Introduction

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Angiotensins have long been known as critical regulators of cardiovascular functions and body water homeostasis and have been the focus of extensive studies for several decades. Recently, studies of the structure-function relationships of the angiotensin peptides have lead to the discovery that angiotensin IV (AngIV), a hexapeptide angiotensin fragment, binds a unique cell surface receptor (AT₄) (Swanson et al., 1992) that modulates specific physiological functions (Wright et al., 1995). Since its discovery, the ever-expanding list of physiological functions attributed to the AngIV/AT₄ system indicates that it plays essential physiological roles that are distinct from those of the classical angiotensins, angiotensin II (AngII) and angiotensin III (AngIII). Among the first discovered was the enhancement of cognitive function as evidenced by augmented memory retrieval and retention in passive avoidance tests (Braszko, 1988; Wright et al., 1993) and the improvement of spatial learning tasks in cognitively compromised rats (Pederson et al., 1998, Stubley-Weatherly et al., 1996). Furthermore, it has been shown that the AT₄ receptor mediates various aspects of renal function including Na⁺ excretion in rat proximal tubules (Handa et al., 1998) and redistribution of blood within the renal cortex (Coleman et al., 1998). In addition to the brain and kidney, recent data suggest that the AngIV/AT₄ system regulates pertinent physiological functions in other organs and tissues. In the heart AngIV, which exhibits apparent antihypertrophic actions, attenuates AngII-dependent increases in protein and RNA synthesis (Baker and Aceto, 1990) and dramatically inhibits increases in immediate early gene expression that accompany mechanical loading (Yang et al., 1997). AT₄ agonists have also been shown to inhibit neurite outgrowth in sympathetic ganglia (Moeller et al., 1996b), increase cerebral blood flow (Kramár et al., 1997, 1998), and increase plasminogen activator inhibitor I expression in vascular endothelial cells (Kerins et al., 1995). Taken together, the physiological functions that are sensitive to AT₄ activators or inhibitors are extraordinarily diverse.

In addition to functional diversity, the AT₄ receptor is characterized by broad tissue and species distributions. The AT₄ receptor has been identified in all mammalian species so far examined including rat, rabbit, guinea pig, monkey, human, and bovine (Wright et al., 1995; Moeller et al., 1996a). The AT₄ receptor is present in a variety of tissues with particularly high concentrations in heart, kidney, vasculature, thymus, bladder, aorta, adrenals, and many regions of the brain (Hanesworth et al., 1993; Hall et al., 1993, 1995; Miller-Wing et al., 1993; Bernier et al., 1994; Moeller et al., 1995).

The AT₄ receptors differ from other angiotensin receptors (AT₁ and AT₂) in terms of tissue distribution, function, and binding affinity to a variety of angiotensin analogs. The AT₁ receptor has high binding affinity for AngII, AngIII, and losartan, whereas the AT₂ receptor, in addition to AngII and AngIII, exhibits higher affinity for CGP 4211A, PD 123177, and related substances. Both bind AngIV with low affinity. Conversely, the AT₄ receptor shows high binding affinity for AngIV and its analogs, but poor affinity to AngII, losartan, CGP 4211A, and PD 123177 (Swanson, 1992). Recent studies on the bovine adrenal AT₄ receptor, in which a particularly higher density of the AT₄ receptor is found, indicate that the AT₄ receptor is structurally different from other known angiotensin receptors (Zhang et al., 1998). Based on native polyacrylamide gel electrophoresis (PAGE) analysis and, reducing and nonreducing denatured SDS-PAGE analysis of the photoaffinity-labeled AT₄ receptor, the AT₄ receptor is predicted to contain multiple subunits that are associated through disulfide and/or other unknown linkages. The -subunit that is specifically cross-linked with ¹²⁵I-benzoyl phenylalamine-angiotensin IV ([¹²⁵I]BP-AngIV; indicating the presence of the AngIV binding site) has a molecular mass of 165 kDa, whereas the native AT₄ receptor complex is estimated to be more than 300 kDa (Zhang et al., 1998). In addition, the molecular mass of the  subunit is approximately 225 kDa when associated with one or more other subunits through disulfide bonds. A similarly sized AT₄ receptor has recently been identified by Bernier and colleagues (1998) in endothelial cells. These distinct binding and structural features of the AT₄ receptor are easily distinguishable from AT₁ and AT₂ receptors, which are G protein coupled and significantly smaller.

