G protein coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-p...
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G protein coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT(1A)) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (Ang-II) and [Sar(1),Ile(4),Ile(8)]AngII, an analog that only activates signaling through the constitutive receptors, Wild type AT(1A) receptors displayed a basal level of phosphorylation, which was stimulated by AngII, Unexpectedly, the constitutively active AT(1A) receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT(1A) receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar(1),Ile(4),Ile(8)]AngII. Paradoxically, [Sar(1),Ile(4),Ile(8)]AngII produced a robust (similar to 85% of AngII), dose-dependent phosphorylation of the wild type AT(1A) receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII, Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar(1),Ile(4),Ile(8)]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT(1A) receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a
Analogues of sarilesin (type I AT1 antagonists), and sarmesin (type II AT1 antagonists) with homoserine (hSer) at position 8 were prepared and bioassayed. The presence of a Tyr(4)-Ile(5)-His(6) bend found in sarmesin ...
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Analogues of sarilesin (type I AT1 antagonists), and sarmesin (type II AT1 antagonists) with homoserine (hSer) at position 8 were prepared and bioassayed. The presence of a Tyr(4)-Ile(5)-His(6) bend found in sarmesin but not in sarilesin was identified. The obtained results coupled with conformational analysis studies, using a combination of NMR spectroscopy and computational chemistry, propose important conformational and stereoelectronic properties for agonist and antagonist activity at ATI receptors. (C) 2000 Elsevier Science Ltd. All rights reserved.
We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT, receptor through an induced-fit mechanism (Noda, K,, Feng, Y. H., Liu, X. P., Saad, Y,, Husain, A., and Karnik, S, S. (1996) Bioc...
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We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT, receptor through an induced-fit mechanism (Noda, K,, Feng, Y. H., Liu, X. P., Saad, Y,, Husain, A., and Karnik, S, S. (1996) Biochemistry 35, 16435-16442). In this activation process, interactions between Tyr(4) and Phe(8) of Ang II with Asn(111) and His(256) Of the AT(1) receptor, respectively, are essential for agonism, Here we show that aromaticity, primarily, and size, secondarily, of the Tyr(4) side chain are important in activating the receptor. Activation analysis of AT(1) receptor position 111 mutants by various Ang II position 4 analogues suggests that an amino-aromatic bonding interaction operates between the residue Asn(111) of the AT(1) receptor and Ty(r)4 of Ang II. Degree and potency of AT(1) receptor activation by Ang II can be recreated by a reciprocal exchange of aromatic and amide groups between positions 4 and 111 of Ang II and the AT(1) receptor, respectively. In several other bonding combinations, set up between Ang II position 4 analogues and receptor mutants, the gain of affinity is not accompanied by gain of function, Activation analysis of position 256 receptor mutants by Ang II position 8 analogues suggests that aromaticity of Phe(8) and His256 Side chains is crucial for receptor activation;however, a stacked rather than an amino-aromatic interaction appears to operate at this switch locus. Interaction between these residues, unlike the Tyr(4):Asn(111) interaction, plays an insignificant role in ligand docking.
I-125-Angiotensin (Ang) IV and I-125-divalinal Ang IV [AT receptor subtype 4 (AT(4))] receptor agonist and putative antagonist, respectively] were used to characterize the AT(4) receptor in Mardin-Darby bovine kidney ...
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I-125-Angiotensin (Ang) IV and I-125-divalinal Ang IV [AT receptor subtype 4 (AT(4))] receptor agonist and putative antagonist, respectively] were used to characterize the AT(4) receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both I-125-Ang IV and I-125-divalinal Ang IV bound to a single high-affinity site (K-D = 1.37 and 1.01 nM, respectively) and to a comparable density of binding sites (B-max = 1335 and 1407 fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated similar displacement affinities in the following affinity order: Ang IV = divalinal Ang IV > Ang III > Ang II > losartan = PD 123177. Guanosine-5'-O-(3-thio)triphosphate or sulfhydryl reducing agents did not affect the binding of either radiolabeled ligand. Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1 nM to 1 mu M) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium response observed with Ang IV at micromolar concentrations. These results indicate that Ang IV and divalinal Ang IV bind with high affinity to the same receptor and that the MDBK AT(4) receptor is not coupled to a classic G protein, nor are sulfhydryl bonds important in regulation of receptor affinity. The MDBK AT(4) receptor appears to be pharmacologically similar to that described in nonrenal tissues. Functional studies suggest that AT(4) receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.
Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for th...
