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Internalization dissociates ?2-adrenergic receptors.

ABSTRACT: G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that ?(2)-adrenergic receptors (?(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of ?(2)ARs between subcellular compartments. BRET between ?(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between ?(2)ARs and endosome markers increases. Energy transfer between ?(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled ?(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that ?(2)ARs associate transiently with each other in the plasma membrane, or that ?(2)AR dimers or oligomers are actively disrupted during internalization.

SUBMITTER: Lan TH

PROVIDER: S-EPMC3043075 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Internalization dissociates β2-adrenergic receptors.

Lan Tien-Hung TH Kuravi Sudhakiranmayi S Lambert Nevin A NA

PloS one 20110222 2

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β(2)-adrenergic receptors (β(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance ...[more]

PMID: 21364942

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Project description:AimsAutoantibodies against second extracellular loops of ?(1)-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function.Methods and resultsIgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to ?(1)-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed.Conclusions(i) Autoantibodies trigger conformation changes in the ?(1)-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

Dataset Information

Internalization dissociates ?2-adrenergic receptors.

Publications

Internalization dissociates β2-adrenergic receptors.

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