The widespread distribution of the AT₄ receptor in various tissues of mammalian species, as well as the diverse biological functions of AngIV in different systems and cell types suggest the possibility that the AT₄ receptor may exert its effects through different isoforms. This article compares the structural features of AT₄ receptors from selected bovine tissues using SDS-PAGE and HPLC analysis subsequent to affinity labeling with a radiolabeled photoprobe. The data presented provide evidence for at least two distinct AT₄ subtypes in different bovine tissues.

	Materials and Methods

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Tissues and Chemicals. Fresh bovine tissues including adrenal, heart, bladder, thymus, kidney, aorta, and hippocampus were obtained locally and frozen at 80°C. AngIV [VYIHPF] and divalinal-AngIV [V(CH₂NH)YV(CH₂NH)HPF] were synthesized in the author's laboratory (J. W. Harding). Dup 753 was a gift from Dr. Ron Smith of DuPont/Merck, Wilmington, DE. PD123319 was a gift from Dr. David Taylor of Warner Lambert-Park-Davis, Ann Arbor, MI. CGP42112A was a gift from Mark DeGasparo of Ciba-Geigy, Basel, Switzerland. AngII and Sar¹-Ile⁸-AngII were purchased from Sigma Chemical Co. (St. Louis, MO). Endopeptidase C was purchased from Wako Chemical (Richmond, VA), glycopeptidase F (PNGase) F deglycosidase, other angiotensin peptides, and general chemicals used were from Sigma and were of the highest quality available unless specified.

Synthesis of BP-AngIV. Ile¹, benzoylphenylalanine⁶, Gly⁷(C-terminal extended) AngIV (BP-AngIV) was synthesized on methylated 1% divinylbenzene cross-linked polystyrene resins substituted with the protected carboxy-terminal amino acid using a semiautomated peptide synthesizer (Vega 250C Coupler; Vega Biotechnologies, Tucson, AZ). The tert.-butoxycarbonyl protection group was used for the -amine of all amino acids. Other protecting groups employed were 2,6-dichlorobenzyl for Tyr and tosyl for His. Amino acids were activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and coupling was monitored for completeness with the Kaiser ninhydrin test (Kaiser et al., 1970). Coupling reactions that tested less than 99.4% complete were repeated. The crude peptide was cleaved from the resin and deprotected utilizing anhydrous hydrogen fluoride containing 10% anisole at 0°C for 45 min. The hydrogen fluoride and anisole were removed under vacuum and the resin was washed with anhydrous diethyl ether. The peptide was extracted with 10% acetic acid and lyophilized.

Iodination of [¹²⁵I]AngIV and BP-AngIV. The peptides were iodinated using the chloramine T method. Peptide (50 µg) was incubated in a total volume of 150 µl of 0.2 M sodium phosphate buffer (pH 7.2), containing 80 µg of chloramine T and 2 mCi Na¹²⁵I (DuPont, Wilmington, DE) for 25 s at room temperature. The reaction was stopped by the addition of 100 µg of Na₂S₂O₅ in 50 µl of sodium phosphate buffer. The mono-¹²⁵I peptides were separated from unlabeled and diiodinated peptides by HPLC (Beckman) using a reversed phase C₁₈ column (5 µm × 250 mm, Microsorb-MV, Rainin Instrument). Solvent A was 80 mM triethylamine phosphate (pH 3.0), whereas ACN was solvent B. [¹²⁵I]AngIV and [¹²⁵I]BP-AngIV were purified using a linear gradient of 9% B-25% B and 15% B-30% B over 90 min, respectively.

Tissue preparation and Solubilization of AT₄ Receptors from Membranes of Selected Bovine Tissues. For each membrane preparation containing the AT₄ receptor, 3 g of each bovine tissue was homogenized in 10 ml of hypotonic buffer (50 mM Tris and 5 mM EDTA, pH 7.4) at 4°C as described by Hanesworth et al. (1993). After homogenization, the concentration of total membrane protein was adjusted to 10 mg/ml. Membrane proteins were solubilized in buffer containing 1% 3-[(3-cholamidopropyl)dimethylammonion]propanesulfonate (CHAPS), 50 mM Tris (pH 6.8), and 5 mM EDTA at 4°C for 2 h. Nonsolubilized material was removed by passing the solubilized mixture through a 0.2-µm filter (Sigma). The amount of AT₄ receptor in each preparation was determined by a radioligand ([¹²⁵I]AngIV) binding as described previously (Hanesworth et al., 1993).

