The use of spin-labeled ligands as biophysical probes to report real-time endocytosis of G protein-coupled receptors in living cells
| Autor: | Aaron M. Shafer, John C. Voss |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
DNA
Complementary G protein Cell Survival Endocytic cycle Biophysics Cell Culture Techniques CHO Cells Biology Endocytosis Ligands Transfection Cell Line Receptors G-Protein-Coupled Radioligand Assay Cricetulus Neurotransmitter receptor Cell surface receptor Computer Systems Heterotrimeric G protein Cricetinae Animals G protein-coupled receptor Electron Spin Resonance Spectroscopy General Medicine Receptors Neurokinin-1 Flow Cytometry Cell biology Molecular Probes Spin Labels Signal transduction Peptides |
| Zdroj: | Science's STKE : signal transduction knowledge environment. 2004(232) |
| ISSN: | 1525-8882 |
| Popis: | Recycling and degradation of plasma membrane receptors and transporters are fundamental mechanisms for regulating cell signaling and metabolic processes. For many membrane proteins, endocytosis reduces the number of molecules available for transport or signal transduction, providing an attenuation response. Fluorescent reporters attached to either the receptor or ligand have been used to monitor the trafficking of internalization; however, these approaches provide poor resolution for the early endocytic response. Here, we describe the use of a spin-labeled ligand for a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor for measuring the kinetics of endocytosis in real time. Included are protocols for designing a nitroxide-labeled ligand and measuring receptor endocytosis in live cells using electron paramagnetic resonance (EPR) spectroscopy. Methods for the evaluation of the receptor binding and activation properties of modified ligands and the generation of a cell line stably expressing high receptor levels are also provided. |
| Databáze: | OpenAIRE |
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