Functional reconstitution of β 2 -adrenergic receptors utilizing self-assembling Nanodisc technology

Abstract

Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the trans-membrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional 2-adrenergic receptor (β2AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain β2AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allows unrestricted acces... Finding Binding Despite the fact that GPCRs have been of intense interest as pharmacologic targets, a significant subset remain orphan receptors, devoid of known function or defined ligands. A means to reconstitute funtional receptors in a defined environment would be of great utility in accelerating understanding of ligand recognition and transmembrane signaling. Nanodiscs, which consist of a nanoscale lipid bilayer core encircled by a membrane scaffold protein, have proven suitable for reconstitution of membrane proteins obtained in purified form or isolated in crude membrane preparations. In work published on p. 601, Leitz et al. describe how Nanodiscs can be used to reconstitute GPCRs that are fully functional. The study focuses on the β2-adrenergic receptor, and provides evidence demonstrating faithful binding of antagonists and agonists. In addition, the β2AR-Nanodisc complex is shown to functionally interact with a soluble G protein, an event that cannot occur when the GPCR is solubilized by deterg...