The third cytoplasmic loop of the angiotensin II type 1 receptor exerts differential effects on extracellular signal-regulated kinase (ERK1/ERK2) andapoptosis via Ras- and Rap1-dependent pathways

The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT( 1)) is important for receptor coupling to G proteins and activation of down stream events. Therefore, we determined whether specific AT(1) sequences we re required for kinase activation and inhibition of apoptosis by transfecti ng wild-type (AT1Rwt) and mutated AT(1) into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERKI/ERK2) ac tivity in 293 cells transfected with AT1Rwt. However, in 293 cells that exp ressed a receptor in which amino acids 221 and 222 were deleted (AT1R[De122 1/222]), Ang II-mediated ERKI/ERK2 activation was inhibited by >85%. In con trast, c-Jun NH2-terminal protein kinase (JNK) activation was similar in AT 1Rwt- and AT1R(De1221/222)-transfected cells. Activation of ERK1/ERK2 by AT 1Rwt was independent of Ca2+, whereas the low level of ERK1/ERK2 activation by AT1R(De1221/222) was completely Ca2+ dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(De1221/222) required Rap1. These resu lts demonstrate the presence of 2 different pathways for ERK1/ERK2 activati on by Ang II, which differ in their requirements for Ca2+ and small G prote ins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-indu ced apoptosis in cells transfected with AT1Rwt but: not AT1R(De1221/222). A KT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpre ssion of constitutively active AKT significantly reduced serum deprivation- induced apoptosis in cells transfected with AT1R(De1221/222). This study sh ows for the first time a direct link between kinase activation and inhibiti on of apoptosis dependent on amino acids 221 and 222 in the third cytoplasm ic loop of the AT(1).