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 (AT2 R), 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 AT2 R activation. Although AT2 R has not been shown to engage Gq/11 , G12 , Gi2 , and β-arrestin (βarr) pathways as does the AT1 R upon angiotensin II (AngII) stimulation, the atypical positioning of helix VIII in the recently published AT2 R structure may play a role in the receptor's capacity to couple to downstream effectors. In the AT2 R 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 AT2 R 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 AT1 R/AT2 R receptor chimeras to validate the roles of ICL1 and helix VIII in AT2 R signaling. Substituting the AT1 R ICL1 into AT2 R led to a mutant receptor that coupled to Gi2 . The substitution of the helix VIII and C-terminal domains of AT2 R into the AT1 R backbone led to a mutant receptor that retained AT1 R-like signaling properties. These results suggest that the C-terminal portion of AT2 R is compatible with canonical GPCR signaling and that ICL1 of AT2 R is involved in repositioning helix VIII, which impedes engagement of classical GPCR effectors such as G proteins or β-arrestins.