A NOVEL PATHWAY FOR ENDOCYTOSIS OF OXIDIZED LDL VIA AN ALLOSTERIC ACTIVATION OF THE ANGIOTENSIN II TYPE 1 RECEPTOR

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Objective: Arrestin-dependent internalization of a G-protein-coupled receptor (GPCR) can intracellularly transport adjacent molecules. We previously proposed that a pattern recognition receptor (PRR), lectin-like oxidized LDL (oxLDL) receptor (LOX-1) physically interacts with the angiotensin II type 1 receptor (AT1) and the interaction mediates oxLDL-induced allosteric activation of AT1 and endothelial dysfunction. In this study, we analyzed direct downstream signaling pathways of AT1 by oxLDL, and investigated if the oxLDL-LOX-1 complex utilizes AT1-beta arrestin internalization to undergo endocytosis. Design and method: We used CHO-cells stably transfected with LOX-1 (CHO-LOX-1), AT1 (CHO-AT1), LOX-1 and AT1 (CHO-LOX-1-AT1), and LOX-1 and mutated AT1 that lacks ability to activate G protein or beta-arrestin pathway (CHO-LOX-1-mAT1). Activation of G(alpha)q and G(alpha)i were quantified by measuring the accumulation of IP1 and the inhibition of forskolin-induced cAMP production, respectively. Endocytosis of the oxLDL-LOX-1 complex through the AT1-beta-arrestin pathway was detected by an AT1-beta-arrestin BRET assay, real-time imaging of the membrane dynamics of LOX-1, and visualization of endocytosis of oxLDL. Endocytosis of oxLDL was assessed by the quantification of fluorescent intensity in cells with treatment and subsequent washout of Dil-labelled oxLDL. Results: Using genetically modified CHO cells, we found that oxLDL induced G(alpha)i-dependent inhibition of cAMP production only in the presence of both LOX-1 and AT1 with intact G protein binding. In contrast, oxLDL did not induce the accumulation of IP1, a downstream of G(alpha)q signaling, in CHO cells expressing both AT1 and LOX-1. The BRET signal between AT1-beta-arrestin was exerted by oxLDL only in the presence of LOX-1. Real-time imaging of cellular membrane revealed that oxLDL induced endocytosis of LOX-1 that depends on the intact beta-arrestin pathway of AT1. oxLDL accumulated in CHO cells expressing both LOX-1 and AT1 more prominently than those expressing LOX-1 or AT1 alone. This enhanced accumulation depended on the beta-arrestin-binding potency of AT1, and was abolished by dominant-negative or pharmacological inhibition of beta-arrestin. Accumulation of oxLDL was similarly attenuated by knockdown of AT1, beta-arrestin or LOX-1 in human vascular endothelial cells. Conclusions: oxLDL triggers selective G protein activation and beta-arrestin-dependent internalization of AT1 whereby the oxLDL-LOX-1 complex undergo endocytosis.