The octapeptide hormone angiotensin II (Ang II) exerts its major biological effects via angiotensin AT1 receptors (AT1Rs). Signaling of AT1Rs is regulated by ß-arrestins, which bind to activated AT1Rs, uncouple them form G proteins, and initiate their internalization via clathrin-coated pits and cause G protein independent MAP kinase activation. It has been shown previously that AT1Rs internalize via ß-arrestin-dependent and independent mechanisms, whereas angiotensin AT2 receptors, which are unable to internalize, do not bind ß-arrestins. To study the role of G protein independent MAP kinase activation in cells, which endogenously express AT1Rs, a mutant receptor (S109Y) was created, which is unable to bind candesartan. On the other hand, the Ang II binding and Ang II-induced functional responses of the S109Y mutant receptor are completely normal. This mutation was combined with a mutation (DRY/AAY), which can bind to ß-arrestin2, but its G protein coupling is completely impaired. The receptors were expressed in C9 cells, which express endogenous AT1Rs. In the presence of candesartan the Ca2+ signal and MAP kinase activation of the endogenous AT1R was completely eliminated. However, the Ca2+ signal generation and MAP-kinase activation of the S109Y mutant receptor was readily detectable. in the presence of candesartan, which inhibits the endogenous AT1Rs, the combined S109Y and DRY/AAY mutant receptor was unable to induce Ca2+ signal generation, whereas it mediated Ang II-induced MAP kinase activation with a slow kinetics. These data suggest that G protein independent MAP kinase activation can occur in C9 cells.
This work was supported by OTKA T46445 and ETT 447/2006.