RESUMO
This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (Szent-Gyorgyi et al., Nat Biotechnol 2010;00:000-000).
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/metabolismo , Proteínas de Membrana/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Membrana Celular/metabolismo , Sobrevivência Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Endocitose , Corantes Fluorescentes/química , Transportador de Glucose Tipo 4/metabolismo , Humanos , Proteínas de Membrana/química , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/metabolismoRESUMO
G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Ligantes , Microscopia de Fluorescência , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genéticaRESUMO
Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.