Reconstitution of Membrane-associated Components of a G-protein Signaling Pathway on Membrane-coated Nanoparticles (Lipobeads)

G-protein coupled signaling pathways are organized into multi-protein complexes called signalosomes that are located within and on cellular membranes. We describe the use of silica nanoparticles coated with a unilamellar phospholipid bilayer (lipobeads) to reconstitute the activated photoreceptor G-protein a-subunit (Gta*) with its cognate effector (phosphodiesterase-6; PDE6) for biochemical and structural studies of the activation mechanism regulating this GPCR signaling pathway. Lipobeads are prepared by resuspending dried-down phospholipid mixtures with monodisperse 70 nm silica particles, followed by extrusion through a 100 nm membrane filter. This uniform and supported liposomal preparation is easily sedimented, permitting the separation of soluble from membrane-associated proteins. Upon loading lipobeads with Gta* and PDE6, we find that activation of PDE6 catalysis by Gta* occurs much more efficiently than in the absence of membranes. Chemical cross-linking of membrane-confined proteins allows detection of changes in protein-protein interactions, resulting from G-protein activation of PDE6. The advantages of using lipobeads over partially purified membrane preparations or traditional liposomal preparations are generally applicable to the study of other membrane-confined signal transduction pathways.

Automated summary

This study describes the use of silica nanoparticles coated with a phospholipid bilayer to reconstitute and study the GPCR signaling pathway. The use of these lipobeads allows for efficient activation of the pathway and detection of protein-protein interactions. The method has advantages over traditional liposome preparations and is applicable to other membrane-confined signaling pathways.