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 (AT₂R), 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 AT₂R activation. Although AT₂R has not been shown to engage G_q/11, G₁₂, G_i2, and β-arrestin (βarr) pathways as does the AT₁R upon angiotensin II (AngII) stimulation, the atypical positioning of helix VIII in the recently published AT₂R structure may play a role in the receptor's capacity to couple to downstream effectors. In the AT₂R 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 AT₂R 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 AT₁R/AT₂R receptor chimeras to validate the roles of ICL1 and helix VIII in AT₂R signaling. Substituting the AT₁R ICL1 into AT₂R led to a mutant receptor that coupled to G_i2. The substitution of the helix VIII and C-terminal domains of AT₂R into the AT₁R backbone led to a mutant receptor that retained AT₁R-like signaling properties. These results suggest that the C-terminal portion of AT₂R is compatible with canonical GPCR signaling and that ICL1 of AT₂R is involved in repositioning helix VIII, which impedes engagement of classical GPCR effectors such as G proteins or βarrs.