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Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [(125)I-Sar(1)-Ile(8)]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to (125)I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [(125)I-Sar(1)-Ile(8)]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands. (C) 1999 Academic Press.
The angiotensin IV receptor (AT(4)) receptor is widely distributed in both species and tissues. This broad distribution appears to be reflected in an equally diverse repertoire of physiological actions that are mediat...
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The angiotensin IV receptor (AT(4)) receptor is widely distributed in both species and tissues. This broad distribution appears to be reflected in an equally diverse repertoire of physiological actions that are mediated through AT(4) receptors. This breadth of location and function of AT(4) receptors encourages speculation that multiple AT(4) isoforms might exist. In this study, we compared the structural properties of bovine AT(4) receptors from adrenals, kidney, heart, thymus, bladder, aorta, and hippocampus. These comparisons were made using polyacrylamide gel electrophoresis or HPLC analysis of AT(4) receptors that had been covalently radiolabeled with the AT(4)-specific photoprobe I-125-benzoyl phenylalamine-angiotensin IV. Except for the hippocampal AT(4) receptor, the binding subunit in all tissues had a molecular mass of approximately 165 kDa and associated with additional subunits via disulfide linkages. The hippocampal receptor was significantly smaller (150 kDa) and did not appear to possess other disulfide-linked subunits. The receptor was highly glycosylated in all tissues examined. Peptide mapping following cleavage of I-125-labeled receptor with endopeptidase C or cyanogen bromide resulted in complex cleavage patterns. Together these mapping studies demonstrated the uniqueness of the hippocampal receptor and further suggested that other AT(4) isoforms may exist and be variably distributed among bovine tissues. In agreement with the peptide mapping studies, differences in the binding pattern of several AngIV analogs were observed among the various tissues.
Angiotensin blockade was established in hypertensive patients with the competitive inhibitor saralasin and the blood pressure response was compared to prior renin determinations. Two patients with subsequently confirm...
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Angiotensin blockade was established in hypertensive patients with the competitive inhibitor saralasin and the blood pressure response was compared to prior renin determinations. Two patients with subsequently confirmed renovascular hypertension had normal peripheral renin and non-lateralizing renal vein renin ratios, yet both showed a clear-cut lowering of blood pressure after administration of the blocking agent, indicating the presence of renin-mediated hypertension. Thus, direct in vivo testing with saralasin appears to offer certain advantages over renin determinations.
The effect of the reversal of the direction of amide bonds in the peptide chain of angiotensin was determined by the synthesis and study of retroenantiomers of the following peptides: 1, [Val5]angiotensin II (angioten...
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The effect of the reversal of the direction of amide bonds in the peptide chain of angiotensin was determined by the synthesis and study of retroenantiomers of the following peptides: 1, [Val5]angiotensin II (angiotensin);2, [Suc1]angiotensin (desamino-angiotensin);3, [Ala7]desamino-angiotensin;4 [.beta.-Ala7]desamino-angiotensin. In all of these retroenantiomers, the N-terminal Phe residue was replaced by a benzylmalonyl moiety in order to maintain the topological features of angiotensin''s C terminus which are important for biological activity. The separation of the diastereomeric peptides containing D- or L-benzylmalonyl residues was possible in the cases of the retroenantiomers of 1 and 2 but not in those of 3 and 4. The retroenantiomers of 1 and 2 were devoid of smooth muscle contracting activities, while those of 3 and 4 contracted the isolated guinea-pig ileum and rat uterus with activities ranging from 8 to 24%, when compared with the respective parent compounds. The sense of the peptide bonds in angiotensin''s backbone is not essential for activity, and the Pro7 residue in angiotensin is important for maintaining an active conformation of the molecule.
Patients with essential hypertension were sodium deprived by five days on a 10 mM sodium diet and were then infused with an incremental infusion of saralasin, a competitive inhibitor of angiotensin II. Patients with n...
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Patients with essential hypertension were sodium deprived by five days on a 10 mM sodium diet and were then infused with an incremental infusion of saralasin, a competitive inhibitor of angiotensin II. Patients with normal renin hypertension showed no change in lying or standing blood pressure during the infusion of saralasin. Angiotensin II is not, therefore, directly maintaining blood pressure in these patients when sodium deprived by diet, and is therefore unlikely to be playing any direct role in maintaining their blood pressure on their normal sodium intake. Patients with low renin hypertension showed a significant rise in blood pressure during saralasin infusion. Saralasin may be a further method of distinguishing low renin hypertensives from other hypertensives if they are infused when sodium deprived by diet.
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