Human Angiotensin II Type 1 Receptor Isoforms Encoded by Messenger RNA Splice Variants Are Functionally Distinct

Mickey M. Martin, Barry M. Willardson, Gregory F. Burton, C. Roger White, Joseph N. McLaughlin, Steven M. Bray, James W. Ogilvie, Jr. and Terry S. Elton

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
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human tissues that express the angiotensin II (Ang II) type 1 receptor (hAT1R) can synthesize four distinct alternatively spliced hAT1R mRNA transcripts. In this study, we show that the relative abundance of these mRNA transcripts varies widely in human tissues, suggesting that each splice variant is functionally distinct. Here we demonstrate, for the first time, that the hAT1R-B mRNA splice variant encodes a novel long hAT1R isoform in vivo that has significantly diminished affinity for Ang II (i.e. >3-fold) when compared with the short hAT1R isoform (encoded by hAT1R-A mRNA splice variant). This reduced agonist affinity caused a significant shift to the right in the dose-response curve for Ang II-induced inositol trisphosphate production and Ca2+ mobilization of the long hAT1R when compared with that of the short hAT1R. The functional differences between these isoforms allows Ang II responsiveness to be fine-tuned by regulating the relative abundance of the long and short hAT1R isoform expressed in a given human tissue.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The peptide hormone, angiotensin II (Ang II), the biologically active component of the renin-angiotensin system, regulates a variety of physiological responses including fluid homeostasis, aldosterone production, renal function, and contraction of vascular smooth muscle (1). Additionally, Ang II has been demonstrated to be a growth-promoting factor in cultured rat vascular smooth muscle cells (2, 3, 4), renal mesangial cells (5), cardiomyocytes (6), and cardiac fibroblasts (7). This mitogenic response requires the rapid activation of one or several mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK 1/2), stress-activated C-Jun N-terminal kinases, and p38 mitogen-activated protein kinase (8).

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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tissue Distribution of hAT1R mRNA Splice Variants
Four known distinct alternatively spliced hAT1R mRNAs are synthesized from a single hAT1R gene. These transcripts are comprised of exons 1 and 4 (hAT1R-A); exons 1, 3, and 4 (hAT1R-B); exons 1, 2, and 4 (hAT1R-C); and exons 1, 2, 3, and 4 (hAT1R-D) (Fig. 1Go) (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. 2Go, 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. 2Go 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 1Go. These results suggest that each alternatively spliced hAT1R mRNA may be functionally distinct since they are differentially expressed in these tissues.



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Figure 1. A Schematic Representation of the Four hAT1R mRNA Splice Variants

Human lung 5'-RACE-ready cDNA was PCR amplified using an anchor and hAT1R-specific primers. PCR products were subcloned and sequenced. Four distinct hAT1R cDNA clones were designated (hAT1R-A -> hAT1R-D). Sequence analysis demonstrated that exons 1, 2, and 3 encode 5'-UTR sequence while exon 4 harbors the entire uninterrupted open reading frame for the hAT1R. Exon 3 also harbors a putative translational start site.

 


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Figure 2. Autoradiograms of RT-PCR Experiments Investigating the Relative Expression of hAT1R mRNA Splice Variants

Total RNA was isolated from various human tissues and subjected to RT-PCR analysis as described in Materials and Methods. The expected sizes of the RT-PCR products were 274 bp (product comprised of exons 1 and 4), 333 bp (product comprised of exons 1, 3, and 4), 359 bp (product comprised of exons 1, 2, and 4), and 418 bp (product comprised of exons 1, 2, 3, and 4), respectively. To visualize the amplified products, the PCR reactions were spiked with [{alpha}-32P]dCTP. The linear range of amplification is shown for human adrenal, kidney, and placenta. PCR products were not observed for non-RT samples (data not shown). Each autoradiogram is representative of at least three separate experiments.