AT₄ Receptor Cross-Linking with [¹²⁵I]BP-AngIV. Twenty-five micrograms of solubilized protein containing AT₄ receptors was incubated with 0.6 nM [¹²⁵I]BP-AngIV in a total volume of 250 µl of isotonic buffer [150 mM NaCl, 50 mM Tris, 5 mM EDTA, 0.1% BSA, 50 µM Plummer's inhibitor (DL-2-mercaptomethyl-3-quanidinoethylthiopropanoic acid), and 20 µM bestatin at pH 7.4]. The AT₄-bound [¹²⁵I]BP-AngIV was separated from free [¹²⁵I]BP-AngIV by using a P-6 spin-column. The P-6 column was prepared by transferring 1 ml of P-6 resin (Bio-Rad Labs., Hercules, CA) into a 1-ml disposable syringe that was then placed in a 12 × 75-mm plastic centrifuge tube and centrifuged at 232g for 10 min at room temperature. After elution from the P-6 column, the effluent was then transferred to a weighing dish and exposed to UV light [312 nm variable intensity Transilluminator FBTIV-88 (Fisher Scientific, Pittsburgh, PA) on ice for 30 min.

Determination of AT₄ Receptor Ligand Specificity Using SDS-PAGE. The ligand specificity of AT₄ receptors from different bovine tissues was examined by cross-linking [¹²⁵I]BP-AngIV to the receptors in the presence of AT₁, AT₂, or AT₄ specific competitors. The binding conditions and the separation of the bound and free ligands were the same as above. After binding and cross-linking of the [¹²⁵I]BP-AngIV with the AT₄ receptor, 100 µl of each sample was methanol/chloroform precipitated (Sealfon, 1995). The protein pellet was dissolved in 40 µl of the Laemmli sample buffer, heated at 57°C for 20 min, and analyzed by SDS-PAGE using the Laemmli buffer system at a constant current of 20 mA for 100 min at room temperature (Sambrook et al., 1989). After electrophoresis, the gel was vacuum dried and autoradiography was performed using Kodak X-ray film (Sigma) at 80°C. The compounds (1 µM) used as competitors of [¹²⁵I]BP-AngIV included Dup 753 (an AT₁ antagonist), PD 123319 (an AT₂ antagonist), AngII, CGP 42112A (an AT₂ ligand), and divalinal-AngIV (an AT₄ antagonist). Nonradiolabeled AngIV was also included as an AT₄ specificity control.

Comparison of Molecular Sizes of AT₄ Binding Subunits from Different Bovine Tissues. The AT₄ receptors solubilized from selected bovine tissues were cross-linked with [¹²⁵I]BP-AngIV and analyzed by reduced SDS-PAGE to compare the molecular sizes of their  subunits. The cross-linking and SDS-PAGE analysis conditions are described above. After electrophoresis, the polyacrylamide gels were vacuum dried and autoradiography performed.

Nonreducing SDS-PAGE Analysis of AT₄ Receptor Subunits Associated through Disulfide Bonds. To test the hypothesis that disulfide bonds exist between the AT₄ receptor subunits from various bovine tissues, the solubilized AT₄ receptors from various bovine tissues were cross-linked with [¹²⁵I]BP-AngIV and analyzed by nonreducing SDS-PAGE and compared with the results obtained above from reducing SDS-PAGE. For each analysis, 20 µl of the cross-linked sample was mixed with 20 µl of Laemmli loading buffer without dithiothreitol and heated at 57°C for 20 min. The samples containing the AT₄ receptors from different bovine tissues were resolved by SDS-PAGE using the standard Laemmli buffer system (see above). After electrophoresis, the gel was vacuum dried and autoradiography performed.

