Mechanism of constitutive activation of the AT1 receptor:: Influence of the size of the agonist switch binding residue Asn111

The AT(1) receptor is a G-protein-coupled receptor (GPCR); its activation from the basal state (R) requires an interaction between Asn(111) in transmembrane helix In (TM-III) of the receptor and the Tyr(4) residue of angiotensin II (Ang II). Asn(111) to Gly(111) mutation (N111G) results in constitutive activation of the AT(1) receptor (Noda et al. (1996) Biochemistry, 35, 16435-16442). We show here that replacement of the AT(1) receptors TM-III with a topologically identical 16-residue segment (Cys(101)-Val(116)) from the AT(2) receptor induces constitutive activity, although Asn(111) 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 Tyr(4) of Ang II. Both the R state of the receptor and the Tyr(4) 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 AT(1) receptor, R' conformation is generated by replacement with residues smaller but not larger than the Asn(111). 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 Asn(111) 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 AT(1) receptor is to increase the entropy of the key agonist-switch binding residue, Asn(111). As a result, the normally agonist-dependent R --> R' transition occurs spontaneously. This mechanism may be applicable to many other GPCRs.