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

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.