Research ArticleInhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors
Introduction
Signaling by β2-adrenergic receptors (β2ARs) upon treatment with β2-agonists is attenuated by several distinct mechanisms. Phosphorylation of receptors by G-protein-coupled receptor (GPCR) kinases and protein kinases A and C [1], [2], [3] causes partial uncoupling from G-proteins. Similar to other GPCRs, phosphorylated β2ARs then bind with high affinity to β-arrestin, which fully uncouples receptors from G proteins [4] and also interacts with clathrin and adaptor proteins, thereby facilitating rapid receptor endocytosis [5], [6]. Internalized receptors traffic through early endosomes, localizing with transferrin receptors [7] and rab5a [8] before returning to the cell surface (recycling). During prolonged exposure to agonists, β2ARs may also sort from early endosomes to lysosomes via late endosomes for degradation [9], [10]. Receptor endocytosis and subsequent intracellular sorting events are thus critical regulators of surface β2AR number, thereby influencing the intensity of tissue responses to β-agonists used as therapeutic agents.
Given the importance of proper postendocytic sorting to maintain adequate surface receptor numbers, much effort has been made to discover intracellular factors that regulate the choice between receptor degradation and receptor recycling. An important advance in the study of protein sorting was the discovery that phosphatidylinositol 3-kinase (PI 3-kinase), which phosphorylates phosphatidylinositol to phosphatidylinositol 3-phosphate (PtdIns3P), is required for the transport of membrane proteins to the yeast vacuole [11]. Subsequent work with intact mammalian cells showed that pharmacologic inhibition of PI 3-kinase by the drugs LY294002 or wortmannin inhibits the intracellular trafficking of receptors, including the recycling of transferrin receptors [12], [13], [15] and the degradation of PDGF receptors [16], [17]. A previous study also showed that wortmannin treatment reduces the recycling kinetics of the G protein-coupled angiotensin-1 receptor [18], indicating that postendocytic trafficking of GPCRs might be regulated by phosphoinositides. However, there is no information regarding the dependence of β2AR degradation or recycling on PtdIns3P production.
Since PI 3-kinase inhibitors affect the postendocytic sorting of other receptors, we sought to determine whether such compounds would affect the degradation or recycling of β2ARs. We thus treated HEK293 cells stably expressing β2ARs with PI 3-kinase inhibitors and then examined agonist-induced receptor endocytosis and postendocytic sorting, including the recycling and degradation of internalized receptors.
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Cells and reagents
12β6 cells, an HEK293 line expressing ∼ 1 pmol/mg of hemagglutinin (HA)-tagged β2ARs, were obtained from B. Kobilka. A rabbit polyclonal antibody to the human β2AR C-terminus (H-20) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A rabbit polyclonal antibody to the human Na+/K+-ATPase was obtained from Upstate Cell Signaling Solutions (Charlottesville, VA), and a monoclonal antibody mHA.11 against the HA tag from Covance (Berkeley, CA). Fluorescent secondary antibodies (goat
Treatment with PI 3-kinase inhibitors augments agonist-induced β2AR internalization
To determine the effect of PI 3-kinase inhibitors on the cellular trafficking of agonist-activated β2ARs, various studies were performed on HEK293 cells stably expressing β2ARs (12β6 cells). Initially, we determined the concentration and effect of the PI 3-kinase inhibitor, LY 294002 on receptor internalization. Cells were treated with varying concentrations of LY294002, followed by a saturating concentration of a full agonist, isoproterenol (ISO) for 20 min as detailed in Materials and methods
Discussion
Our major finding is that inhibitors of PI 3-kinase disrupt the postendocytic sorting of β2ARs. This is manifested in three ways: (1) internalized β2ARs are prevented from recycling normally to the cell surface (Fig. 3C); (2) the rate of agonist-induced receptor degradation is slowed (Fig. 5); and (3) β2ARs accumulate in EGFP-rab7-positive vesicles (Fig. 6). The effect on recycling is different for β2ARs compared with transferrin receptors (Fig. 4), as the latter appear to enter a recycling
Acknowledgments
We thank D. Eikenburg and A. Bean for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (R01HL064934, R.H.M. and R01 HL50047, B.J.K.) and the American Heart Association, Texas Affiliate (0455072Y, B.J.K.). Important technical assistance was provided by L. Iles.
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These authors contributed equally.