| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Chemistry and Biochemistry (M.M.M., B.M.W., J.N.M.,
S.M.B., J.W.O., T.S.E.) Department of Microbiology (G.F.B.)
Brigham Young University Provo, Utah 84602
University of
Alabama at Birmingham Vascular Biology and Hypertension Program
(C.R.W.) Birmingham, Alabama 35294
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The biological responses to Ang II are mediated by its interaction with high affinity G protein-coupled receptors (GPCRs) localized on the surface of target cells (9). Two main Ang II receptor subtypes, AT1R and AT2R, have been pharmacologically identified (10). AT1R activation by Ang II stimulates phosphatidylinositol-specific phospholipase C, leading to the generation of inositol trisphosphate and diacylglycerol, which are involved in intracellular Ca2+ mobilization (11, 12) and protein kinase C activation (13). AT1R activation by Ang II also stimulates the ERK 1/2 cascade (14); however, the coupling mechanisms between the AT1R and the ERK 1/2 cascade are still incompletely characterized. Recent investigations suggest that Ang II activates ERK 1/2 through transactivation of tyrosine kinase receptors, which appears to be mediated by several nonreceptor tyrosine kinases, including the proline-rich tyrosine kinase 2 (PYK2) and Src family tyrosine kinases (14, 15, 16, 17, 18, 19, 20, 21). Transactivation results in Shc-Grb2-SOS complex formation and RAS activation, which in turn initiates a kinase cascade culminating in ERK 1/2 activation (22, 23). In contrast, the signaling pathways of the AT2R are not well defined. Although the exact physiological function of the AT2R is not clear, studies utilizing vascular smooth muscle cells (24) or coronary endothelial cells (25) suggest that the AT2R inhibits proliferation. Thus, the AT2R may antagonize the growth-promoting effects of the AT1R.
Recently, our laboratory (26, 27) and others (28, 29) have demonstrated that the human AT1R (hAT1R) gene is comprised of at least four exons and spans greater than 60 kilobases (kb). Exons 1, 2, and 3 have been presumed to constitute the 5'-untranslated region (UTR) mRNA sequence, while exon 4 harbors the entire uninterrupted open reading frame, for the hAT1R. A comparison of several published hAT1R cDNA sequences revealed that although these cDNA clones shared identical open reading frames, they differed in portions of their presumed 5'-UTR (30, 31, 32). These results suggested that alternative splicing events combine various 5'-UTR exons (i.e. exons 1–3) with the same coding region exon (i.e. exon 4). In support of this hypothesis, our laboratory demonstrated by 5'-rapid amplification of cDNA ends (RACE) experiments that four distinct hAT1R mRNA splice variants are synthesized in human lung tissues (i.e. hAT1R mRNA transcripts are comprised of exons 1 and 4; exons 1, 3, and 4; exons 1, 2, and 4; or exons 1, 2, 3, and 4) (26, 27). Sequence analysis has shown that an AUG triplet located in exon 3 is in frame with the downstream open reading frame located in exon 4 (29). Therefore, hAT1R mRNA transcripts containing exons 3 and 4 may encode a novel hAT1R with an amino-terminal extension of 32 amino acids (long hAT1R) when compared with the short receptor encoded by exon 1, 4 hAT1R mRNA.
Curnow et al. (29) have previously demonstrated that the exon 1,3,4 hAT1R mRNA transcript was expressed in a number of human tissues. Additionally, they demonstrated that human kidney 293 cells transfected with an exon 1,3,4/hAT1R expression construct produced a functional hAT1R (29). Although these investigators demonstrated that transfected 293 cells express hAT1Rs, they were unable to determine whether these cells were actually expressing the long hAT1R isoform. This is a critical consideration since the AUG codon harbored in exon 3 is not a consensus Kozak translation initiation start site (33). Therefore, it is possible that the hAT1R mRNA exon 1,3,4 splice variant does not encode the long hAT1R, but rather encodes the short hAT1R, since translation may only be initiated at the previously characterized AUG start codon harbored in exon 4 (26, 27, 28, 29). Therefore, the following study was initiated to determine whether the long hAT1R is actually expressed in vivo and, if so, to determine whether the long and short hAT1R isoforms are pharmacologically and functionally distinct.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
)
(26, 27). These alternatively spliced mRNAs differ only in the lengths
of their 5'-UTR encoded by exons 1, 2, and 3 while exon 4 harbors the
open reading frame for the hAT1R. To determine
the relative abundance of each hAT1R mRNA splice
variant, total RNA was isolated from various human tissues and
subjected to RT-PCR analysis as described in Materials and
Methods. A hAT1R-specific amplimer set was
used that allowed for the simultaneous amplification of all four
endogenous hAT1R transcripts. To directly compare
the relative abundance of the hAT1R mRNA splice
variants, RT-PCR reactions were terminated and the products quantified
when the samples were in the linear range of amplification. As shown in
Fig. 2, the hAT1R
PCR products remain in the linear range for only a limited number of
cycles (i.e. human adrenal, 34 cycles; kidney, 34 cycles;
and placenta, 30 cycles). Additionally, the results in Fig. 2 clearly
demonstrate that all four hAT1R splice variants
are expressed in the human tissues investigated; however, each is
expressed in dramatically distinct quantities, relative to one another,
within a given tissue. The relative quantitation of the RT-PCR
experiments are summarized in Table 1.
