Ligand Binding to G Protein-Coupled Receptors in Tethered Cell Membranes

Abstract

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 compounds. 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.

Supported Cell‐Membrane Sheets for Functional Fluorescence Imaging of Membrane Proteins

“…The orientation of native membranes on the support has been controlled via specific mutual interaction between functionalities on the surfaces of the membrane and of the support, such as biotin/ streptavidin using biotinylated proteins, [18] or less-specific interactions but with multiple interaction sites rendering the immobilization more stable and uniform, for instance using cellulose. [19] However, no system has been proposed for imaging diffusion and other dynamic cellular processes in the same way as on a cell, but taking advantage of performing the experiments in vitro.…”

Kinetics of the Initial Steps of G Protein‐Coupled Receptor‐Mediated Cellular Signaling Revealed by Single‐Molecule Imaging

“…Previous experiments with ensembles of GPCRs in reconstituted systems aimed at the exploitation of GPCRs for sensing purposes [4,5] and allowed the elucidation of molecular interactions in vitro. [6,7] In vivo investigations, in which fluorescent proteins are exploited to study important phenomena related to GPCR function, such as dimerization, [8,9] desensitization, [10] and G-protein behavior upon activation, are also being performed.…”

Applications of silica supports in affinity chromatography

“…Another approach for the immobilization of membrane-bound receptors is to entrap such proteins in a silica sol-gel [96]. Additional examples can be found in the literature [99][100][101][102].…”