Abstract—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 AT2receptor 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 aKd=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT2receptor caused a rapid activation of &agr;iand &agr;obut not of &agr;qand &agr;s, as measured by radioactive guanosine 5′-3-O-(thio)triphosphate (GTP&ggr;S) binding. Activation required the presence of activated receptors, &bgr;&ggr;, and &agr; subunits. As a first step aimed at developing an in vitro assay to examine AT2receptor pharmacology, we tested a battery of Ang II–related ligands for their ability to promote AT1or AT2receptor–catalyzed Giactivation. Two proteolytic fragments of Ang II, Ang III and Ang1–7, also promoted activation of &agr;ithrough the AT2receptor. Furthermore, we found that [Sar1,Ala8]Ang II is an antagonist for both AT1and AT2receptors and that CPG42112 behaves as a partial agonist for the AT2receptor. In combination with previous observations, these results show that the AT2receptor is fully capable of activating Giand provides a new tool for exploring AT2receptor pharmacology and interactions with G-protein trimers.