nelson, F.-T. Lin, T. A. Kohout, W. E. Miller, S. T. Exum, and N. J. Freedman The Adaptor Protein {beta}-Arrestin2 Enhances Endocytosis of the Low Density Lipoprotein Receptor J. Biol. Chem., November 7, 2003; 278(45): 44238 - 44245. [Abstract] [Full Text] [PDF]
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T. Seck, R. Baron, and W. C. Horne Binding of Filamin to the C-terminal Tail of the Calcitonin Receptor Controls Recycling J. Biol. Chem., March 14, 2003; 278(12): 10408 - 10416. [Abstract] [Full Text] [PDF]
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L. Gendron, J.-F. Oligny, M. D. Payet, and N. Gallo-Payet Cyclic AMP-independent Involvement of Rap1/B-Raf in the Angiotensin II AT2 Receptor Signaling Pathway in NG108-15 Cells J. Biol. Chem., January 31, 2003; 278(6): 3606 - 3614. [Abstract] [Full Text] [PDF]
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U. Rueckschloss, M. T. Quinn, J. Holtz, and H. Morawietz Dose-Dependent Regulation of NAD(P)H Oxidase Expression by Angiotensin II in Human Endothelial Cells: Protective Effect of Angiotensin II Type 1 Receptor Blockade in Patients With Coronary Artery Disease Arterioscler Thromb Vasc Biol, November 1, 2002; 22(11): 1845 - 1851. [Abstract] [Full Text] [PDF]
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R. E. Widdop, K. Matrougui, B. I. Levy, and D. Henrion AT2 Receptor-Mediated Relaxation Is Preserved After Long-Term AT1 Receptor Blockade Hypertension, October 1, 2002; 40(4): 516 - 520. [Abstract] [Full Text] [PDF]
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 L. Hunyady, A. J. Baukal, Z. Gaborik, J. A. Olivares-Reyes, M. Bor, M. Szaszak, R. Lodge, K. J. Catt, and T. Balla Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor J. Cell Biol., June 24, 2002; 157(7): 1211 - 1222. [Abstract] [Full Text] [PDF]
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Y. Xu, D. Kumar, J. R. B. Dyck, W. R. Ford, A. S. Clanachan, G. D. Lopaschuk, and B. I. Jugdutt AT1 and AT2 receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1206 - H1215. [Abstract] [Full Text] [PDF]
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S. Miserey-Lenkei, C. Parnot, S. Bardin, P. Corvol, and E. Clauser Constitutive Internalization of Constitutively Active Angiotensin II AT1A Receptor Mutants Is Blocked by Inverse Agonists J. Biol. Chem., February 15, 2002; 277(8): 5891 - 5901. [Abstract] [Full Text] [PDF]
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J. L. Zhuo, J. D. Imig, T. G. Hammond, S. Orengo, E. Benes, and L. G. Navar Ang II Accumulation in Rat Renal Endosomes During Ang II-Induced Hypertension: Role of AT1 Receptor Hypertension, January 1, 2002; 39(1): 116 - 121. [Abstract] [Full Text] [PDF]
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C. M Filipeanu, R. H Henning, S A. Nelemans, and D. de Zeeuw Review: Intracellular angiotensin II: from myth to reality? Journal of Renin-Angiotensin-Aldosterone System, December 1, 2001; 2(4): 219 - 226. [PDF]
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L. B. Dale, M. Bhattacharya, J. L. Seachrist, P. H. Anborgh, and S. S. G. Ferguson Agonist-Stimulated and Tonic Internalization of Metabotropic Glutamate Receptor 1a in Human Embryonic Kidney 293 Cells: Agonist-Stimulated Endocytosis Is beta -Arrestin1 Isoform-Specific Mol. Pharmacol., December 1, 2001; 60(6): 1243 - 1253. [Abstract] [Full Text] [PDF]
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J. G. Modrall, M. Nanamori, J. Sadoshima, D. C. Barnhart, J. C. Stanley, and R. R. Neubig ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling Am J Physiol Cell Physiol, September 1, 2001; 281(3): C801 - C809. [Abstract] [Full Text] [PDF]
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 S. S. G. Ferguson Evolving Concepts in G Protein-Coupled Receptor Endocytosis: The Role in Receptor Desensitization and Signaling Pharmacol. Rev., March 1, 2001; 53(1): 1 - 24. [Abstract] [Full Text] [PDF]