Glycosylation Analysis of AT₄ Receptors Isolated from Different Bovine Tissues. The relative amount of the N-linked carbohydrate side chains on the AT₄ receptors of selected bovine tissues was determined by SDS-PAGE following removal of N-linked sugars with PNGase F. The AT₄ receptors were first cross-linked with [¹²⁵I]BP-AngIV and then digested with PNGase F (Coligan et al., 1995). For each reaction, 100 µl of sample was precipitated in a 1.5-ml microcentrifuge tube using the methanol/chloroform method (Sealfon, 1995). The protein pellet was thereafter dissolved in 35 µl of a resolving buffer containing 0.5% SDS and 0.1% -mercaptoethanol. Once the proteins went into solution, 45 µl of the digestion buffer containing 25 µl of 0.5 M Tris-Cl (pH 8.0), 10 µl of 0.1 M 1,10-phenanthroline in methanol, 10 µl of 10% of Nonidet P-40, and 5 µl of PNGase F (Sigma) was added and incubated at 37°C overnight. To each reaction mixture, 15 µl of distilled water was added and the deglycosylated proteins were precipitated as described above. The protein pellets containing deglycosylated AT₄ receptors were then dissolved in 40 µl of Laemmli loading buffer and analyzed by SDS-PAGE. After electrophoresis, the polyacrylamide gel was vacuum dried and autoradiography performed.

HPLC Analysis of Ligand Binding Site of AT₄ Receptors from Selected Bovine Tissues. To analyze for structural variations among the AT₄ receptors from different bovine tissues, the receptors were solubilized from the tissue membranes, cross-linked with [¹²⁵I]BP-AngIV, and digested with either endopeptidase C or cyanogen bromide. For endopeptidase C digestion, 1000 µl of cross-linked AT₄ receptor solution was vacuum dried and dissolved in 30 µl of digestion buffer containing 0.5% SDS, 0.1 M Tris base, and 10 µg of the Endopeptidase C. The digestion reaction was carried out at 37°C for 20 h. After digestion, each sample was mixed with 100 µl of distilled water and vacuum dried. The dried sample was redissolved in 100 µl of 0.1% TFA/10% ACN and applied to a C₁₈ column, which was pre-equilibrated with 0.1% TFA/10% ACN. Labeled fragments were then eluted with a linear gradient of 10 to 80% ACN (containing 0.1% TFA), which encompassed 90 min. Two-minute fractions were collected and counted, using a gamma counter (¹²⁵I efficiency 77%; ICN Isomedic 10/800, ICN Radiochemicals, Irvine, CA). For cyanogen bromide cleavage, a 1000 µl of the AT₄ sample was vacuum dried and dissolved in 100 µl of 70% formic acid containing cyanogen bromide (~70 mg/ml). The cleavage was conducted in the dark at room temperature for 20 h. The reaction mixture following cyanogen bromide digestion was diluted 10× and vacuum dried. The dried protein fragments were then dissolved in 100 µl of 0.1% TFA, 10% ACN, and analyzed by HPLC, using a C₄ column. The peptides were separated using a gradient of 10 to 80% of ACN containing 0.1% TFA over 80 min. One-minute fractions were collected into 80 tubes. After HPLC separation, the fractions of each were counted using a gamma counter.

Comparison of Glycosylated and Deglycosylated Receptor Fragments. To determine whether differences in the HPLC elution pattern observed for radiolabeled fragments of AT₄ receptors that were generated by cyanogen bromide (CNBr) cleavage were attributable to variations in primary sequence and/or glycosylation level, individual peaks were collected, dried down under vacuum, and deglycosylated with PNGase F as described above. The deglycosylated AT₄ fragments were then rechromatographed under conditions identical with those employed for the fully glycosylated CNBr fragments. Individual HPLC fractions were again collected and evaluated.

Receptor Binding Assays. Binding assays were performed at 37°C in a total volume of 250 µl isotonic assay buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 50 µM Plummer's inhibitor, 20 µM bestatin, and 0.1% heat-treated BSA, pH 7.4, at 37°C). Bound and free ligands were separated at the conclusion of each experiment by vacuum filtration using (Brandel Cell Harvester, Schleicher & Schuell, Keene, NH) 32 glass fiber filters following the addition of room temperature PBS (pH 7.2) and repeated washing (4 × 4 ml) with PBS. Radioactivity retained by the filters was determined using an ICN 10/880 gamma counter (77% efficiency).

