Constitutive Internalization of Constitutively Active Angiotensin II AT1A Receptor Mutants Is Blocked by Inverse Agonists

Stéphanie Miserey-Lenkei,Charles Parnot,Sabine Bardin,Pierre Corvol,Eric Clauser

doi:10.1074/jbc.m108398200

Abstract

As constitutively active mutants (CAMs) mimic an active conformation, they can be used to characterize the process of G protein-coupled receptor activation. Here, we used CAMs to study the link between activation and internalization of the angiotensin II AT(1A) receptor. The cellular localization of fluorescently tagged N111A, I245T, and L305Q mutants was determined by confocal microscopy. In the absence of ligand, CAMs were mostly located in intracellular vesicles, whereas the wild-type AT(1A) was found at the cell surface. After 2 h incubation with inverse agonist, losartan, CAMs were translocated to the plasma membrane. Similar observations were made in H295, a human adrenocortical cell line which expresses physiologically the AT(1) receptor. This phenomenon, which was not dependent on protein synthesis and the pharmacology and kinetics of which were similar to the recycling of the wild-type receptor, was called "externalization". After externalization and losartan removal, the L305Q CAM underwent rapid ligand-independent endocytosis, with the same kinetics and temperature sensitivity as the angiotensin II-induced internalization of the wild-type AT(1A). Moreover, the addition of a second mutation known to block internalization (Delta 329 truncation) prevented intracellular localization of the CAM. These data show that AT(1A) CAMs are constitutively and permanently internalized and recycled. This mechanism is different from the down-regulation observed for CAMs of other G protein-coupled receptors and thus defines a new paradigm for the cellular regulation of CAMs.

Highlights

G protein-coupled receptors (GPCR)1 form one of the largest protein families, with several hundred members in humans [1]
Characterization of the EGFP-tagged constitutively active mutants (CAMs) of the angiotensin II (AngII) AT1A Receptor—The CAM N111A, I245T, L305Q, and the wildtype (WT) AT1A receptors were tagged at the N terminus with EGFP to determine their subcellular localization and trafficking
The Double Mutant, EGFP-L305Q/⌬329, Is Localized at the Plasma Membrane—To confirm the role of internalization in the cellular localization of the CAM AT1A receptors, we used a AT1A receptor mutant truncated at residue 329, which presents a default of internalization [19]

Summary

Introduction

G protein-coupled receptors (GPCR) form one of the largest protein families, with several hundred members in humans [1]. GPCRs are supposed to isomerize spontaneously between an inactive (R) and an active state (R*), the latter being responsible for G protein coupling and subsequent intracellular signaling This two-state model is probably oversimplified but is helpful for the interpretation of mutagenesis and pharmacological data. The arrestins prevent further interaction with the G proteins They promote internalization via clathrincoated pit-dependent endocytosis, which results in the disappearance of the receptor from the plasma membrane [2]. The AT1 receptor regulates the contraction and hypertrophy of vascular smooth muscle cell contraction and the secretion of aldosterone It plays a critical role in the control of blood pressure and sodium homeostasis.

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