Relação estrutura-atividade entre os receptores AT1 e AT2 da angiotensina II à luz da estrutura do receptor CXCR4 da quimiocina
Arquivos
Data
2014
Autores
Martin, Renan Paulo 
Orientadores
Shimuta, Suma Imura
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A angiotensina II (AngII) e um potente agente do sistema renina-angiotensina participando da regulacao da pressao arterial e na homeostase hidromineral. Seu efeitos fisiologicos sao mediados pelos receptores tipo 1 (AT1) e tipo 2 (AT2) que possuem respostas contrarias nas suas vias de sinalizacao. Alguns modelos de interacao agonista-receptor vem sendo propostos onde e possivel destacar alguns residuos importantes para interacao do agonista ao receptor como Asp1, Arg2 e Tyr4 alem das extremidades N e C-terminais. Numa etapa inicial foi proposta para avaliar a possiblidade da substituicao do metodo de estudo de ligacao tradicional baseado em uso de radioligantes por um nao radioativo (fluoroforo). Foi entao utilizado o lantanideo Europio (Eu3+) como marcador do ligante nos ensaios de competicao pelo sitio de ligacao comparando ao uso do 3H. Depois disso, o projeto foi dividido em duas etapas, sendo a primeira, uma abordagem pratica, onde diferentes ligantes foram testados e as afinidades de ligacao de cada um comparada com aquelas dos receptores AT1 e AT2. Posteriormente foram obtidas 3 mutacoes sitio dirigidas no receptor AT2 onde a primeira causou o rompimento da segunda ponte SS (C35S), a segunda a substituicao das Asn127 por Gly no meio da helice III (N127G) e a terceira a substituicao da Asp297 por Ala (D297A). Alem disso foi proposto um metodo teorico de estudo usando modelagem molecular dos receptores AT1 e AT2 por homologia ao CXCR4. Os testes de ligacao usando os dois metodos propostos mostraram resultados equivalentes, razao pela qual se adotou o metodo fluorimetrico para o prosseguimento dos experimentos. E interessante destacar que a substituicao da Tyr4 por Ile provocou uma queda de afinidade de cerca de 100 vezes enquanto o uso da Phe na mesma posicao quase nao afetou a afinidade do ligante ao receptor AT1. Por outro lado o mesmo experimento conduzido com o receptor AT2 mostrou nao haver diferenca significativa. Esses resultados estao de acordo com os da literatura, uma vez que o residuo Asn111 do AT1 parece funcionar como botao que controla as mudancas do estado nao ativado para um estado propicio a ativacao que e um estado essencial para a correta acomodacao do ligante no receptor. Entretanto o receptor AT2 parece ja estar nesse estado independentemente da presenca desse residuo. Alem disso, o receptor AT2 possui um sitio de interacao mais flexivel do que o do AT1 que parece ser mais critico as mudancas no ligante. Na analise mutacional abordada, tanto o mutante C35S quanto o N127G nao apresentaram mudancas significativas no perfil de resposta aos ligantes estudados. Por outro lado o receptor AT2 carregando a mutacao D297A sofreu uma queda drastica na capacidade de ligacao com todos os analogos testados, indicando assim que esse residuo e crucial para a interacao com o segmento N-terminal da AngII. Os resultados de dinamica corroboram com os dados experimentais reforcando a melhor acomodacao da molecula de AngII ao receptor AT2 quando comparada ao receptor AT1
Angiotensin II (AngII) is the main agent of the renin-angiotensin system to regulate blood pressure and hydro-electrolytic homeostasis. Its physiological actions are mainly mediated by the type1 (AT1) and type2 (AT2) receptors that present almost always an opposite response in their signaling pathway. Several receptor-agonist interaction models have been proposed, and it is noteworthy that several AngII residues are involved in the peptide interaction, such as, Asp1, Arg2, Tyr4 and Phe4 beyond the N and C terminal segments. As an initial step it was proposed to evaluate the possibility to replace the traditional radioactive binding method to a fluorimetric one. So it was tried to use a peptide labeled with the lanthanide Europium (Eu3+) as competitive agent in the binding experiments in comparison to the 3H-AngII radioligand. For the following step, a practical study protocol was applied based on binding affinity data of AngII analogs towards AT1 and AT2 receptors. In addition it was proposed to obtain three site-directed mutagenesis on the AT2 receptor. These mutations would be due to: disruption of the second SS-bond between the N-terminal and EC3-Loop (C35S); the substitution of Asn127 to Gly at middle of helix III (N127G) and the replacement of Asp297 to Ala (D297A). Moreover, a theoretical study was included using receptors modeling built from the CXCR4 homology. From our experiments