Functional Reconstitution of the Angiotensin II Type 2 Receptor and Gi Activation

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 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 [I] angiotensin (Ang) II revealed a single binding site with a Kd=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT2 receptor caused a rapid activation of αi and αo but not of αq and αs, 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 Gi activation. Two proteolytic fragments of Ang II, Ang III and Ang1–7, also promoted activation of αi through the AT2 receptor. Furthermore, we found that [Sar,Ala]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 Gi and provides a new tool for exploring AT2 receptor pharmacology and interactions with G-protein trimers.