These results suggest that each alternatively spliced
hAT1R mRNA may be functionally distinct since
they are differentially expressed in these tissues.
|
|
|
). These mRNAs were chosen for
further investigation since sequence analysis demonstrated that exon 3
harbors an AUG translation initiation start site that is in frame with
the major open reading frame located in exon 4. Therefore, if
hAT1R-B mRNA were to be translated, a long
hAT1R isoform with an amino-terminal extension of
32 amino acids would be synthesized when compared with the short
hAT1R encoded by hAT1R-A
(Fig. 3) (29).
|
To establish the specificity of the antibodies to be used in the flow
cytometric experiments, Western blot analyses were performed using
glutathione-S-transferase (GST) fusions of the long/short
hAT1R sequence (MILNSST... ) or the
long-specific hAT1R sequence (MNHKSTD... )
(see Fig. 3). The results in Fig. 4
demonstrate that the anti-long/short hAT1R
antibody cross-reacts exclusively with the GST-long/short fusion
protein (lane 3) while the anti-long antibody only recognizes the
GST-long fusion protein (lane 6). These results show that the
antibodies tested specifically recognize the expected epitope.
|
76% of transfected CHO cells
labeled positive), whereas these same cells were not labeled when the
anti-long hAT1R antibody was used (Fig. 5A). These results demonstrate that
pCR/hAT1R-A transfected CHO cells only express
the short hAT1R isoform. Mock transfected CHO
cells were not labeled with either antibody (data not shown).
|
). However, the
hAT1R-B transfected cells were not labeled to the
same extent with each antibody (see Fig. 5B), suggesting that these
cells were expressing a heterogeneous population of long and short
hAT1R consistent with our hypothesis that the
hAT1R-B mRNA (i.e. exon 1,3,4) is
bicistronic and can initiate translation from the AUG start codon
present in either exon 3 or 4. Thus, these data suggest that both
receptor isoforms can be synthesized from the same mRNA. To test this
hypothesis, the AUG start codon in exon 4 of
pCR/hAT1R-B was mutated to AUA (see
Materials and Methods). This new expression construct was
designated pCR/hAT1R-mut B. As seen in Fig. 5C, pCR/hAT1R-mut B-transfected CHO cells showed a
significant increase in the number of cells that were recognized by the
long hAT1R antibody (71%), supporting the
hypothesis that the hAT1R-B mRNA is
bicistronic.
To further investigate whether the long hAT1R
isoform was expressed in vivo, a human adrenocortical
carcinoma-derived (H295-R) cell line that expresses high levels of
hAT1Rs (39) was also subjected to flow cytometric
analysis. Importantly, approximately 42% of H295-R cells labeled
positive for the long hAT1R isoform (Fig. 5D),
supporting our hypothesis that the start codon harbored in exon 3 can
initiate translation in vivo.
To validate the results obtained from the flow cytometric
experiments, CHO cells were transiently transfected with expression
constructs [pcDNA3/hAT1R (N4, 176, 188D),
pCR/hAT1R-B(N26, 36, 208, 220D), or
pCR/hAT1R/hAT1R-mut B(N26,
36, 208, 220D)] that generate aglycosylated
hAT1Rs in vivo (see Materials
and Methods). Transfected CHO cells were subsequently
photoaffinity labeled with
[125I][Bpa8]Ang II and
analyzed by SDS-PAGE and autoradiography. As shown in Fig. 6, a radiolabeled protein(s) was
identified (lanes 1, 3, and 5) in transfected CHO cells. The labeling
of the AT1R by the photoreactive analog was
completely abolished by 10 µM Losartan, an
AT1R selective antagonist (lanes 2, 4, and 6),
thereby confirming the specificity and selectivity of the labeling.
Since the expressed hAT1Rs are not glycosylated,
the Mr of long hAT1R (lane
5) and the short hAT1R (lane 1) are clearly
distinguishable, approximately 38 kDa vs. approximately 34
kDa. Importantly, an aglycosylated hAT1R-B mRNA
results in the synthesis of both the long and short
hAT1R isoforms (lane 3), again supporting the
hypothesis that this splice variant is bicistronic.
|
. These results demonstrate that the
long hAT1R has a significantly diminished
affinity for Ang II, and most of the Ang II antagonists investigated,
when compared with the short hAT1R
(IC50, 19.8 ± 2.2 nM and
5.9 ± 0.3 nM, P < 0.001, for Ang II,
respectively). Interestingly, the AT1R-selective
nonpeptide antagonist, Losartan, cannot discriminate between the two
hAT1R isoforms (Table 2).
|
. The dose-response curve for Ang
II-induced IP3 production of the long
hAT1R was shifted to the right when compared with
that of the short hAT1R, consistent with the
reduced agonist affinity of this isoform (Fig. 7). The
EC50 values determined from three separate
experiments for the long and short hAT1Rs were
16.5 ± 5.4 nM and 2.44 ± 1.11
nM (P < 0.001), respectively.
