title: Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors.
abstract: Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P(1) receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands.
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.
authors.forename: Maikel
authors.initials: M
authors.lastname: Jongsma
authors.forename: Urszula M
authors.initials: UM
authors.lastname: Florczyk
authors.forename: Mariëlle C
authors.initials: MC
authors.lastname: Hendriks-Balk
authors.forename: Martin C
authors.initials: MC
authors.lastname: Michel
authors.forename: Stephan L M
authors.initials: SL
authors.lastname: Peters
authors.forename: Astrid E
authors.initials: AE
authors.lastname: Alewijnse
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D008024:Ligands; D008246:Lysophospholipids / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D008246:Lysophospholipids; D049349:Receptors, Lysosphingolipid; C060506:sphingosine 1-phosphate; D013110:Sphingosine