11

4

	Results

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AT₄ Receptors of Selected Bovine Tissues Bound Specifically to AngIV and Its Analogs. To establish the binding specificity of AT₄ receptors in each of the tissues examined, as defined by [¹²⁵I]BP-AngIV binding, membranes from representative bovine tissues were solubilized and their binding affinity for both AngII or AngIV analogs was examined. After the binding reaction that contained [¹²⁵I]BP-AngIV and an angiotensin competitor (1 µM), the AT₄ receptor was photo cross-linked and analyzed by SDS-PAGE. Figure 1 shows the autoradiography of AT₄ receptors from selected tissues including bladder (Fig. 1a), adrenal gland (Fig. 1b), aorta (Fig. 1c), hippocampus (Fig. 1 day), kidney (Fig. 1e), and thymus (Fig. 1f). For all the samples, lanes 1 to 4 depict the binding between the AT₄ receptors and [¹²⁵I]BP-AngIV in the presence of PD 123319 (an AT₂ antagonist), Dup 753 (an AT₁ antagonist), divalinal-AngIV (an AT₄ antagonist), and nonradiolabeled AngIV, respectively. Lane 5 illustrated the binding between the AT₄ receptor and [¹²⁵I]BP-AngIV in the absence of competitors. For all the tissues examined, lanes 3 and 4 showed no labeled band, whereas lanes 1, 2, and 5 contained a band of the same size. These data thus indicate that only AngIV and divalinal-AngIV were effective competitors of [¹²⁵I]-BP-Ang IV binding to AT₄ receptors, whereas PD 123319 and Dup 753 did not compete.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Reducing SDS-PAGE analysis of ability of various competitors to block binding and cross-linking of [125I]BP-AngIV to AT4 receptors of selected bovine tissues. Tested bovine tissues were bladder (a), aorta (b), heart (c), hippocampus (d), kidney (e), and thymus (f). Compounds used for treatments of each tissue sample were: lane 1, Dup 753 (an AT1 antagonist); lane 2, CGP 42112A (an AT2 ligand); lane 3, AngIV; lane 4, divalinal-AngIV (an AT4 antagonist); lane 5, no addition of competitors (total binding). Bands displayed for each tissue sample have estimated molecular masses of 165 kDa except those of hippocampus, which have a molecular mass of 150 kDa.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Comparison of AT4 -subunits from various bovine tissues by SDS-PAGE analysis. Tissues included for AT4 -subunit analysis are bladder (lane A), adrenal (lane B), aorta (lane C), hippocampus (lane D), kidney (lane E), thymus (lane F), and heart (lane G). Numbered arrows indicate estimated molecular mass of [125I]BP-AngIV-labeled AT4 receptors based on molecular mass standards.

AT₄ Receptors Contained N-Linked Carbohydrate Side Chains of Similar Size. Previous studies have established that the bovine adrenal AT₄ receptor contains N-linked carbohydrate chains that can be removed by PNGase F digestion (Zhang et al., 1998). To determine whether N-linked glycosylation is universally present on the AT₄ receptors from other tissues, the AT₄ receptors solubilized from bovine bladder, adrenal, aorta, hippocampus, kidney, thymus, and heart were cross-linked with [¹²⁵I]BP-AngIV and treated with PNGase F. After deglycosylation, the proteins were resolved by reduced SDS-PAGE and the [¹²⁵I]BP-AngIV-labeled bands were visualized by autoradiography (Fig. 3). After deglycosylation, the apparent molecular mass of the [¹²⁵I]BP-AngIV-labeled protein band was shifted from 165 to 130 kDa for all tissues except the hippocampus, where it shifted from 150 to 130 kDa (see Fig. 2 for comparison). Thus, it is estimated that the AT₄ receptor of hippocampus contained approximately 13.3% N-linked glycosylation, whereas those of other tested tissues contained about 21.2% N-linked glycosylation.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Comparison of deglycosylated AT4 receptors from various bovine tissues by SDS-PAGE analysis. Tissues included for AT4 receptor analysis are bladder (lane A), adrenal (lane B), aorta (lane C), hippocampus (lane D), kidney (lane E), thymus (lane F), and heart (lane G). Numbered arrows indicate estimated molecular mass of [125I]BP-AngIV-labeled AT4 receptors based on molecular mass standards.

Subunits of AT₄ Receptor Are Associated through Disulfide Bonds. The  subunit of the bovine adrenal AT₄ receptor was found to associate with one or more unidentified subunits via disulfide linkages (Zhang et al., 1998). To establish the presence of disulfide-linked subunits of the AT₄ receptor in other bovine tissues, the AT₄ receptors of bladder, heart, hippocampus, aorta, kidney, and thymus were solubilized from membranes and cross-linked with [¹²⁵I]BP-AngIV and analyzed by nonreducing SDS-PAGE and visualized by autoradiography. As shown in Fig. 4 besides the 165-kDa protein band, the AT₄ receptors from all tested tissues but hippocampus contained an additional [¹²⁵I]BP-AngIV-labeled protein band with a molecular mass of approximately 225 kDa under nonreducing conditions. Under reducing conditions, however, the 225-kDa protein band disappeared as shown in Fig. 2.