The EC50 values for IP3
responses of long and short hAT1R showed good
correlation with the respective IC50 values for
inhibition of radioligand binding by Ang II (Table 2). Time course
experiments of Ang II-induced IP3 stimulation
utilizing stably transfected CHO cells demonstrated that at
subsaturating amounts of Ang II, (10-8
M), the rate of IP3
production was significantly less in cells expressing the long
hAT1R isoform (Fig. 8). These data suggest that although both
receptor isoforms stimulate IP3 production, the
long hAT1R will produce a diminished Ang II
response at low concentrations of Ang II. In contrast, at higher
concentrations of Ang II, the IP3 production
elicited by both receptor isoforms was essentially identical. At
10-6 M Ang II, a decrease
in [IP3] was observed after the initial
response (data not shown). This phenomenon has been previously observed
(40) and is caused, at least in part, by the inactivation of the
receptor. The inactivation mechanism involves receptor phosphorylation,
ß-arrestin binding, and receptor internalization (41). This process
appears to be equivalent in both the long and short
hAT1R isoforms.
|
|
). The EC50 values
determined from 10 separate experiments for the long and short
hAT1Rs were 3.2 ± 0.45 nM and
1.0 ± 0.21 nM (P < 0.001),
respectively.
|
and
Figs. 7–9) were not due to clonal variability. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and Table 1). Alternative splicing of GPCR pre-mRNA is
a mechanism observed in many receptor subtypes to further increase the
number of receptor isoforms that can be synthesized from a single
receptor gene. For example, within the GPCR superfamily, alternative
splicing may produce structural variation in the amino terminus
[pituitary adenylate cyclase-activating polypeptide (PACAP) receptor
(42)], the third intracellular loop [dopamine
D2 receptor (43)] or the carboxyl terminus [PG
EP3 receptor (44)]. Structural variation produced by alternative
splicing in GPCRs may result in differences in ligand specificity (42),
modes of signal transduction (43, 44), G protein coupling efficiency
(43), and desensitization (44).
We have focused our current study on the potential functional
differences between two of these mRNA splice variants,
hAT1R-A (comprised of exons 1 and 4) and
hAT1R-B (comprised of exons 1, 3, and 4) since
hAT1R mRNAs containing exons 3 and 4 encode a
novel hAT1R with an amino-terminal extension of
32 amino acids (long hAT1R) when compared with
the short hAT1R encoded by mRNA comprised of
exons 1 and 4 (Fig. 3). According to the scanning model of eukaryotic
translation (33), the 40S ribosomal subunit with its associated factors
engages the mRNA at or near the cap and then scans in a 3'-direction.
Upon encountering the first AUG initiation codon in an optimal context,
the 60S subunit joins the 40S subunit to form a complete 80S ribosome,
and polypeptide synthesis commences. An optimal sequence,
GCCRCCAUGG, for initiator codons has been defined by a
survey of vertebrate mRNAs (33). The most important positions for
efficient translation are a purine at position –3 and a G at position
+4 where A of the AUG codon is defined as position +1 (33). If these
features are absent, the initiation codon is said to be in a suboptimal
context; therefore, the 40S ribosomal complex may skip the first
initiation codon from the 5'-end and begin translation at a subsequent
AUG (45). Alternatively, the suboptimal AUG may be inefficiently
recognized; thus, a fraction of the 40S ribosomal complexes would
initiate translation while another portion of these complexes would
ignore the first AUG and proceed to a subsequent optimal AUG
(i.e. a leaky scanning mechanism). This scenario would
result in two or more proteins being synthesized by the translation of
a single mRNA. The relative abundance of the multiple products would be
determined by the relative strength of the initiation sites.
With respect to the AUG codon harbored in exon 3 of the
hAT1R-B mRNA splice variant, a purine is at
position –3; however, an A is at position +4 (29); thus, this
initiation codon is in a suboptimal context. Our data demonstrate that
although the AUG in exon 3 is in a suboptimal context, translation is
initiated at this start codon in CHO cells stably, or transiently,
transfected with expression constructs that synthesize the
hAT1R-B or hAT1R-mut B
splice variants (Fig. 5, A–C, and Fig. 6). More importantly, flow
cytometric experiments demonstrated that a nontransfected human
adrenocortical carcinoma-derived cell line, H295-R, expressed the long
hAT1R isoform, suggesting that the long
hAT1R isoform is synthesized in human tissues
from endogenously expressed hAT1R-B mRNA.
Additionally, our flow cytometric and photo cross-linking data suggest
that the hAT1R-B mRNA splice variant is
bicistronic since it encodes both the long and short
hAT1R isoform (Fig. 5, B and C, and Fig. 6). The
synthesis of both isoforms by this mRNA may occur through a leaky
scanning mechanism since the AUG codon harbored in exon 3 is in a
suboptimal context or through an internal ribosome entry site.
