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

TY - JOUR

T1 - Ligand binding to G protein-coupled receptors in tethered cell membranes

AU - Martinez, Karen L.

AU - Meyer, Bruno H.

AU - Hovius, Ruud

AU - Lundstrom, Kenneth

AU - Vogel, Horst

PY - 2003/12/23

Y1 - 2003/12/23

N2 - 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.

AB - 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.

U2 - 10.1021/la035354t

DO - 10.1021/la035354t

M3 - Journal article

AN - SCOPUS:0348196678

SN - 0743-7463

VL - 19

SP - 10925

EP - 10929

JO - Langmuir

JF - Langmuir

IS - 26

ER -