View larger version (79K): [in this window] [in a new window]   Fig. 4.   SDS-PAGE analysis of AT4 receptors of selected bovine tissues. Solubilized AT4 receptors of selected bovine tissues were resolved by SDS-PAGE under nonreducing reducing conditions. AT4 receptors were analyzed from the following tissues: bladder (lane A), adrenal (lane B), aorta (lane C), hippocampus (lane D), kidney (lane E), thymus (lane F), and heart (lane G). Numbered arrows indicate estimated molecular mass of [125I]BP-AngIV-labeled protein bands based on molecular mass standards.

Fragments of Bovine AT₄ Receptors that Contain Ligand Binding Site Vary According to Tissue of Origin. Data presented above indicate that the bovine hippocampal AT₄ receptor is different, at least in terms of glycosylation level, from the receptor present in other bovine tissues. To further probe potential heterogeneity among bovine AT₄ receptors, receptors were photolabeled with [¹²⁵I]BP-AngIV, fragmented by either cyanogen bromide or endopeptidase C, and analyzed by HPLC. Only the elution of radiolabeled fragments, which presumably contain the ligand binding site, was monitored and compared.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Cyanogen bromide mapping of the [125I]BP-AngIV-labeled AT4 receptors by HPLC. AT4 receptors solubilized from selected bovine tissues and cross-linked with [125I]BP-AngIV were cleaved with cyanogen bromide and separated by C4 HPLC. Major radioactivity-containing fractions were deglycosylated and rechromatographed. Data from glycosylated receptors are indicated by closed symbols (), whereas data from deglycosylated receptors are indicated by open symbols (, , ). X-axes represent individual fractions, whereas y-axes represent relative amount of radioactivity for each fraction. Data were normalized based upon the total counts recovered. Individual peaks contained from 10,000 to 200,000 cpm. Data shown are representative of duplicate analyses. See text for details of method.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Endopeptidase C mapping of the [125I]BP-AngIV-labeled AT4 receptors by HPLC. AT4 receptors solubilized from selected bovine tissues and cross-linked with [125I]BP-AngIV were digested with endopeptidase C and separated by C18 HPLC. X-axes represent individual fractions, whereas y-axes represent relative amount of radioactivity for each fraction. Data were normalized based upon total counts recovered. Individual peaks contained from 6,000 to 180,000 cpm. Data shown are representative of duplicate analysis. See text for details of method.

Binding Characteristics of AT₄ Ligands Varies Depending on Tissue of Origin. A comparison of the binding affinities of five AT₄ ligands is presented in Table 1. AngIV bound with similar affinities to receptors from all tissues (K  2 nM) while exhibiting a one-site fit. The ligands, NleYIamide, NleYI-6-aminohexmide, and VYPHPF also exhibited one-side fits in all the tissues. The adrenal receptor, however, consistently possessed higher affinity than the AT₄ receptor in other tissues for each of above-mentioned ligands, with differences near 35-fold for adrenal-hippocampus and adrenal-thymus. The most distinguishing difference among tissues was observed for VYIGGdF, the binding of which fit a one-site model with similar affinity in aorta, heart, and thymus but a two-site model in adrenal and hippocampus where subnanomolar and subpicomolar sites were observed. The adrenal possessed mostly the low-affinity site (88%), whereas the hippocampus had a fairly even split between sites (60% high affinity and 40% low affinity). These data again emphasized the unique properties of the hippocampal AT₄ receptor.

