title: Quantifying GPCR internalization: a focus on the Kisspeptin receptor.
abstract: GPCR internalization is a critical regulatory step in determining receptor activity. While internalization terminates G protein-coupled signaling, it might be required for G protein-independent signaling. A large number of clinical therapies are based on preventing or promoting GPCR internalization. Thus, for any given GPCR, it is important to characterize its internalization and understand the factors that regulate such internalization. Here we describe different experimental protocols to evaluate the internalization of any GPCR transiently expressed in HEK 293 cells. The protocols describe the use of immunofluorescence and imaging techniques as well as flow cytometry. The techniques described use the FLAG-tagged kisspeptin receptor (KISS1R) as an example but are equally applicable to any other GPCR.
authors.affiliation: The Children's Health Research Institute, Victoria Research Laboratories, The University of Western Ontario, A4-140, 800 Commissioners Road East, London, ON, Canada, N6C 2V5.
authors.forename: Macarena
authors.initials: M
authors.lastname: Pampillo
authors.forename: Andy V
authors.initials: AV
authors.lastname: Babwah
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D060807:Bioluminescence Resonance Energy Transfer Techniques; D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D049408:Luminescent Agents / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D021381:Protein Transport; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D000074502:Receptors, Kisspeptin-1; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; C118688:KISS1R protein, human; D049408:Luminescent Agents; D008164:Luminescent Proteins; D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D000074502:Receptors, Kisspeptin-1; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D008156:Luciferases