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Ligand binding to G protein-coupled receptors in tethered cell membranes

Publikation: Forskning - peer reviewTidsskriftartikel

Karen L. Martinez, Bruno H. Meyer, Ruud Hovius, Kenneth Lundstrom, Horst Vogel

G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the molecular details of ligand binding is important for the understanding of receptor function and in turn for the design and development of novel therapeutic compound. Here we show how ligand-receptor interaction can be investigated in situ with high sensitivity on sensor surfaces by total internal reflection fluorescence (TIRF) measurements. A generally applicable method for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification.

OriginalsprogEngelsk
TidsskriftLangmuir
Vol/bind19
Tidsskriftsnummer26
Sider (fra-til)10925-10929
Antal sider5
ISSN0743-7463
DOI
StatusUdgivet - 23 dec. 2003

ID: 131611292