Functional reconstitution of the angiotensin II type 2 receptor and G(i) activation

On the basis of the patterns of conserved amino acid sequence, the angioten sin II type 2 (AT(2)) receptor belongs to the family of serpentine receptor s, which relay signals from extracellular stimuli to heterotrimeric G prote ins. However, the AT(2) receptor signal transduction mechanisms are poorly understood. We have measured AT(2)-triggered activation of purified heterot rimeric proteins in urea-extracted membranes from cultured COS-7 cells expr essing the recombinant receptor. This procedure removes contaminating GTP-b inding proteins without inactivating the serpentine receptor. Binding studi es using [I-125] 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 AT(2) receptor caused a rapid activation of alpha(i) and alpha(o), but not of alpha(q) and alpha(s), as measured by radioactive guanosine 5'-3-O- (thio)triphosphate (GTP gammaS) binding. Activation required the presence o f activated receptors, beta gamma, and alpha subunits. As a first step aime d at developing an in vitro assay to examine AT(2) receptor pharmacology, w e tested a battery of Ang II-related ligands for their ability to promote A T(1) or AT(2) receptor-catalyzed G(i) activation. Two proteolytic fragments of Ang II, Ang III and Ang1-7, also promoted activation of alpha(i) throug h the AT(2) receptor. Furthermore, we found that [Sar(1),Ala(8)]Ang II is a n antagonist for both AT(1) and AT(2) receptors and that CPG42112 behaves a s a partial agonist for the AT(2) receptor. In combination with previous ob servations, these results show that the AT(2) receptor is fully capable of activating G(i) and provides a new tool for exploring AT(2) receptor pharma cology and interactions with G-protein trimers.