(L5092) A Limited or a Transient AT1R Internalization Induced by Two Discriminating Agonists Result in Distinct Downstream AT1R Signaling: ERK1/2 and Akt/PKB

Jesus Castillo-Hernandez (Unidad Académica Multidisciplinaria Zona Media UASLP)| Rafael Rubio (Facultad de Medicina de la UASLP)| Martha Maldonado-Cervantes (Unidad Académica Multidisciplinaria Zona Media UASLP)| Jesus Olivares-Reyes (Centro de Investigación y Estudios Avanzados CINVESTAV_IPN)

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.

Support or Funding Information

CONACYT 101850 to Rafael Rubio, CONACYT CB-2005-01-48777 to J. Alberto Olivares-Reyes