Interplay between intracellular loop 1 and helix VIII of the angiotensin II type 2 receptor controls its activation.

The signaling mechanisms of the angiotensin II type 2 receptor (ATR), a heptahelical receptor, have not yet been clearly and completely defined. In the present contribution, we set out to identify the molecular determinants involved in ATR activation. Although ATR has not been shown to engage G, G, G, and β-arrestin (βarr) pathways as does the ATR upon angiotensin II (AngII) stimulation, the atypical positioning of helix VIII in the recently published ATR structure may play a role in the receptor's capacity to couple to downstream effectors. In the ATR structure, helix VIII points inwards and towards intracellular loop 3 (ICL3) to form tertiary interactions with transmembrane domain 6 (TM6), possibly impeding access to signaling effectors. On the other hand, in most class A GPCRs, helix VIII is found to be engaged in tertiary interactions with ICL1 and away from the effector binding site. Upon closer examination of the ATR structure, we found that the residues contained within intracellular loop 1 (ICL1) may be involved in driving this unusual conformation of helix VIII. To explore this hypothesis, we designed a series of ATR/ATR receptor chimeras to validate the roles of ICL1 and helix VIII in ATR signaling. Substituting the ATR ICL1 into ATR led to a mutant receptor that coupled to G. The substitution of the helix VIII and C-terminal domains of ATR into the ATR backbone led to a mutant receptor that retained ATR-like signaling properties. These results suggest that the C-terminal portion of ATR is compatible with canonical GPCR signaling and that ICL1 of ATR is involved in repositioning helix VIII, which impedes engagement of classical GPCR effectors such as G proteins or βarrs.