RESUMO
Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.
Assuntos
Angiotensina II/agonistas , Bradicinina/agonistas , Peptídeos/química , Receptor Tipo 1 de Angiotensina/química , Receptores da Bradicinina/química , Sequência de Aminoácidos , Angiotensina II/genética , Angiotensina II/metabolismo , Sítios de Ligação , Bradicinina/genética , Bradicinina/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismoRESUMO
Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.
Assuntos
Angiotensina II/metabolismo , Sinalização do Cálcio/fisiologia , Peptidil Dipeptidase A/metabolismo , Análise de Variância , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Citometria de Fluxo , Lisinopril/farmacologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Previous studies on angiotensin II (AngII) AT(1) receptor function have revealed that the N-terminal residues of AngII may modulate receptor activation by binding at the receptor extracellular site. A remarkable feature of this site is an insertion of 8 amino acids in the middle of the EC-3 loop including the Cys(274) residue that supposedly makes a disulfide bond with N-terminal Cys(18). As demonstrated by assays with Del(267-275)AT(1), the role of the Cys(18)-Cys(274) disulfide bridge is to keep a conformation of the inserted residues that allows a normal binding of the AngII N-terminal residues. C18S AT(1) receptor mutant, supposedly having a dissociated disulfide bridge, but an intact residue insertion, is constitutively activated and can less efficiently bind AngII. Similar results were observed when the S-S disulfide bond was disrupted in (C18S,C274S) AT(1) receptor. The importance of the free N-terminal amino group of Asp(1) and of the Arg(2) guanidino group for the binding of AngII to C18S mutant with EC-3 loop insertion was investigated by means of assays using AngII peptide analogues bearing a single mutation of Asp(1) for Sar(1) or Arg(2) for Lys(2), as ligands. This study showed that like AngII, [Sar(1)]-AngII can bind the C18S mutant receptor with low affinity whereas [Lys(2)]-AngII binding is still more reduced. Interestingly, when (125)I-AngII instead of (3)H-AngII was used, no significant binding of this mutant was observed although wild type AT(1) receptor was shown to bind all AngII analogues.
Assuntos
Angiotensina II/metabolismo , Cisteína/metabolismo , Radioisótopos do Iodo/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Ligação Proteica , Ensaio RadioliganteRESUMO
Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.
Assuntos
Mutação , Proto-Oncogenes/genética , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Angiotensina II/metabolismo , Animais , Células CHO , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Cricetulus , Fluoresceínas , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes , Indóis , Concentração Inibidora 50 , Fosfatos de Inositol/análise , Fosfatos de Inositol/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptor Tipo 1 de Angiotensina/química , Receptores Acoplados a Proteínas G/genética , TransfecçãoRESUMO
An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.
Assuntos
Cistina/fisiologia , Dissulfetos/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Angiotensina II/metabolismo , Animais , Compostos de Boro , Células CHO , Cricetinae , Cricetulus , Citometria de Fluxo , Proteínas de Fluorescência Verde/biossíntese , Fosfatos de Inositol/biossíntese , Microscopia Confocal , Modelos Moleculares , Estrutura Terciária de Proteína , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/genética , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de SequênciaRESUMO
To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT(1) receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2+ influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation.
Assuntos
Proliferação de Células , Forma Celular , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Colforsina/metabolismo , Cricetinae , AMP Cíclico/metabolismo , Leucina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The manifestation of tachyphylaxis to angiotensin II in Chinese hamster ovary (CHO) cells expressing the rat angiotensin II AT(1) receptor was investigated. The cells were transfected with a cDNA fragment containing the complete coding region of the angiotensin II AT(1A) receptor gene, as well as 56 bp of its 3'- and 52 bp of its 5'-untranslated regions. These cells (CHO-AT(1)) responded to angiotensin II by increases in intracellular Ca(2+) concentration and inositol phosphate turnover, which were inhibited upon repeated administrations, characterizing the tachyphylaxis phenomenon. In contrast to smooth muscle cells, which are rendered tachyphylactic to angiotensin II but not to [2-lysine]angiotensin II ([Lys(2)]angiotensin II), this analogue induced responses in CHO-AT(1) cells that were also inhibited upon repeated administrations. A smooth muscle cell line, which showed tachyphylaxis only to angiotensin II, became tachyphylactic also to [Lys(2)]angiotensin II after transfection with the angiotensin II AT(1) receptor gene. Our findings suggest that posttranscriptional control directed by the 3'- or the 5'-untranslated regions in the angiotensin II AT(1) receptor gene may play a role in modulating the signal transduction pathways involved in the mechanism of angiotensin II tachyphylaxis.