In the current model of receptor activation, the given hormone is not involved in the conversion of the inactive receptor (R) to the fully active state (R*). Rather, it preferentially selects the activated receptor conformation, thereby shifting the equilibrium toward R*. The hormone angiotensin II (Ang II) contains two residues, Tyr4 and Phe8, that are essential for agonism. We show that the conserved Asn111 in transmembrane helix III of the AT1 angiotensin receptor directly interacts with the Tyr4 side chain. A decrease in the size of the Asn111 side chain induces an intermediate activated receptor conformation (R'). The Ang II analogue [Sar1,Ile4,Ile8]Ang II fully activates the N111G mutant, indicating that either the transition from R' to R* or the stabilization of the R* state requires binding by Ang II but not its Tyr4 and Phe8 side chains. In contrast, [Sar1,Ile4,Ile8]Ang II binds to but does not activate the wild-type AT1 receptor (R), suggesting that in the wild-type receptor spontaneous occurrence of R' and R* states is rare. Thus, Ang II through interactions involving Tyr4 and Phe8 induces a transition from R to R' and through unspecified interactions induces transition from R' to R* states rather than stabilizing the spontaneously generated R* state by "conformational, selection".

The AT1 receptor is a G-protein-coupled receptor (GPCR); its activation from the basal state (R) requires an interaction between Asn111 in transmembrane helix III (TM-III) of the receptor and the Tyr4 residue of angiotensin II (Ang II). Asn111 to Gly111 mutation (N111G) results in constitutive activation of the AT1 receptor (Noda et al. (1996) Biochemistry, 35, 16435-16442). We show here that replacement of the AT1 receptors TM-III with a topologically identical 16-residue segment (Cys101-Val116) from the AT2 receptor induces constitutive activity, although Asn111 is preserved in the resulting chimera, CR18. Effects of CR18 and N111G mutations are neither additive nor synergistic. The conformation(s) induced in either mutant mimics the partially activated state (R'), and transition to the fully activated R conformation in both no longer requires the Tyr4 of Ang II. Both the R state of the receptor and the Tyr4 Ang II dependence of receptor activation can be reinstated by introduction of a larger sized Phe side chain at the 111 position in CR18, suggesting that the CR18 mutation generated an effect similar to the reduction of side chain size in the N111G mutation. Consistently in the native AT1 receptor, R' conformation is generated by replacement with residues smaller but not larger than the Asn111. However, size substitution of several other TM-III residues in both receptors did not affect transitions between R, R', and R states. Thus, the property responsible for Asn111 function as a conformational switch is neither polarity nor hydrogen bonding potential but the side chain size. We conclude that the fundamental mechanism responsible for constitutive activation of the AT1 receptor is to increase the entropy of the key agonist-switch binding residue, Asn111. As a result, the normally agonist-dependent R --> R' transition occurs spontaneously. This mechanism may be applicable to many other GPCRs.