Competition binding experiments utilizing membranes isolated from CHO
cells transfected with the pCR/hAT1R-B expression
construct always resulted in displacement curves intermediate
(IC50, 12.1 ± 0.9 nM
vs. 19.8 ± 2.2 nM for the long
and 5.9 ± 0.3 nM for the short
hAT1R, data not shown) of CHO cells expressing
homogeneous populations of long or short hAT1R
isoforms, suggesting that the relative abundance of the isoforms being
synthesized from this splice variant is approximately equal.
Additionally, these data also suggest that the Met
Ile mutation
present in the long hAT1R encoded by the
pCR/hAT1R-mut B construct is not responsible for
the decreased affinity for Ang II since shifts in the displacement
curves were also observed with pCR/hAT1R-B
transfected cells (IC50 12.1 ± 0.9
nM vs. 5.9 ± 0.3
nM for the short hAT1R,
data not shown).
The bicistronic nature of the hAT1R-B mRNA splice
variant is significant because it is atypical of eukaryotic transcripts
(46). Polycistronic transcripts are well described in prokaryotes where
they commonly encode proteins involved in the same functional pathway,
thereby constituting an operon (47). Examples of eukaryotic bicistronic
mRNAs are nearly exclusively known for viral systems. However, there
are increasing reports of mammalian bicistronic mRNAs including
molybdopterin synthase subunits MOCO1-A and MOCO1-B (48), osteogenic
growth peptide (49), c-myc (50), fibroblast growth factor-2
(51), SNRPN (small nuclear ribonucleoprotein N) (52), and
CCAAT/enhancer binding protein-
(53). One possible reason for the
development of such gene fusions in mammals might be the colinear
regulation of gene expression and also to produce guaranteed proximity
of interacting proteins. This may be a critical consideration in light
of the recent observations that some GPCRs can undergo homo- and
heterodimerization (54, 55, 56, 57). Therefore, the coexpression of both
hAT1R isoforms may result in a receptor comprised
of a heterodimer that may be functionally distinct from the
hAT1R monomer.
To accurately assess the pharmacological properties of the long and
short hAT1R isoforms, we used expression
constructs that would synthesize homogenous populations of either the
long or short hAT1R isoform. This was
accomplished by mutating the translational start site located in exon 4
of the hAT1R-B mRNA splice variant (see Fig. 3).
Our binding data, utilizing membranes isolated from CHO cells stably
transfected with pCR/hAT1R-A or
pCR/hAT1R-mut B expression constructs
demonstrated that the long hAT1R isoform has a
diminished affinity for Ang II (>3-fold), when compared with the short
hAT1R isoform (Table 2). Additionally, the
dose-response curve for Ang II-induced IP3
production and Ca2+ mobilization of the long
hAT1R was shifted to higher Ang II concentration
when compared with that of the short hAT1R,
consistent with the reduced agonist affinity of this isoform (Figs. 7 and 9). The reduced affinity of the long hAT1R
for Ang II is not due to the mutation of the translational start site
(MetIle) located in exon 4, since more dramatic nonconserved amino
acid changes (i.e. MetSer or Asp) did not alter the
IC50 of the long isoform for Ang II (data not
shown).
It was recently demonstrated from two complementing, yet independent, lines of mutagenesis that the binding of Ang II involves a number of discontinuously located residues in the extracellular domain of the rat AT1AR sequence, particularly at the N-terminus adjacent to the first transmembrane domain (TM-I), a tyrosine in extracellular loop 1, and two neighboring aspartate residues in the C-terminal part of the third extracellular loop (58). Taken together with our data, it is reasonable to speculate that the addition of 32 amino acids to the N terminus of the long hAT1R would interfere with the important Ang II binding site located in the N terminus of the short hAT1R (i.e. equivalent to the rat AT1AR), therefore reducing this isoform’s affinity for Ang II. Alternatively, the reduced affinity for Ang II may result from the N-linked glycosylation of Asn26 present in the N terminus of the long hAT1R. Preliminary competition binding experiments utilizing membranes isolated from CHO cells transfected with an expression construct which generates a long hAT1R isoform that cannot be glycosylated at Asn26 (i.e. Asn was mutated to Gln) demonstrated an increased affinity for Ang II (IC50 of 11.9 ± 1.3 nM vs. 19.8 ± 2.2 nM). Thus, these data suggest that glycosylation of Asn26 can reduce the affinity of the long hAT1R for Ang II, but glycosylation cannot entirely account for the decrease in affinity since the short receptor has an IC50 of 5.9 ± 0.3 nM.
Interestingly, we have also demonstrated that the long and short hAT1R isoforms bind the nonpeptide AT1R-specific antagonist, Losartan, with identical affinities. These results suggest that the Losartan binding epitopes in the AT1R are distinct from Ang II and that these contact points are not compromised by the additional 32 amino acids present in the long hAT1R isoform. In support of this hypothesis, mutagenesis studies demonstrated that residues located deep within TM-III, IV, VI, and VII, particularly Asn295 in TM-VII, impaired the binding of Losartan without affecting Ang II binding (59, 60).
