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

Walter G Thomas,Hongwei Qian,Chang-Sheng Chang,Sadashiva Karnik

doi:10.1074/jbc.275.4.2893

Abstract

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 (AT(1A)) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar(1),Ile(4),Ile(8)]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT(1A) receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT(1A) 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 AT(1) receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar(1),Ile(4),Ile(8)]AngII. Paradoxically, [Sar(1),Ile(4), Ile(8)]AngII produced a robust ( approximately 85% of AngII), dose-dependent phosphorylation of the wild type AT(1A) 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 [Sar(1),Ile(4),Ile(8)]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT(1A) 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.

Highlights

Heterotrimeric guanyl nucleotide-binding proteins (G proteins)1 are activated by an array of sensory and hormonal stimuli
We observed that the constitutively active AT1A receptors (N111A and N111G) are poor substrates for phosphorylation and that this cannot be modulated by angiotensin II (AngII) or [Sar1,Ile4,Ile8]AngII stimulation or by prior conversion of the constitutive receptors to the basal state by pretreatment with the inverse agonist, EXP3174
The wild type receptor was efficiently phosphorylated by the supposedly inactive analog, [Sar1,Ile4,Ile8]AngII, indicating that the molecular switches required for phosphorylation are distinct from those necessary for G protein-mediated signaling

Summary

Introduction

Heterotrimeric guanyl nucleotide-binding proteins (G proteins)1 are activated by an array of sensory and hormonal stimuli. The non-signaling AngII analog, [Sar1,Ile4,Ile8]AngII, induced robust phosphorylation of the wild type AT1A receptor, but this analog did not stimulate internalization of either wild type or constitutively active mutant AT1A receptors. 4. [Sar1,Ile4,Ile8]AngII-stimulated phosphorylation of wild type and constitutively active AT1A receptors.

Results

Conclusion