Asunto(s)
Arrestinas/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Arrestinas/clasificación , Arrestinas/metabolismo , Humanos , Ligandos , Ratones , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , beta-ArrestinasRESUMEN
BACKGROUND AND PURPOSE: The angiotensin II type 1 (AT(1)) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or beta-arrestins often induces higher binding affinity for agonists. Here, we examined interactions between AT(1A) receptors and beta-arrestins to look for differences between the AT(1A) receptor interaction with beta-arrestin1 and beta-arrestin2. EXPERIMENTAL APPROACH: Ligand-induced interaction between AT(1A) receptors and beta-arrestins was measured by Bioluminescence Resonance Energy Transfer 2. AT(1A)-beta-arrestin1 and AT(1A)-beta-arrestin2 fusion proteins were cloned and tested for differences using immunocytochemistry, inositol phosphate hydrolysis and competition radioligand binding. KEY RESULTS: Bioluminescence Resonance Energy Transfer 2 analysis showed that beta-arrestin1 and 2 were recruited to AT(1A) receptors with similar ligand potencies and efficacies. The AT(1A)-beta-arrestin fusion proteins showed attenuated G protein signalling and increased agonist binding affinity, while antagonist affinity was unchanged. Importantly, larger agonist affinity shifts were observed for AT(1A)-beta-arrestin2 than for AT(1A)-beta-arrestin1. CONCLUSION AND IMPLICATIONS: beta-Arrestin1 and 2 are recruited to AT(1A) receptors with similar ligand pharmacology and stabilize AT(1A) receptors in distinct high-affinity conformations. However, beta-arrestin2 induces a receptor conformation with a higher agonist-binding affinity than beta-arrestin1. Thus, this study demonstrates that beta-arrestins interact with AT(1A) receptors in different ways and suggest that AT(1) receptor biased agonists with the ability to recruit either of the beta-arrestins selectively, would be possible to design.
Asunto(s)
Arrestinas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Animales , Arrestinas/clasificación , Línea Celular , Proteínas de Unión al GTP/metabolismo , Humanos , Conformación Proteica , Transducción de Señal , beta-Arrestina 1 , beta-ArrestinasAsunto(s)
Analgesia , Analgésicos Opioides , Arrestinas/antagonistas & inhibidores , Morfina , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Analgésicos Opioides/efectos adversos , Analgésicos Opioides/farmacología , Animales , Arrestinas/clasificación , Arrestinas/fisiología , Humanos , Morfina/efectos adversos , Morfina/farmacología , Receptores Acoplados a Proteínas G/clasificación , Receptores Acoplados a Proteínas G/fisiología , beta-ArrestinasRESUMEN
Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for thrombin, is irreversibly proteolytically activated. beta-Arrestin1 and beta-arrestin2 have been reported to have different effects on signal desensitization and transduction of PAR1. In this study, we investigated whether beta-arrestin1 and beta-arrestin2 regulate Src-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) induced by PAR1 in HEK 293 cells. Our results show that PAR1-mediated activation of Src and ERK1/2 in HEK 293 cells was increased with overexpression of beta-arrestin1 or depletion of beta-arrestin2. PAR1-mediated activation of Src and ERK1/2 in HEK 293 cells was decreased or eliminated with depletion of beta-arrestin1 or overexpression of beta-arrestin2. Furthermore, depletion of beta-arrestin2 blocked PAR1-induced degradation of Src. Thus, beta-arrestin1 and beta-arrestin2 have opposing roles in regulating the activation of Src induced by PAR1. beta-Arrestin2 also appears to promote PAR1-induced degradation of Src. This degradation of Src provides a possible mechanism for terminating PAR1 signaling.