To investigate potential functional differences between the long and
short hAT1R isoforms, several steps in the Ang
II-induced signal transduction pathways were investigated (
Figs. 7–9).
Our results suggest that both receptor isoforms can be activated by Ang
II to stimulate IP3 production and mobilize
Ca2+. Importantly, however, the reduced affinity
of the long hAT1R for Ang II leads to significant
decreases in the amount of IP3 and
Ca2+ produced at subsaturating levels of Ang II
(i.e. 10-8
M) (Figs. 7 and 9). Taken together, our results
clearly demonstrate that dependent upon the concentration of Ang II,
the percentage of the long and short hAT1R
isoforms activated will differ, and therefore the potency of the Ang II
response will vary.
Since the only difference between the hAT1R-A
(encodes the short hAT1R isoform) and the
hAT1R-B (encodes the long
hAT1R isoform) mRNA splice variants is the
presence or absence of exon 3 (Fig. 1), we computer analyzed this
sequence utilizing the BLAST program (National Center for Biotechnology
Information, Bethesda, MD) to determine whether
AT1R cDNAs cloned from other animal species
(bovine, dog, mouse, pig, rabbit, rat, sheep, and Xenopus)
shared homology with the hAT1R exon 3 mRNA
sequence. Interestingly, none of these AT1R cDNAs
harbored sequences that were homologous to exon 3 (data not shown).
This analysis suggests that the long and short
hAT1R isoforms described in this study may be a
human-specific phenomenon even though AT1R mRNA
splice variants have been described in other species
(61).
In summary, our results support the hypothesis that in human tissues, Ang II responsiveness can be fine-tuned by regulating the relative expression of the long and short hAT1R in a given tissue. Since the short hAT1R has the highest affinity for Ang II, abnormally high expression of this receptor would lead to exaggerated Ang II responsiveness. Therefore, aberrant regulation of the production of hAT1R isoforms may play a pivotal role in the pathogenesis of hypertension as well as other cardiovascular disorders such as cardiac hypertrophy, coronary artery disease, and atherosclerosis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cell Culture
CHO cells [American Type Culture Collection
(ATCC), Manassas, VA] were grown in DMEM (Life Technologies, Inc.) supplemented with 10% FBS (HyClone Laboratories, Inc. Logan, UT), 80 U/ml penicillin/80 µg/ml
streptomycin (Life Technologies, Inc.), and 0.0175 mg/ml
L-proline (Sigma). Stably transfected CHO cell
lines were propagated in the same medium with 1,000 µg/ml geneticin
(G418, Life Technologies, Inc.). Human adrenocortical
carcinoma-derived (H295-R) cells (ATCC) were maintained in
DMEM/F12 medium supplemented with 5% NuSerum (Collaborative
Biomedical, Becton Dickinson and Co., Bedford, MA)
and ITS supplement (Collaborative Biomedical). Cell lines were
maintained in a humidified atmosphere at 5% CO2
and 37 C.
RT-PCR
Total RNA was isolated from various human tissues obtained from
the Cooperative Human Tissue Network (Cleveland, OH) utilizing
Tri-Reagent (Molecular Research Center, Inc., Cincinnati,
OH). First-strand cDNA synthesis was performed using SuperScript II
Reverse Transcriptase (Life Technologies, Inc.) and random
primers, according to the manufacturer’s protocol. A sense primer was
synthesized (5'-ACCCGCACCAGCGCAGCCGGCCCTC-3') that corresponded to
hAT1R nucleotide sequence +194 to +219 bp, with
respect to transcription initiation. An antisense primer was
synthesized (5'-CATAGCTGTGTAGACAGCCCATAGTG-3') that corresponded to
nucleotide sequence +709 to +683 bp (i.e. exon 4, see Fig. 1A). PCR experiments were performed utilizing various human tissue cDNA
templates and the above described amplimer set. The PCR reactions were
spiked with 1 µl of [-32P]dCTP (10 µCi
of 3,000 Ci/mM). The linear range of amplification for each tissue was
determined by removing 20 µl of the 100 µl reaction volume at
various cycles. The radiolabeled PCR products were separated by 6%
polyacrylamide gel electrophoresis at 250 V for 3 h. The gel was
then transferred to 3 mm paper, dried, and quantified by
phosphorimaging. The expected sizes of the RT-PCR products were 274 bp
(exons 1 and 4), 333 bp (exons 1, 3, and 4), 359 bp (exons 1, 2, and 4)
and 418 bp (exons 1, 2, 3, and 4), respectively.