 

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Table 1. Comparison of the Relative Percent Expression of the hAT1R mRNA Splice Variants in Various Human Tissues

 
Cell Surface Expression of the Long hAT1R Isoform
To begin to investigate the potential functional differences between the hAT1R mRNA splice variants, we have focused our current study on hAT1R-A mRNA comprised of exons 1 and 4 and hAT1R-B mRNA comprised of exons 1, 3, and 4 (Fig. 1Go). 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. 3Go) (29).



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Figure 3. Putative Amino-Terminal Extension of the Long hAT1R Isoform Encoded by hAT1R mRNAs Containing Exon 3

The nonshaded nucleotide sequence of exon 4 and the first seven amino acids (1 -> 7 labeled in bold type) of the short hAT1R isoform encoded by this sequence is shown. The nucleotide sequence of exon 3 is shaded. If translation is initiated at the AUG codon located in exon 3 (shown in bold type), a long hAT1R isoform with an amino-terminal extension of 32 amino acids would be synthesized. The suboptimal Kozak sequence located in both exons 3 and 4 is designated with a double underline. 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 ).

 
One major obstacle to the detection of in vivo GPCR expression has been the well established inability to produce antibodies that detect the hAT1R by Western blotting or immunoprecipitation (29). To circumvent these experimental shortcomings, the cell surface expression of the long and short hAT1R isoforms was investigated by flow cytometric analysis utilizing an antibody that recognizes both isoforms (anti-long/short hAT1R) or an antibody that recognizes only the long isoform (anti-long hAT1R).

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. 3Go). The results in Fig. 4Go 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.



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Figure 4. Western Blot Analysis of GST-hAT1R Fusion Proteins

Purified GST-long/short hAT1R sequence (MILNSST... ) (10 µg), lanes 1, 3, and 5; and GST-long-specific hAT1R sequence (MNHKSTD... ) (10 µg), lanes 2, 4, and 6 were resolved by 10% SDS-PAGE and analyzed by Western blot with rabbit preimmune, anti-long/short or anti-long antiserum. Positions of molecular mass markers are shown on the left in kilodaltons. The data are representative of three independent experiments.

 
Flow cytometric analysis of Chinese hamster ovary (CHO) cells stably transfected with the pCR/hAT1R-A construct (i.e. only the short receptor is synthesized) demonstrated that they were labeled by the anti-long/short hAT1R antibody (~76% of transfected CHO cells labeled positive), whereas these same cells were not labeled when the anti-long hAT1R antibody was used (Fig. 5AGo). 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).



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Figure 5. Surface Expression of Long and Short hAT1R Isoforms on Transfected CHO or H295-R Cells

The overlay histogram plots show the flow cytometric analysis of CHO cells stably transfected with pCR/hAT1R-A (panel A), pCR/hAT1R-B (panel B), or pCR/hAT1R-mut B (panel C) or human adrenocortical, H295-R (panel D) cells, respectively. Cells were labeled with anti-long/short hAT1R (L/S) (histogram outlined in gray), anti-long hAT1R (L) (histogram outlined in black), or preimmune serum (PI) (shaded histogram) antibodies. The cells were subsequently labeled with fluorescein isothiocyanate-conjugated second antibody and subjected to flow cytometric analysis. Anti-long/short hAT1R will label either receptor isoform whereas anti-long hAT1R will label only the long receptor isoform. The data are representative of three independent experiments.

 
In contrast, CHO cells stably transfected with the pCR/hAT1R-B construct (i.e. harbors potential translation initiation codons in both exons 3 and 4) labeled positive for the long hAT1R. Approximately 42% of the CHO cells that were recognized by the long hAT1R antibody and about 98% of the cells that were recognized by the long/short hAT1R antibody, clearly demonstrating that the AUG harbored in exon 3 can indeed initiate translation in vivo (Fig. 5BGo). However, the hAT1R-B transfected cells were not labeled to the same extent with each antibody (see Fig. 5BGo), 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. 5CGo, 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. 5DGo), 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. 6Go, 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.