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A. Gonzalez Iglesias, C. Suarez, C. Feierstein, G. Diaz-Torga, and D. Becu-Villalobos Desensitization of angiotensin II: effect on [Ca2+]i, inositol triphosphate, and prolactin in pituitary cells Am J Physiol Endocrinol Metab, March 1, 2001; 280(3): E462 - E470. [Abstract] [Full Text] [PDF]
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G. Vauquelin, F. L. Fierens, I. Verheijen, and P. M. Vanderheyden Distinctions between non-peptide angiotensin II AT1-receptor antagonists Journal of Renin-Angiotensin-Aldosterone System, March 1, 2001; 2(1_suppl): S24 - S31. [Abstract] [PDF]
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L G. Navar, K. D Mitchell, L. M Harrison-Bernard, H. Kobori, and A. Nishiyama Review: Intrarenal angiotensin II levels in normal and hypertensive states Journal of Renin-Angiotensin-Aldosterone System, March 1, 2001; 2(1_suppl): S176 - S184. [PDF]
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Z. Gáborik, M. Szaszák, L. Szidonya, B. Balla, S. Paku, K. J. Catt, A. J. L. Clark, and L. Hunyady {beta}-Arrestin- and Dynamin-Dependent Endocytosis of the AT1 Angiotensin Receptor Mol. Pharmacol., February 1, 2001; 59(2): 239 - 247. [Abstract] [Full Text]
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P. H. Anborgh, J. L. Seachrist, L. B. Dale, and S. S. G. Ferguson Receptor/{beta}-Arrestin Complex Formation and the Differential Trafficking and Resensitization of {beta}2-Adrenergic and Angiotensin II Type 1A Receptors Mol. Endocrinol., December 1, 2000; 14(12): 2040 - 2053. [Abstract] [Full Text]
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J. A. Olivares-Reyes, S. Jayadev, L. Hunyady, K. J. Catt, and R. D. Smith Homologous and Heterologous Phosphorylation of the AT2 Angiotensin Receptor by Protein Kinase C Mol. Pharmacol., November 1, 2000; 58(5): 1156 - 1161. [Abstract] [Full Text]
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Y. Xu, A. S. Clanachan, and B. I. Jugdutt Enhanced Expression of Angiotensin II Type 2 Receptor, Inositol 1,4,5-Trisphosphate Receptor, and Protein Kinase C{epsilon} During Cardioprotection Induced by Angiotensin II Type 2 Receptor Blockade Hypertension, October 1, 2000; 36(4): 506 - 510. [Abstract] [Full Text] [PDF]
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Yi Xu, V. Menon, and B. I Jugdutt Cardioprotection after angiotensin II type 1 blockade involves angiotensin II type 2 receptor expression and activation of protein kinase C-{varepsilon} in acutely reperfused myocardial infarction in the dog: Effect of UP269-6 and losartan on AT1- and AT2-receptor expression and IP3 receptor and PKC{varepsilon} proteins Journal of Renin-Angiotensin-Aldosterone System, June 1, 2000; 1(2): 184 - 195. [Abstract] [PDF]
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A. Oksche, G. Boese, A. Horstmeyer, J. Furkert, M. Beyermann, M. Bienert, and W. Rosenthal Late Endosomal/Lysosomal Targeting and Lack of Recycling of the Ligand-Occupied Endothelin B Receptor Mol. Pharmacol., June 1, 2000; 57(6): 1104 - 1113. [Abstract] [Full Text]
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W. C. De Mello and A. H. J. Danser Angiotensin II and the Heart : On the Intracrine Renin-Angiotensin System Hypertension, June 1, 2000; 35(6): 1183 - 1188. [Abstract] [Full Text] [PDF]
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S. Gallinat, S. Busche, M. K. Raizada, and C. Sumners The angiotensin II type 2 receptor: an enigma with multiple variations Am J Physiol Endocrinol Metab, March 1, 2000; 278(3): E357 - E374. [Abstract] [Full Text] [PDF]
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S. L. Ferrari, V. Behar, M. Chorev, M. Rosenblatt, and A. Bisello Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves beta -Arrestin2. REAL-TIME MONITORING BY FLUORESCENCE MICROSCOPY J. Biol. Chem., October 15, 1999; 274(42): 29968 - 29975. [Abstract] [Full Text] [PDF]
