The constitutively active N111G-AT1 receptor for angiotensin II maintains a high affinity conformation despite being uncoupled from its cognate G protein Gq/11α

Asn111, localized in the third transmembrane domain of the AT(1) receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller ( Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT(1), recognized I-125-Ang II with high affinities comparable to that of the wild-typeAT(1) receptor. In phospholipase C assays, the four mutants encompassed the entire spectrum of functional states, ranging from constitutive activity ( without agonist) for N111A-AT(1) and N111G-AT(1) to a significant loss of activity ( upon maximal stimulation) for N111Q-AT(1) and a major loss of activity for N111W-AT(1). In Ca2+ mobilization studies, N111W-AT(1) produced a weak Ca2+ transient and, unexpectedly, N111G-AT(1) also produced a Ca2+ transient that was much weaker than that of the wild-type AT(1). The agonist binding affinity of N111W-AT(1) was not modified in the presence of GTPgamma S, suggesting that this receptor is not basally coupled to a G protein. GTPgamma S did not modify the high agonist-binding affinity of N111G- AT(1) but abolished the coimmunoprecipitation of G(q/11alpha) with this constitutively active mutant receptor. These results are a direct demonstration that the N111G- AT(1) receptor maintains a high affinity conformation despite being uncoupled from the G protein G(q/11).