title: Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins.
abstract: Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a fluorogen activating protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors. Fluorescence microscopy and flow cytometry demonstrate that this dye does not cross the plasma membrane, binds with high affinity to a dL5** FAP-GPCR fusion construct, activating tagged surface receptors within seconds of addition. The ability to rapidly and selectively label cell surface receptors with a fluorogenic genetically encoded tag allows quantitative imaging and analysis of highly dynamic processes like receptor endocytosis and recycling.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA. bruchez@cmu.edu.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yan
authors.forename: Brigitte F
authors.initials: BF
authors.lastname: Schmidt
authors.forename: Lydia A
authors.initials: LA
authors.lastname: Perkins
authors.forename: Matharishwan
authors.initials: M
authors.lastname: Naganbabu
authors.forename: Saumya
authors.initials: S
authors.lastname: Saurabh
authors.forename: Susan K
authors.initials: SK
authors.lastname: Andreko
authors.forename: Marcel P
authors.initials: MP
authors.lastname: Bruchez
mesh_terms: D002478:Cells, Cultured; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008697:Methane / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D043562:Receptors, G-Protein-Coupled; D008697:Methane