Generation of hAT1R Expression
Constructs
To amplify alternatively spliced hAT1R
mRNAs, total RNA was isolated from H295-R cells (ATCC)
using Tri-Reagent (Molecular Research Center, Inc.,
Cincinnati, OH) following the manufacturer’s protocol. First-strand
cDNA synthesis was performed utilizing Superscript II reverse
transcriptase (Life Technologies, Inc.) and oligo
(dT)12-18. PCR experiments
were performed using H295-R cDNA as template and an amplimer set
specific for the hAT1R. The
hAT1R sense primer synthesized
(5'-ACTTCCAGCGCCTGACAGCCAGG-3') corresponded to nucleotide sequence +1
to +23 bp, with respect to transcription initiation. The
hAT1R antisense primer synthesized
(5'-TCACTCAACCTCAAAACATGGTGC-3') corresponded to nucleotide sequence
+1,362 to +1,338 bp, which encompasses the hAT1R
stop codon. The amplified PCR products were subcloned into the
eukaryotic expression vector, pCR3.1 (Invitrogen,
Carlsbad, CA) and sequenced to ensure authenticity and proper
orientation with respect to the cytomegalovirus (CMV) immediate-early
promoter. Two distinct cDNA clones were isolated and characterized that
corresponded to the hAT1R-A (i.e.
comprised of exons 1 and 4) and hAT1R-B
(i.e. comprised of exons 1, 3, and 4) alternatively spliced
mRNA transcripts (Fig. 1). These expression constructs were designated
pCR/hAT1R-A and
pCR/hAT1R-B. The expression construct designated
pCR/hAT1R-mut B was generated in a cDNA
containing exons 1, 3, and 4 by site-directed mutagenesis of the AUG
translational start codon located in exon 4 to AUA using the QuikChange
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).
Briefly, a forward mutagenic primer
(5'-CAGGTGATCAAAATAATTCTCAACTCT-3') and a complementary
reverse mutagenic primer (5'-AGAGTTGAGAATTATTTTGATCACCTG-3')
were synthesized (the mutant base is shown in bold type) and
used in a PCR experiment containing pCR/hAT1R-B
template. Twelve PCR cycles were performed, and the amplification
reaction was treated with the DpnI restriction enzyme to
eliminate the parental DNA template. Competent XL-1 Blue cells were
then transformed with DpnI-treated DNA and plated on
LB-ampicillin agar plates. Ampicillin-resistant colonies were
subsequently grown, plasmid DNA was isolated, and the GA mutation
was confirmed by dideoxy chain termination sequencing.
Generation of CHO Cells Stably Expressing the
hAT1R
The eukaryotic expression constructs,
pCR/hAT1R-A, pCR/hAT1R-B,
and pCR/hAT1R-mut B, were individually
transfected into CHO cells by electroporation (400V, 500 µF, 0.4 cm
cuvette). Pure clonal cell lines were selected by the antibiotic, G418
(1000 µg/ml, Life Technologies, Inc.) and purified by
the limited dilution technique (27). These clonal cell lines were
subsequently assayed for hAT1R expression by
[125I][Sar1,Ile8]AngII
radioreceptor binding experiments. CHO cells that were stably
expressing the hAT1R (e.g. 400–2,000
fmol/mg protein) were selected for further study. Nontransfected CHO
cells do not express AT1Rs.
Western and Flow Cytometric Analysis of
hAT1R Expression
A peptide was synthesized that corresponded to the first 15
amino acids of the long hAT1R isoform (see Fig. 3). A cysteine residue was added at the carboxyl end to facilitate
cross-linking of hemocyanin. Rabbits were immunized with this
conjugated peptide using a standard immunization protocol (Genemed
Biotechnologies, Inc., San Francisco, CA). Peptide-specific antibody
(designated anti-long hAT1R) was obtained by
purifying the sera over a long hAT1R-specific
peptide affinity column. Anti-long/short (N10) antibody, synthesized
against the first 10 amino acids of the short
hAT1R, was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-long/short
hAT1R antibody will react with both the long and
short hAT1R since this amino acid sequence is
present in either receptor.
Western blot analysis was performed utilizing GST-long/short or GST-long hAT1R fusion proteins. These GST fusion proteins were generated by subcloning PCR products corresponding to the first 32 amino acids of the long hAT1R or short hAT1R into the pGEX-4T-3 vector (Amersham Pharmacia Biotech, Piscataway, NJ). The new constructs were sequenced to ensure that the hAT1R fusion proteins were in frame with the GST. The GST fusion proteins were overexpressed in BL-21 DE3 bacteria, induced with 0.1 mM isopropyl-ß-D-thio galactopyranoside (Calbiochem), and purified utilizing glutathione agarose (Sigma), eluting with 10 mM reduced glutathione in 100 mM Tris, pH 8.0, on a Biological chromatography system (Bio-Rad Laboratories, Inc. Hercules, CA). Purified GST-long/hAT1R N terminus (1 µg) and GST-short hAT1R N terminus (1 µg) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked by incubation for 30–60 min in 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Tween-20 (TBS-T) with 5% (wt/vol) nonfat dry milk. Blots were incubated overnight at 4 C in primary antibody (1:1,000) in TBS-T with 5% milk. The membranes were then washed and incubated with a horseradish peroxidase-conjugated goat antirabbit secondary antibody at 1:1,000 dilution in TBS-T. Blots were developed utilizing ECL Western detection system (Amersham Pharmacia Biotech).
