dc.contributor.author |
Grimsey, Natasha |
en |
dc.contributor.author |
Narayan, Pritika |
en |
dc.contributor.author |
Dragunow, Michael |
en |
dc.contributor.author |
Glass, Michelle |
en |
dc.coverage.spatial |
Adelaide, AUSTRALIA |
en |
dc.date.accessioned |
2012-01-11T23:38:59Z |
en |
dc.date.issued |
2008-11 |
en |
dc.identifier.citation |
Clinical and Experimental Pharmacology and Physiology 35(11):1377-1382 2008 |
en |
dc.identifier.issn |
0305-1870 |
en |
dc.identifier.uri |
http://hdl.handle.net/2292/10463 |
en |
dc.description.abstract |
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 internalization 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. |
en |
dc.language |
EN |
en |
dc.publisher |
Blackwell Publishing |
en |
dc.relation.ispartofseries |
Clinical and Experimental Pharmacology and Physiology |
en |
dc.rights |
Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. Details obtained from http://www.sherpa.ac.uk/romeo/issn/0305-1870/ |
en |
dc.rights.uri |
https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm |
en |
dc.subject |
cannabinoid CB1 receptor |
en |
dc.subject |
downregulation |
en |
dc.subject |
endocytosis |
en |
dc.subject |
G-protein-coupled receptors |
en |
dc.subject |
high-throughput screening |
en |
dc.subject |
internalization |
en |
dc.subject |
protein trafficking |
en |
dc.subject |
CB1 CANNABINOID RECEPTOR |
en |
dc.subject |
RAT-BRAIN |
en |
dc.subject |
INTERNALIZATION |
en |
dc.subject |
AGONIST |
en |
dc.subject |
QUANTIFICATION |
en |
dc.subject |
ENDOCYTOSIS |
en |
dc.subject |
SYSTEM |
en |
dc.title |
A novel high-throughput assay for the quantitative assessment of receptor trafficking |
en |
dc.type |
Journal Article |
en |
dc.identifier.doi |
10.1111/j.1440-1681.2008.04991.x |
en |
pubs.issue |
11 |
en |
pubs.begin-page |
1377 |
en |
pubs.volume |
35 |
en |
dc.rights.holder |
Copyright: 2008 The Authors. Journal compilation; 2008 Blackwell Publishing Asia Pty Ltd. |
en |
dc.identifier.pmid |
18565191 |
en |
pubs.end-page |
1382 |
en |
pubs.publication-status |
Published |
en |
dc.rights.accessrights |
http://purl.org/eprint/accessRights/RestrictedAccess |
en |
pubs.subtype |
Article |
en |
pubs.elements-id |
79928 |
en |
pubs.org-id |
Medical and Health Sciences |
en |
pubs.org-id |
Medical Sciences |
en |
pubs.org-id |
Pharmacology |
en |
pubs.org-id |
Science |
en |
pubs.org-id |
Biological Sciences |
en |
pubs.record-created-at-source-date |
2010-09-01 |
en |
pubs.dimensions-id |
18565191 |
en |