Abstract P257: Angiotensin Ii Type 1 Receptor Autoantibodies Decrease Angiotensin Ii Type 1 Receptor Internalization Through Attenuating The Recruitment Of β-arrestin1/2

Introduction: Angiotensin II type 1 receptor autoantibody (AT1-AA) can continuously activate angiotensin II type 1 receptor (AT 1 R), which is related to cardiovascular disease, but the exact mechanism remains obscure. The activated AT 1 R signaling is adjusted or terminated effectively via the internalization, which is mediated by β-arrestin1/2 dependent-endocytic pathways. The hypothesis is that AT1-AA can attenuate β-arrestin1/2 recruitment, leading to the decreased AT 1 R internalization and the sustained AT 1 R activation. Methods and Results: Firstly, subcellular protein fractionation and Total Internal Reflection Fluorescence were used to examine the AT 1 R internalization induced by AT1-AA. The results confirmed that AT1-AA limited the AT 1 R internalization. Then, by co-expressing YFP-labeled AT 1 R and RFP-labeled β-arrestin1/2 in HEK293 cells with different stimulant for 10 min, the image showed that AT 1 R and β-arrestin1/2 were co-localized in the cytoplasm with Ang II. However, there were rare co-localization with AT1-AA. To further investigate the AT1-AA-induced recruiting of β-arrestin1/2, we recorded the relative change of BRET ratio of the interaction between YFP-labeled AT 1 R with Rluc-labeled β-arrestin1/2. The relative change of BRET ratio was significantly elevated with the time [(the BRET ratio with Ang II for 10 min were considered as 100%; 0 (β-arrestin1/2: 0.33 ±0.58/0.00 ±1.46), 2 (89.10 ±19.36/69.27 ±4.47), 5 (107.08 ±12.17/89.27 ±1.46), 10 (100.22 ±0.38/100.00 ±13.12), 20 (93.45 ±11.59/102.93 ±10.38) and 30 min (101.45 ±16.93/114.15±8.15)] and with the concentration (EC50 β-arrestin1/2 =6.69 ±4.09/6.34 ±2.29 nmol/L) after Ang II treatment. However, AT1-AA could not induce the recruitment of β-arrestin1/2. Furthermore, pre-incubation with an inhibitor of β-arrestin1/2 dependent-endocytic pathways prolonged the duration of AT 1 R downstream PKC, ERK1/2 phosphorylation, elevate intracellular Ca 2+ and vasoconstriction caused by Ang II, which simulated AT1-AA-caused persistent AT 1 R activation. Conclusions: Our data suggested that reduced AT 1 R internalization caused by AT1-AA was attributed to the inhibition of β-arrestin1/2 recruitment, which played a key role in how AT1-AA prolonged receptor activation.