CHO cells (1 x 106) stably transfected with either pCR/hAT1R-A, pCR/hAT1R-B, pCR/hAT1R-mut B, or H295-R cells were subjected to flow cytometric analysis. Cells (1 x 106) were washed and labeled with either anti-long or anti-long/short hAT1R antibodies or preimmune serum for 30 min at 4 C. Cells were then washed with PBS and incubated with 50 µl of a 1:35 dilution [complete RPMI-1640 (Life Technologies, Inc.) was used as the diluent] of fluorescein isothiocyanate-conjugated donkey antirabbit antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min at 4 C. Cells were again washed and resuspended in 1 ml PBS and subsequently analyzed on a Coulter EPICS-XL cytometer (Coulter Corp., Hialeah, FL) equipped with System II software. Immediately before examination, 10 µl propidium iodide solution were added to each tube to allow discrimination of dead cells. Control experiments were performed as described above utilizing mock-transfected CHO cells.
Construction of hAT1R Glycosylation
Mutants and Photoaffinity Labeling
The three N-glycosylation sites (N4, 176, and 188) present in
the short hAT1R were previously modified (ND)
by site-directed mutagenesis and functionally characterized by Lanctot
et al. (35). These investigators designated their expression
construct pcDNA3/hAT1R(N4, 176, 188D). For ease
of discussion in this manuscript, we have renamed this construct
pcDNA3/hAT1R-A(N4, 176, 188D). To generate a long
aglycosylated hAT1R, the
pCR/hAT1R-B and
pCR/hAT1R-mut B expression constructs were used
as template, and the N-glycosylation sites were mutated as previously
described (35). Since the long hAT1R harbors one
additional N-glycosylation site (N-26) in the amino-terminal extension,
this site was also modified ND. Due to the fact that the long
hAT1R has the amino-terminal extension, the
designation of the mutated Asn residues (N26, 36, 208, 220D) is
different than those of the short hAT1R even
though the same Asn residues are mutated. The new mutant expression
constructs have been designated pCR/hAT1R-B(N26,
36, 208, 220D) and pCR/hAT1R-mut B(N26, 36, 208,
220D).
CHO cells were transiently transfected with either pcDNA3/hAT1R-A(N4, 176, 188D), pCR/hAT1R-B(N26, 36, 208, 220D), or pCR/hAT1R-mut B(N26, 36, 208, 220D) and the cells were subsequently photolabeled as previously described (35, 36). Briefly, cells were incubated with 5 nM [125I][L-Bpa8]Ang II in the presence or absence of 10 µM Losartan (an AT1R-selective nonpeptide analog) in 1 ml of medium containing 25 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2, and 0.1% BSA. After 1 h incubation at room temperature, cells were washed twice with PBS and irradiated for 60 min at 0 C under filtered UV light (365 nm). Labeled cells were then solubilized in 150 µl of medium containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The supernatant was mixed with an equal volume of 2x Laemmli solution and incubated for 60 min at 37 C followed by electrophoresis on a 10% polyacrylamide gel at 200 V. Gels were dried under vacuum and subjected to autoradiography.
Ang II Binding to CHO Cell Membranes
Membranes were isolated from CHO cells stably expressing the
hAT1R isoforms as previously described (37).
Membrane pellets were resuspended in binding buffer (i.e.
PBS containing 100 µg/ml PMSF) and protein concentration was
determined. Competition binding experiments were performed utilizing
20–30 µg of membrane protein samples incubated with 0.5
nM
[125I][Sar1,
Ile8]Ang II and increasing concentrations
(10-11 to 10-5
M) of various angiotensin antagonists as
indicated. These experiments were conducted at 25 C for 60 min in a 300
µl total volume of PBS, 0.5% BSA, 100 µg/ml PMSF, 1 µg/ml
leupeptin, and 1 µg/ml aprotinin. The membrane-bound radioligand was
separated from the free radioligand by filtration over glass filters
(GF/B) using a cell harvester (Brandel, Inc., Gaithersburg, MD), and
radioactivity was measured by 
-spectrometry. The
IC50 values of the displacement curves were
estimated by nonlinear least squares curve fitting using the computer
program Kaleidagraph (Synergy Software, Reading, PA).
Inositol Trisphosphate Determination
CHO cells stably expressing the long or short
hAT1R isoforms were seeded in six-well plates and
grown to 80–90% confluence (24–36 h). Cells were washed and
incubated for 72 h in serum-free medium and treated with
increasing concentrations of Ang II as indicated for 15 sec in 600 µl
of serum-free medium. For time course experiments, cells were incubated
0–120 sec at the Ang II concentrations indicated. After exposure to
Ang II, the reaction was stopped by the addition of 120 µl of 100%
ice-cold trichloroacetic acid to the plates. The plates were
immediately placed on ice and cells were harvested by scraping and
transferring to polyethylene tubes. Cell extract was centrifuged for 10
min at 14,000 x g. IP3 levels in
the resulting supernatant were determined using a
[3H]radioreceptor-based kit from NEN Life Science Products-DuPont (Boston, MA) following the
manufacturer’s protocol.
