The role of hydrophobic plasma membrane interior in DOR regulation of trimeric G protein activity

The present understanding of the role of membrane organization in functional coupling between δ-opioid receptor (DOR) and the cognate G proteins is unclear. G protein activation is impaired by cholesterol depletion but receptor agonist binding is unchanged. The aim of this project is the detailed correlation between structural and dynamic parameters of plasma membranes gained by fluorescence spectroscopy and microscopy and functional state of DOR-initiated signaling cascade (ligand binding studies, high-affinity GTPγS binding, GTPase assays, adenylyl cyclase). These studies will be performed in intact cells, isolated plasma membranes (PM) and their compartments (membrane domains) prepared from brain cortex and stable cell lines expressing DOR-Gi1α (Cys351-Ile351), DOR and its fluorescent analog DOR-YFP. PM will be reconstituted into model membranes, e.g. into giant unilamellar vesicles. Using cutting edge microscopy techniques (raster image correlation spectroscopy, FLIM) membrane organisation and mobility of fluorescent lipids and DOR-YFP will be followed.