	Discussion

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For many years before the discovery of the AngIV receptor, AT₄, studies of the renin-angiotensin system have focused on the integrative functions of AngII and AngIII, the angiotensin peptides that play critical roles in cardiovascular and body water regulation. One outcome of these studies was the impression that angiotensin peptides smaller than AngIII were biologically inactive. This notion was based on the observation that smaller angiotensin fragments lacked the ability to bind either AT₁ or AT₂ receptors and were unable to initiate "classic" angiotensin-dependent physiologies. Recently, however, at least two angiotensin fragments including AngII (1-7) (Ferrario et al., 1991) and AngIV (Swanson et al., 1992), have been shown to be biologically active, mediating physiological responses distinct from those typically associated with angiotensins. A corresponding receptor, AT₄, which specifically bound Ang IV, was thereafter identified in various tissues, including brain, heart, kidney, thymus, and adrenal from multiple species (Wright et al., 1995.). Purification and characterization of the AT₄ receptor from bovine adrenals indicated that the receptor is a protein complex containing multiple subunits. The  subunit that contains the AngIV binding site has a molecular mass of approximately 165 kDa and associates with other subunits through disulfide or other unknown linkages (Zhang et al., 1998). Reported in this study is a structural analysis and comparison of the  subunit of AT₄ receptors from different bovine tissues. Results revealed that the AT₄ receptor of bovine hippocampus was dissimilar from those of other tested tissues. The  subunit of the hippocampal AT₄ receptor had an estimated molecular mass of 150 kDa, whereas  subunits from other tissues were approximately 165 kDa in size.

One of the most common post-translational modifications seen with cell surface receptors is N-linked glycosylation. Deglycosylation analysis of the AT₄ receptors from various tissues revealed that the deglycosylated hippocampus AT₄ receptor was similar in size to deglycosylated AT₄ receptors from other tissues, suggesting that the differences in molecular mass between the  subunit of the hippocampus receptors and those of other tissues may be due to N-linked glycosylation. To further elucidate the source of the differences, the photolabeled  subunits of the AT₄ receptors of different bovine tissues were digested with either cyanogen bromide or endopeptidase C and the fragmentation pattern of radiolabeled peptides analyzed by HPLC. The HPLC profiles of the photolabeled fragments of the tested receptors showed that the radioactive AT₄ fragments resulting from the digestion with either cyanogen bromide or endopeptidase C varied based on their tissue origins. Cleavage with cyanogen bromide, which cuts adjacent to methionine, a rare amino acid, typically produces large fragments and, as such, is less sensitive to the detection of small variations in primary sequence following subsequent HPLC analysis. Despite this relative insensitivity of CNBr fragmentation to variable primary sequence, major differences were observed between hippocampal AT₄ receptors and AT₄ receptors from other tissues. These differences persisted following deglycosylation, suggesting that the primary sequence of the hippocampal receptor is considerably different from the AT₄ receptors that predominate in other tissues.

In an attempt to detect smaller differences in the primary sequence, radiolabeled AT₄ receptors were cleaved with endopeptidase C, which cleaves adjacent to lysine residues, a common amino acid, and thus generates smaller fragments. Analysis of the AT₄ receptor from the various tissues again highlighted the uniqueness of the hippocampal receptor but also uncovered smaller differences among the receptors form the other tissues. Whether these differences indeed reflect complex mixtures of multiple isoforms will ultimately require DNA sequence data.

The existence of multiple receptor isoforms or subtypes is typical of most receptor systems, thus providing a potential mechanism for varying cellular response to a single extracellular signal. A diversity of receptor isoforms imparts a breadth of cellular responses as a result of different ligand affinities, distinctive regulatory properties, and/or linkage to different intracellular signaling systems. This initial demonstration of AT₄ receptor subtypes has, however, yet to be placed in a functional context. Nonetheless, initial studies comparing structure-binding characteristics of multiple AT₄ ligands from several AT₄-containing tissue sources indicate that differences in binding characteristics do exist among tissues. The data shown in Table 1, which compares the binding affinity of five angiotensin IV analogs, indicate that although AngIV bound with similar affinity to each of the tissues examined, binding of the remaining analogs was characterized by higher affinity to adrenal receptors. Additionally, the ligand VYIGGdF exhibited two-site binding in hippocampus, encouraging speculation that the two sites correspond to two different receptors, as initially suggested by the presence of two distinct labeled CNBr cleavage fragments. The significance of a small amount of high-affinity binding for VYIGGdF in adrenals is presently unknown. Interestingly, adrenals and hippocampus shared a common endopeptidase c-generated peak at fraction 10. Thus, the primary conclusion of this study is that the predominant AT₄ receptor subtype found in hippocampus is different from that seen in several peripheral tissues. Given the broad physiological impact of AT₄ activation elaboration and expansion of our knowledge concerning AT₄ receptor subtypes may be expected to have considerable therapeutic value.