it was found that both radioactive and non-radioactive methods resulted in similar binding data, so that the fluorimetric method was chosen to be used in the affinity binding experimental procedures. It is noteworthy that in the practical study, the substitution of Tyr4 to Ile provoked a 100 times lower affinity, while the use of Phe caused almost no difference in the AT1 receptor binding. On the other hand, the substitution of Tyr4 for both Phe and Ile, in the AT2 receptor, change was observed. Our findings are in accordance with the literature data, since the Asn111 AT1 receptor residue has been considered to be a switch that control the change of a nonactivated state R, to an activated state R’, and this state is essential to the correct docking of the ligand. However the AT2 receptor seems to be on the R’ state in the absence of the ligand. Thus, AT2 receptor has a more flexible interaction site than AT1, ergo changes in AngII are more critical in the AT1 receptor binding. In the mutational experiments the both mutants, C35S and N127G, did not present changes in their affinity profiles to the different ligands when compared with the wild type receptor. On the other hand, the mutation D297A induced a reduction in the affinity indicating that this residue is crucial in the N-terminal agonist binding with the EC-3 receptor´s segment. The results obtained from the molecular dynamics reinforce the idea that AT2 receptor presents a better accommodation of AngII molecule when compared with AT1 receptor.
Angiotensin II (AngII) is the main agent of the renin-angiotensin system to regulate blood pressure and hydro-electrolytic homeostasis. Its physiological actions are mainly mediated by the type1 (AT1) and type2 (AT2) receptors that present almost always an opposite response in their signaling pathway. Several receptor-agonist interaction models have been proposed, and it is noteworthy that several AngII residues are involved in the peptide interaction, such as, Asp1, Arg2, Tyr4 and Phe4 beyond the N and C terminal segments. As an initial step it was proposed to evaluate the possibility to replace the traditional radioactive binding method to a fluorimetric one. So it was tried to use a peptide labeled with the lanthanide Europium (Eu3+) as competitive agent in the binding experiments in comparison to the 3H-AngII radioligand. For the following step, a practical study protocol was applied based on binding affinity data of AngII analogs towards AT1 and AT2 receptors. In addition it was proposed to obtain three site-directed mutagenesis on the AT2 receptor. These mutations would be due to: disruption of the second SS-bond between the N-terminal and EC3-Loop (C35S); the substitution of Asn127 to Gly at middle of helix III (N127G) and the replacement of Asp297 to Ala (D297A). Moreover, a theoretical study was included using receptors modeling built from the CXCR4 homology. From our experiments it was found that both radioactive and non-radioactive methods resulted in similar binding data, so that the fluorimetric method was chosen to be used in the affinity binding experimental procedures. It is noteworthy that in the practical study, the substitution of Tyr4 to Ile provoked a 100 times lower affinity, while the use of Phe caused almost no difference in the AT1 receptor binding. On the other hand, the substitution of Tyr4 for both Phe and Ile, in the AT2 receptor, change was observed. Our findings are in accordance with the literature data, since the Asn111 AT1 receptor residue has been considered to be a switch that control the change of a nonactivated state R, to an activated state R’, and this state is essential to the correct docking of the ligand. However the AT2 receptor seems to be on the R’ state in the absence of the ligand. Thus, AT2 receptor has a more flexible interaction site than AT1, ergo changes in AngII are more critical in the AT1 receptor binding. In the mutational experiments the both mutants, C35S and N127G, did not present changes in their affinity profiles to the different ligands when compared with the wild type receptor. On the other hand, the mutation D297A induced a reduction in the affinity indicating that this residue is crucial in the N-terminal agonist binding with the EC-3 receptor´s segment. The results obtained from the molecular dynamics reinforce the idea that AT2 receptor presents a better accommodation of AngII molecule when compared with AT1 receptor.
Descrição
Citação
MARTIN, Renan Paulo. Relação estrutura-atividade entre os receptores AT1 e AT2 da angiotensina II à luz da estrutura do receptor CXCR4 da quimiocina. 2014. 125 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2014.