On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT2) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT2 receptor signal transduction mechanisms are poorly understood. We have measured AT2-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [125I] angiotensin (Ang) II revealed a single binding site with a K(d)=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT2 receptor caused a rapid activation of α(i) and α(o) but not of α(q) and α(s), as measured by radioactive guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding. Activation required the presence of activated receptors, βγ, and α subunits. As a first step aimed at developing an in vitro assay to examine AT2 receptor pharmacology, we tested a battery of Ang II-related ligands for their ability to promote AT1 or AT2 receptor-catalyzed G(i) activation. Two proteolytic fragments of Ang II, Ang III and Ang 1-7, also promoted activation of α(i) through the AT2 receptor. Furthermore, we found that [Sar1,Ala8]Ang II is an antagonist for both AT1 and AT2 receptors and that CPG42112 behaves as a partial agonist for the AT2 receptor. In combination with previous observations, these results show that the AT2 receptor is fully capable of activating G(i) and provides a new tool for exploring AT2 receptor pharmacology and interactions with G-protein trimers.
- Angiotensin II type 2 receptor
- G(i) activation