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Figure 6. Photoaffinity Labeling of CHO Cells Expressing Aglycosylated hAT1R Isoforms

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 subsequently labeled with [125I][Bpa8]Ang II as described in Materials and Methods. After photolabeling, cellular proteins were solubilized and resolved by 10% SDS-PAGE followed by autoradiography. Lanes 1 and 2, [125I][Bpa8]Ang II-labeled proteins from CHO cells transfected with pcDNA-3/hAT1R-A(N4, 176, 188D) (i.e. only the short aglycosylated hAT1R can be synthesized). Lanes 3 and 4, [125I][Bpa8]Ang II-labeled proteins from CHO cells transfected with pCR/hAT1R-B(N26, 36, 208, 220D) (i.e. both the long and short aglycosylated hAT1R can be synthesized). Lanes 5 and 6, [125I][Bpa8]Ang II-labeled proteins from CHO cells transfected with pCR/hAT1R-mut B(N26, 36, 208, 220D) (i.e. only the long aglycosylated hAT1R can be synthesized). Lanes 2, 4 and 6, Labeling reactions were performed in the presence of 10 µM Losartan, an AT1R selective ligand. Protein standards of the indicated molecular masses (kilodaltons) were run in parallel. These results are representative of three separate experiments.

 
Binding Properties of the Long and Short hAT1R Isoforms
To determine the binding affinities of the long and short hAT1R isoforms, membranes isolated from cells stably transfected with pCR/hAT1R-A or pCR/hAT1R-mut B were subjected to radioreceptor competition binding experiments. The IC50 values from competition binding experiments are summarized in Table 2Go. 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 2Go).


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Table 2. Compared Binding Affinities of Angiotensin Antagonists for Long or Short hAT1R Isoforms Expressed in CHO Cells

 
Functional Properties of the Long and Short hAT1R Isoforms
To begin to assess the functional properties of the long and short hAT1R with respect to the initiation of signal transduction pathways, CHO cell lines stably expressing approximately the same number of receptor (i.e. CHO/long/no. 95, 2,000 fmol/mg vs. CHO/short/no. 151, 1,800 fmol/mg, respectively) were used. Since AT1R activation by Ang II stimulates phosphatidylinositol-specific phospholipase C, we investigated whether Ang II activation of both isoforms stimulated inositol trisphosphate (IP3) production. Importantly, activation of either isoform led to a significant increase in IP3 levels over basal IP3 production (data not shown). Since binding studies revealed significant differences in Ang II affinities for the hAT1R isoforms, a detailed analysis of the dose dependence of their IP3 signaling responses was performed. Representative curves for the IP3 responsiveness of hAT1R isoforms are shown in Fig. 7Go. 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. 7Go). 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 2Go). 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. 8Go). 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.



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Figure 7. Dose-Response Curve for Ang II-Stimulated Production of IP3 from CHO Cells Expressing Long or Short hAT1R Isoforms

Increasing concentrations of Ang II were incubated with CHO cells expressing the long ({square}) 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.

 


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Figure 8. Time Course for Ang II-Stimulated Production of IP3 from CHO Cells Expressing Long or Short hAT1R Isoforms

CHO cells expressing the long ({square}) or short (•) hAT1R were incubated with 10-8 M Ang II at 25 C for the indicated times. Data are shown as means of triplicate determinations ± SE.

 
Since the generation of IP3 and diacylglycerol are involved in intracellular Ca2+ mobilization, Ang II-induced Ca2+ mobilization in the long and short hAT1R stably expressing CHO cells was also examined by measuring the changes in intracellular free Ca2+ using Fura-2 as a Ca2+ indicator. The dose-response curve for Ang II-induced Ca2+ mobilization of the long hAT1R was shifted to the right when compared with that of the short hAT1R, again consistent with the reduced agonist affinity of this isoform (Fig. 9Go). 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.



