A Limited Or A Transient At1r Internalization Induced By Two Discriminating Agonists Result In Distinct Downstream At1r Signaling: Erk1/2 And Akt/Pkb

Background Agonist-induced AT1R Internalization, results from sequestration of the agonist-receptor complex into “clathrin vesicles”, depending on the internalization proteins: β-arrestin, clathrin. In this regard, we have shown that a 15,000 kDa polymer of Ang II (Ang II-POL) at maximal concentration, upon a sustained action on the AT1R of rat coronary endothelial luminal membrane (CELM) produced: a sustained inotropic effect, a lack of endothelial AT1R internalization, enhanced membranal recruitment of β-arrestin1/2, without clathrin recruitment a lack of clathrin-vesicles formation. In contrast, the monomer Ang II (1 kDa) inotropic effects shows desensitization, paralleled by endothelial AT1R internalization, and the well-known gradual membranal recruitment of the internalization proteins β-arrestin1/2 and clathrin. These results clearly show that Ang II and Ang II-POL distinctly activate AT1R as reflected by their different recruitment ways of the internalization proteins, which likely result in distinct downstream AT1R signaling: ERK1/2 and Akt/PKB phosphorylation. We studied this canonical signaling pathways triggered by AT1R activation, with both agonists. Hypothesis The distinct rates of membrane-recruitment of the internalization proteins; β-arrestin and clathrin by Ang II and Ang II-POL likely results also in distinct downstream AT1R signaling: ERK1/2, Akt/PKB phosphorylation. Hypothesis test C9 cells were used as a model of cytoplasmic events. Cells were stimulated either with maximal concentrations of Ang II or Ang II-POL and under various conditions were determined the changes in ERK1/2 and Akt/PKB phosphorylation: 1) at various times, 2) in the presence of effect of AT1R and AT2R antagonist and 3) in the presence of various PI3K and MEK1 inhibitors. Results 1) Ang II as well as Ang II-POL caused, via an AT1R activation, a time dependent phosphorylation of ERK1/2 and Akt/PKB. These time-dependent responses, 2) in the case of Akt the response by Ang II was transient while for Ang II-POL rose to a plateau and sustained, but, 3) in the case of ERK1/2’s for both agonists rose followed by a gradual drop and were equally transient. In addition, 4) PI3K inhibitors blocked the Akt responses induced Ang II and Ang II-POL, however, in the case of ERK1/2, the inhibitors only blocked the effects of Ang II-POL but not Ang II's. Conclusion The distinct rates of membrane-recruitment of the internalization proteins; β-arrestin and clathrin by Ang II and Ang II-POL are associated with distinct downstream AT1R signaling: ERK1/2, Akt/PKB. For example, a sustained AT1R activation or recruitments, enhanced for β-arrestin1/2 and inhibited for clathrin prevent desensitization of Akt/PKB; in addition, PI3K dependent phosphorylation of ERK. Clearly, AngII-POL constitutes a powerful tool for understanding the internalization and intracellular signaling mechanisms, induced by sustained AT1R activation.