Fabricating a New Stabilized Lipid-Based Platform for Handling and Presenting GPCRs

Abstract

G protein-coupled receptors (GPCRs) are members of large family of signaling molecules. They have a key function in neuronal biology and play a critical rule in transmitting extracellular signals in eukaryotic cells. GPCRs include receptors for many neurotransmitters like serotonin. The serotonin receptor 5HT1A has an important role in mental disorders like anxiety, and is the target of some anti anxiety drugs. Here we constructed a novel biomimetic lipid membrane-based platform to be used for screening molecules that interact with GPCRs. Since fragility and short life time limit the applicability of liposomes that are normally used to present membrane proteins, we have developed a platform based on nanoscale liposomes with enhanced longevity and stability. We developed two approaches to make stable liposomes: liposomes containing a UV-initiated poly(ethylene glycol) (PEG) hydrogel and conjugated hydrogel liposomes made from lipids covalently anchored to the hydrogel network. Stability of nanoscale liposomes was confirmed by addition of high concentration of sodium dodecyl sulfate (SDS) to the liposome suspension. The liposome/detergent micelle mixture was passed over a size exclusion chromatography (SEC) column, separating intact liposomes from micelles. Using dynamic light scattering (DLS), we could confirm the existence of intact 160 nm liposomes. This result was in good agreement with the SEC result for polymerized liposomes without detergent. Since our goal is making GPCR-bearing liposomes, serotonin receptor 5HT1A was incorporated into liposomes using a detergent-mediated method. GPCR incorporation was confirmed by binding labeled HTR1A antibody to liposomes containing biotinylated lipids and subsequently separating them from bulk with streptavidin-coated magnetic beads. We are currently undertaking further studies on antibody-liposome binding by fluorescence anisotropy to validate our approach.