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C. Baratti-Elbaz, N. Ghinea, O. Lahuna, H. Loosfelt, C. Pichon, and E. Milgrom Internalization and Recycling Pathways of the Thyrotropin Receptor Mol. Endocrinol., October 1, 1999; 13(10): 1751 - 1765. [Abstract] [Full Text]
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L. Gendron*, L. Laflamme*, N. Rivard, C. Asselin, M. D. Payet, and N. Gallo-Payet Signals from the AT2 (Angiotensin Type 2) Receptor of Angiotensin II Inhibit p21ras and Activate MAPK (Mitogen-Activated Protein Kinase) to Induce Morphological Neuronal Differentiation in NG108-15 Cells Mol. Endocrinol., September 1, 1999; 13(9): 1615 - 1626. [Abstract] [Full Text]
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K. Nakamura, M. d. F. M. Lazari, S. Li, C. Korgaonkar, and M. Ascoli Role of the Rate of Internalization of the Agonist-Receptor Complex on the Agonist-Induced Down-Regulation of the Lutropin/ Choriogonadotropin Receptor Mol. Endocrinol., August 1, 1999; 13(8): 1295 - 1304. [Abstract] [Full Text]
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J. D. Imig, G. L. Navar, L.-X. Zou, K. C. O'Reilly, P. L. Allen, J. H. Kaysen, T. G. Hammond, and L. G. Navar Renal endosomes contain angiotensin peptides, converting enzyme, and AT1A receptors Am J Physiol Renal Physiol, August 1, 1999; 277(2): F303 - F311. [Abstract] [Full Text] [PDF]
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J. Y. A. Lehtonen, L. Daviet, C. Nahmias, M. Horiuchi, and V. J. Dzau Analysis of Functional Domains of Angiotensin II Type 2 Receptor Involved in Apoptosis Mol. Endocrinol., July 1, 1999; 13(7): 1051 - 1060. [Abstract] [Full Text]
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L. Daviet, J. Y. A. Lehtonen, K. Tamura, D. P. Griese, M. Horiuchi, and V. J. Dzau Cloning and Characterization of ATRAP, a Novel Protein That Interacts with the Angiotensin II Type 1 Receptor J. Biol. Chem., June 11, 1999; 274(24): 17058 - 17062. [Abstract] [Full Text] [PDF]
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B. Adams, T. S. Obertone, X. Wang, and T. J. Murphy Relationship between Internalization and mRNA Decay in Down-Regulation of Recombinant Type 1 Angiotensin II Receptor (AT1) Expression in Smooth Muscle Cells Mol. Pharmacol., June 1, 1999; 55(6): 1028 - 1036. [Abstract] [Full Text]
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Z. Huang, T. Bambino, Y. Chen, J. Lameh, and R. A. Nissenson Role of Signal Transduction in Internalization of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH) and PTH-Related Protein Endocrinology, March 1, 1999; 140(3): 1294 - 1300. [Abstract] [Full Text]
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M. Tamura, Y. Wanaka, E. J. Landon, and T. Inagami Intracellular Sodium Modulates the Expression of Angiotensin II Subtype 2 Receptor in PC12W Cells Hypertension, February 1, 1999; 33(2): 626 - 632. [Abstract] [Full Text] [PDF]
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M. Vrecl, L. Anderson, A. Hanyaloglu, A. M. McGregor, A. D. Groarke, G. Milligan, P. L. Taylor, and K. A. Eidne Agonist-Induced Endocytosis and Recycling of the Gonadotropin-Releasing Hormone Receptor: Effect of {beta}-Arrestin on Internalization Kinetics Mol. Endocrinol., December 1, 1998; 12(12): 1818 - 1829. [Abstract] [Full Text]
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L. Hunyady, H. Ji, G. Jagadeesh, M. Zhang, Z. Gáborik, B. Mihalik, and K. J. Catt Dependence of AT1 Angiotensin Receptor Function on Adjacent Asparagine Residues in the Seventh Transmembrane Helix Mol. Pharmacol., August 1, 1998; 54(2): 427 - 434. [Abstract] [Full Text]
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