Agonist-induced Phosphorylation of the Angiotensin II (AT1A) Receptor Requires Generation of a Conformation That Is Distinct from the Inositol Phosphate-signaling State*

G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT1A) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar1,Ile4,Ile8]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT1A receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT1A receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT1 receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar1,Ile4,Ile8]AngII. Paradoxically, [Sar1,Ile4,Ile8]AngII produced a robust (∼85% of AngII), dose-dependent phosphorylation of the wild type AT1A receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar1,Ile4,Ile8]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT1A receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.