Clinical and Experimental Pharmacology and Physiology (2008) 35, 1377–1382 doi: 10.1111/j.1440-1681.2008.04991.x Blackwell Publishing Asia High-throughput assay for receptor trafficking High-throughput assay for receptor trafficking Annual Scientific Meeting of ASCEPT 2007 A NOVEL HIGH-THROUGHPUT ASSAY FOR THE QUANTITATIVE ASSESSMENT OF RECEPTOR TRAFFICKING Natasha L Grimsey,* Pritika J Narayan,* † Mike Dragunow* † and Michelle Glass* *Department of Pharmacology and Clinical Pharmacology and † National Research Centre for Growth and Development, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand SUMMARY 1. Receptor transport between intracellular compartments has important consequences for receptor function and is an exciting area of current study. Existing methods for studying receptor trafficking often require labour-intensive techniques or are difficult to quantify reliably. We report a novel high-throughput method that uses automated imaging and analysis tools to accurately quantify cannabinoid CB1 receptor trafficking. 2. Haemagglutinin (HA)-tagged CB1 was stably expressed in HEK-293 cells and cell surface or total receptors were detected immunocytochemically. Images of receptor and nuclear staining were acquired with an automated fluorescent microscope (Discovery-1; Molecular Devices, Sunnyvale, CA, USA) and quantified at high throughput with MetaMorph (Molecular Devices) software. The ‘Granularity’ assay measured internaliza- tion by counting receptor clusters that appear during receptor endocytosis, a well-established approach. Our assay, referred to as ‘Total Grey Value per Cell’ (TGVC), measures the total fluorescence above background, normalized to cell count. 3. Incubation with the cannabinoid agonist HU-210 (100 nmol/L) resulted in rapid CB1 internalization, reaching a maximum within 20 min. Whether quantified by Granularity or TGVC, the time-course of endocytosis could be modelled with exponentially derived curves and with similar half-lives. We demonstrate the sensitivity of our TGVC method by measuring the concentration dependence of CB1 internalization and its versatility by measuring downregulation following chronic agonist exposure, whereby total CB1 was reduced to approximately 55% of basal after 3 h. 4. The TGVC quantification method described is efficient, accurate and versatile and is likely to provide a valuable tool in receptor trafficking studies. Key words: cannabinoid CB1 receptor, downregulation, endocytosis, G-protein-coupled receptors, high-throughput screening, internalization, protein trafficking. INTRODUCTION Receptor trafficking between intracellular compartments is an important and popular area of current research. Regulation of cell surface receptor expression via synthesis, constitutive or agonist- induced internalization, recycling and downregulation have multiple roles in G-protein-coupled receptor (GPCR) signalling and regula- tion. 1 In particular, these pathways are intrinsically linked to drug sensitivity and tolerance owing to their effect on receptor availability and, consequently, the ability of agonists to generate an effective response. 2 To date, approaches to quantifying changes in receptor localization or expression have included radioligand binding with membrane- impermeable or -permeable ligands (to measure cell surface or total receptors, respectively), 3 ratiometric analysis of confocal images 4 and biotin labelling with immunoprecipitation and western blotting. 5 Although these techniques have been applied successfully, limitations can include the availability of appropriate radioligands and the labour-intensive nature of the techniques, resulting in relatively small sample sizes or test conditions. The use of ELISA 6 or flow cytometry 7 to detect immunocytochemical labelling overcomes some of these problems, offering the advantages of larger sampling capacity and more automated quantification. As technologies improve and facilities become more widely avail- able, high-content and high-throughput assays are increasingly being used by basic researchers and pharmaceutical companies alike. 8,9 Such approaches offer attractive advantages, including rapid data acquisition and high-quality outputs, 10 and are applicable to many facets of cell biology, including receptor trafficking. One well-established high-throughput assay for measuring receptor internalization is based on the quantification of clusters of endocytic vesicles that appear in the cytoplasm as receptors internalize from the cell surface, usually as a result of ligand-induced receptor activation. When identified with fluorescent probes, these clusters appear as bright puncta or ‘granules’, the abundance of which has been demonstrated to correlate with receptor endocytosis. 11,12 A limitation of this approach is that its use is restricted to the detection of early endocytosis and it does not lend itself well to recycling or degradation assays because a change in granule number could indicate either process. Correspondence: Associate Professor Michelle Glass, Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Email: m.glass@auckland.ac.nz Presented at the 2007 SEAWP-RMP-ASCEPT joint meeting, Adelaide, December 2007. Received 3 February 2008; revision 7 April 2008; accepted 10 April 2008. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Asia Pty Ltd