Elucidating GPCR Functional Dependence on Plasma Membrane Composition using Giant Unilamellar Protein-Vesicles

Abstract

Using an agarose hydration technique for protein incorporation into vesicular bilayers, we elucidate the effects of membrane composition and ordering on G Protein Coupled Receptors (GPRCs). We successfully incorporate GPCRs into model membranes in the form of giant unilamellar proteinvesicles (GUPs). Using this completely in vitro platform we observe that the functional rate of the human serotonin receptor, GPCR 5-HT 1A , and the A 2 a Adenosine GPCR is dependent on membrane composition and ordering. We use BODIPY-GTPgS as our fluorescent marker to track the irreversible exchange between GDP and GTP on G proteins over time in GUPs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), brain sphingomyelin (BSM), and cholesterol (Chol) as well as synthetic lamellar phase diblock copolymer. Furthermore, using this approach we demonstrate that the incorporated receptors display a biased orientation with the N-terminus located on the exterior (extracellular) and the C-terminus on the interior (cytosolic).