Measurement of Intracellular Ca2+
Concentration
([Ca2+]i)
[Ca2+]i was
measured using the Ca2+-sensitive dye, Fura-2
(Molecular Probes, Inc., Eugene, OR) (38). CHO cells
expressing either the long or short hAT11R
isoform were grown to confluence (5 x 106
cells) on 100-mm diameter dishes and harvested using HBSS with 2
mM EDTA and 1% BSA. Cells were rinsed once with serum-free
DMEM and incubated for 30 min at 37 C in the same medium with 2
µM Fura-2/AM, which was dissolved in dimethyl sulfoxide.
The resulting Fura-2-loaded cells were washed once with serum-free DMEM
and 1% BSA, and then resuspended in the same medium. Approximately
106 cells/ml were used in each experiment. After
stimulation with increasing concentrations of Ang II, fluorescence was
measured using a spectrofluorimeter (Fluorolog model, Spex Industries,
Edison, NJ) with excitation at 340 and 380 nM and
emission at 500 nM.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This work was supported by NIH Research Grant 48848.
Received for publication March 13, 2000. Revision received May 24, 2000. Accepted for publication November 1, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
isoforms by angiotensin II in vascular smooth muscle cells. Role of
q/11, 12, and ß G protein subunits. J Biol Chem 273:19772–19777Ser mutations at Cys101 and Cys236. J
Biol Chem 274:11629–11634This article has been cited by other articles:
 |
|
 |
|
  M. M. Martin, J. A. Buckenberger, J. Jiang, G. E. Malana, G. J. Nuovo, M. Chotani, D. S. Feldman, T. D. Schmittgen, and T. S. Elton The Human Angiotensin II Type 1 Receptor +1166 A/C Polymorphism Attenuates MicroRNA-155 Binding J. Biol. Chem., August 17, 2007; 282(33): 24262 - 24269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. S. Elton and M. M. Martin Angiotensin II Type 1 Receptor Gene Regulation: Transcriptional and Posttranscriptional Mechanisms Hypertension, May 1, 2007; 49(5): 953 - 961. [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. Yates and M. Stafford-Smith The genetic determinants of renal impairment following cardiac surgery. Seminars in Cardiothoracic and Vascular Anesthesia, December 1, 2006; 10(4): 314 - 326. [Abstract] [PDF]
|
|
|
|
 |
|
 R. T. Cowling, X. Zhang, V. C. Reese, M. Iwata, D. Gurantz, W. H. Dillmann, and B. H. Greenberg Effects of cytokine treatment on angiotensin II type 1A receptor transcription and splicing in rat cardiac fibroblasts Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1176 - H1183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wu, H. Kamma, M. Fujiwara, Y. Yano, H. Satoh, H. Hara, T. Yashiro, E. Ueno, and Y. Aiyoshi Altered Expression Patterns of Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in the Adrenal Cortex J. Histochem. Cytochem., April 1, 2005; 53(4): 487 - 495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kanaide, T. Ichiki, J. Nishimura, and K. Hirano Cellular Mechanism of Vasoconstriction Induced by Angiotensin II: It Remains To Be Determined Circ. Res., November 28, 2003; 93(11): 1015 - 1017. [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhou, Y. Chen, W. P. Dirksen, M. Morris, and M. Periasamy AT1b Receptor Predominantly Mediates Contractions in Major Mouse Blood Vessels Circ. Res., November 28, 2003; 93(11): 1089 - 1094. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Johren, C. Golsch, A. Dendorfer, F. Qadri, W. Hauser, and P. Dominiak Differential Expression of AT1 Receptors in the Pituitary and Adrenal Gland of SHR and WKY Hypertension, April 1, 2003; 41(4): 984 - 990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. McLaughlin, C. D. Thulin, S. M. Bray, M. M. Martin, T. S. Elton, and B. M. Willardson Regulation of Angiotensin II-induced G Protein Signaling by Phosducin-like Protein J. Biol. Chem., September 13, 2002; 277(38): 34885 - 34895. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
) or short (•) hAT1R for 15 sec.
IP3 production was measured as described in
Materials and Methods. Percent maximal IP3
responses were calculated from the equation: [([IP3] –
[IP3]o)/[IP3]max –
[IP3]o)]*100, where
[IP3]o is the [IP3] in the
absence of Ang II and [IP3]max is the
[IP3] concentration at saturating [Ang II].
Lines represent nonlinear least squares fits of the data
to the equation [([IP3]max –
[IP3]o)*[Ang II]/(EC50 + [Ang
II])] + [IP3]o. EC50 values
were determined from the data fit. Data points represent means ±
SE of triplicate determinations. A representative example
of three experiments with similar results is shown.
