Lysosomal localisation of a novel human arrestin-related protein and its potential role in G protein coupled receptor trafficking

David Lake, Simon Dawson, Nick Holliday. University of Nottingham, School of Biomedical Sciences, NG9 2UH, Nottingham, United Kingdom.

β-Arrestins are essential adaptors for G-protein coupled receptor (GPCR) trafficking1. Recently, arrestin-related proteins have been identified in yeast/fungi that, similar to β-arrestins, recognise cargo proteins and mediate their intracellular trafficking2-4. In mammals, five Arrestin-related domain containing (ARRDC) proteins display the arrestin fold, but their function is largely unknown. Here we examine cellular localisation of ARRDC2 by confocal microscopy and its relationship to unoccupied and agonist-stimulated β2-adrenoceptors (β2AR).

Human osteosarcoma (U2OS) cells, stably expressing β2AR-GFP as appropriate, were transiently transfected with ARRDC2 constructs (lipofectamine). Confocal images of living cells (Zeiss LSM 510M/710M) were obtained using a Plan-Apochromat 63X NA 1.4 oil objective, and suitable laser excitation/emission filter sets. Colocalisation was quantified using Zeiss LSM Image Examiner or ZEN 2009 software. Statistical analysis was by one-way ANOVA and Tukey post test (Prism v5.01).

ARRDC2 (GFP- or mCherry-tagged) was present in punctate vesicular structures throughout the cytoplasm and at the plasma membrane. ARRDC2-mCherry was largely distinct from early endosomal antigen 1 (EEA1)-GFP or GFP-Rab5 labelled early endosomes, and from GFP-Rab11 or transferrin labelled recycling endosomes (13.7±3.0%, 13.6±3.4%, 15.5±4.5%, 11.1±2.2% ARRDC2-mCherry colocalised with EEA1-GFP, GFP-Rab5, GFP-Rab11, internalised transferrin, respectively; n ≥ 3). A higher proportion of ARRDC2 colocalised with the late endosomal marker GFP-Rab7, immunostaining for the lysosomal marker LAMP1, or LysoTracker-stained acidic compartments (33.0±5.3%, 43.0±4.1%, 58.6±6.8%, respectively; P < 0.001, n ≥ 3), indicating a predominantly late endosomal/lysosomal localisation for ARRDC2. In addition, ARRDC2-mCherry colocalised with the prototypical GPCR, the β2-adrenergic receptor (β2AR-GFP), upon stimulation with 10 µM isoprenaline (ISO) for 1 h (67.2±2.3% ARRDC2-mCherry colocalised with β2AR-GFP, n = 5). ARRDC2-mCherry was found to redistribute from LAMP1-positive, transferrin-negative compartments (non-stimulated: 54.3±5.1%, 18.2±3.1% ARRDC2-mCherry colocalised with LAMP1, transferrin, respectively; P < 0.001, n = 5) to LAMP1-negative, transferrin-positive compartments (10 µM ISO 1h: 18.9±3.1%, 42.6±3.8% ARRDC2-mCherry colocalised with LAMP1, transferrin, respectively; P < 0.001, n = 5). Thus, ARRDC2 is localised the endocytic system, predominantly to lysosomes. The recruitment of ARRDC2 from lysosomes to early/recycling endosomal compartments containing internalised β2AR implicates ARRDC2 in a potential role in GPCR cargo protein trafficking. Considering the recent identification of another human arrestin-related protein, ARRDC3, as an adaptor for β2AR ubiquitination and degradation5, the apparent involvement of ARRDC2 is worth further investigation.

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2. Herranz, S et al. (2005) PNAS. 102, 12141-12146.

3. Lin, CH et al. (2008) Cell. 135, 714-725.

4. Nikko, E and Pelham, HRB (2009) Traffic. 10, 1856-1867

5. Nabhan, JF et al. (2010) EMBO Rep. 11, 605-611.