MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β2-adrenergic receptors

Abstract

Lysosomal degradation of ubiquitinated β2-adrenergic receptors (β2ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the βAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed β2ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-β2ARs in HEK-293 cells. Carvedilol prevented β2AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography–tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound β2AR. The association of MARCH2 with internalized β2ARs was stabilized by carvedilol and did not involve β-arrestin. Small interfering RNA–mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous β2ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to β blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface β2AR expression and, consequently, its signaling.

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

“…Transient expression of YFP-tagged ␤arr2 constructs in double knockout MEFs was achieved with Lipofectamine 2000 TM transfection using a modified protocol (36). The use of YFPtagged ␤arr2 constructs allowed assessment of transfection efficiency: 30 -50% for the ␤arr2-WT and ␤arr2-0K constructs and 20 -30% for the ␤arr2-Ub construct.…”

“…RNA Interference-Non-targeting control siRNA and siRNA targeting ␤arr2, USP20, or USP33 were purchased from Dharmacon GE Healthcare Life Sciences and described previously (25,36,37). For rescue experiments, siRNA specifically targeting human ␤arr2 or human USP20 were co-transfected along with siRNA-resistant, YFP-tagged rat ␤arr2 (2 g in a 100-mm dish) or HA-tagged mouse USP20 (2 g in a 100-mm dish).…”

Carvedilol binding to β2-adrenergic receptors inhibits CFTR-dependent anion secretion in airway epithelial cells

“…Earlier studies with Calu-3 cells showed that the signaling complex formed by the ␤ 2 -AR, CFTR, and associated scaffolding proteins dissociates following PKA-dependent phosphorylation of CFTR (21). Moreover, carvedilol was shown to induce ␤ 2 -AR ubiquitination and internalization independently of G protein activation and ␤-arrestin recruitment by a mechanism involving interactions with MARCH2-E3 ligase, thus regulating apical expression and signaling of ␤ 2 -ARs (14). Previous investigations suggest that the effect of carvedilol on CFTR surface expression observed in the present study may result from internalization of the ␤ 2 -AR and CFTR, since carvedilol binding to the ␤ 2 -AR does not induce G protein signaling and subsequent PKA-dependent phosphorylation of CFTR, which appears to be necessary for uncoupling of the ␤ 2 -AR-CFTR signaling complex (21).…”

“…As a result, the hemodynamic effects of carvedilol are similar to those of renin-angiotensin system blockers and significantly greater than those of conven-tional ␤-AR antagonists such as atenolol (15,17). Furthermore, carvedilol has been shown to function as an inverse agonist by blocking G protein activation while stimulating ␤-arrestinassociated signaling pathways (14). Structural studies involving the use of site-specific 19 F-nuclear magnetic resonance labels within the ␤ 2 -AR showed that the cytoplasmic ends of helicies 6 and 7 assume distinct conformational states, depending on the activating ligand, with carvedilol primarily affecting the conformational states of helix 7 (18).…”

Regulation of G Protein-Coupled Receptors by Ubiquitination

“…Treatment of the β2AR with β-arrestin biased agonist (carvedilol) also resulted in ubiquitination and lysosomal sorting of the receptor. This appears to be a distinct type of ubiquitination, rather than the one induced by a balanced agonist in which another E3 ligase is involved and where ubiquitin is probably attached to other non-lysine residues [38]. Upon balanced agonist (isoproterenol) treatment, β2AR is ubiquitinated by the HECT-type E3 ligase Nedd4 [36]; while upon β-arrestin biased agonist treatment (carvedilol), the RING-type ubiquitin ligase MARCH2 ubiquitinates the wild type receptor as well as the lysine-lacking mutant [38].…”