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Figure 9. Dose-Response Curve for Ang II-Stimulated Ca2+ Activation in CHO Cells Expressing Long or Short hAT1R Isoforms

CHO cells expressing the long ({square}) or short (•) hAT1R were loaded with Fura-2 as described in Materials and Methods. The cells were subsequently incubated with increasing concentrations of Ang II and [Ca2+]i was determined. Percent maximal [Ca2+]i was calculated from the equation: [([Ca2+] – [Ca2+]o)/[Ca2+]max – [Ca2+]o)]*100, where [Ca2+]o is the [Ca2+] in the absence of Ang II, and [Ca2+]max is the [Ca2+] concentration at saturating [Ang II]. Lines represent nonlinear least squares fits of the data to the equation [([Ca2+]max – [Ca2+]o)*[Ang II]/(EC50 + [Ang II)] + [Ca2+]o. EC50 values were determined from the data fit. Data points represent means ± SE of 10 individual experiments.

 
Identical experiments were performed utilizing additional long and short clonal CHO cell lines expressing approximately 400 fmol/mg (CHO/long/no. 3 and CHO/short/no. 126) or 1,000 fmol/mg (CHO/long/no. 47 and CHO/short/no. 154) receptor, respectively (data not shown). Importantly, all of the long hAT1R-expressing clonal cell lines investigated showed a decreased affinity for Ang II, and this reduced agonist affinity resulted in a significant shift to the right in the dose-response curve for Ang II-induced IP3 production and Ca2+ mobilization when compared with that of the short hAT1R-expressing clonal cell lines. Taken together, these results suggest that the observed differences between the long and short hAT1R isoforms (Table 1Go and Figs. 7–9GoGoGo) were not due to clonal variability.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we have investigated the relative distribution of the four known hAT1R mRNA splice variants in human tissues by utilizing RT-PCR. Our results demonstrate, for the first time, that the relative abundance of each transcript varies widely in human tissues, suggesting that hAT1R mRNA splice variants may be functionally distinct (Fig. 2Go and Table 1Go). 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. 3Go). 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. 5Go, A–C, and Fig. 6Go). 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. 5Go, B and C, and Fig. 6Go). 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-{alpha} (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. 3Go). 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 2Go). 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. 7Go and 9Go). The reduced affinity of the long hAT1R for Ang II is not due to the mutation of the translational start site (Met->Ile) located in exon 4, since more dramatic nonconserved amino acid changes (i.e. Met->Ser 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. 7–9GoGoGo). 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. 7Go and 9Go). 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. 1Go), 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Ang I, Ang II, Ang III, and Ang II antagonists were purchased from Sigma (St. Louis, MO). The AT1R nonpeptide antagonist, Losartan, was a generous gift from Merck & Co., Inc. (Rahway, NJ). The L-Bpa8-Ang II was a generous gift from Dr. Emanuel Escher (University of Sherbrooke, Sherbrooke, Quebec, Canada). [125I]Ang II, [125I][Sar1, Ile8]Ang II, and [125I][L-Bpa8]Ang II ligands were iodinated at the Peptide Radioiodination Center (Washington State University, Pullman, WA). Oligonucleotides were purchased from Life Technologies, Inc. (Gaithersburg, MD). The cDNA clone encoding the aglycosylated short hAT1R (i.e. pcDNA3/hAT1R-A mut (N4, 176, 188D) was kindly provided by Dr. Richard Leduc (University of Sherbrooke).

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. 1AGo). 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 [{alpha}-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. 1Go). 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 G->A 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. 3Go). 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 (N->D) 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 N->D. 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 {gamma}-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
 
The authors would like to thank Dr. Daniel Simmons (Brigham Young University) for his contributions in the preparation of this manuscript and Drs. Emanuel Escher and Richard Leduc at the University of Sherbrooke for the [Sar1, Bpa8]Ang II compound and the pcDNA/hAT1R (N4, 176, 188D) expression construct.


    FOOTNOTES
 
Address requests for reprints to: Terry S. Elton, Ph.D., Department of Chemistry and Biochemistry, Brigham Young University, C206 Benson Building, P.O. Box 25700, Provo, Utah 84602-5700. E-mail: terry_elton{at}byu.edu

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.


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