title: Lipid-dependent GPCR dimerization.
abstract: It has been widely demonstrated that G protein-coupled receptors (GPCRs) can form dimers both in vivo and in vitro, a process that has functional consequences. These receptor-receptor interactions take place within a phospholipid bilayer, yet, generally, little is known of the requirements for specific lipids that mediate the dimerization process. Studying this phenomenon in vivo is challenging due to difficulties in modulating the lipid content of cell membranes. Therefore, in this chapter, we describe techniques for reconstitution of GPCRs into model lipid bilayers of defined composition. The concentrations of specific lipids and sterols can be precisely controlled in these liposomes, as well as maintaining an appropriate lipid-protein ratio to avoid artifactual interactions. Receptor dimerization in this system is monitored via Förster resonance energy transfer (FRET), which requires the use of fluorescently labeled receptors. We therefore also include protocols for labeling with appropriate fluorophores and determining the apparent FRET efficiency, a measurement of the extent of receptor dimerization. Understanding the lipid dependence of GPCR dimerization will be key in understanding how this process is regulated in the dynamic heterogeneous environment of the cell membrane.
authors.affiliation: School of Life Sciences, University of Lincoln, Lincoln, United Kingdom.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Goddard
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Dijkman
authors.forename: Roslin J
authors.initials: RJ
authors.lastname: Adamson
authors.forename: Anthony
authors.initials: A
authors.lastname: Watts
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D018028:Receptors, Neurotensin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013010:Sonication
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D008081:Liposomes; D008164:Luminescent Proteins; D010743:Phospholipids; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; C114036:neurotensin type 1 receptor; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins
keywords: Dimer; FRET; G protein-coupled receptor (GPCR); Lipid dependence; Liposome; NTS1