title: Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology.
abstract: Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein-protein interaction at the surface of living cells. This technology is based on the use of specific antibodies directed against each partner and labeled either with europium cryptate or with Alexa Fluor 647. This allows the measurement of a fluorescence resonance energy transfer (FRET) signal in a time-resolved manner if both antibodies are in close proximity. This approach is here validated using the heterodimeric gamma-aminobutyrate B receptor as a model. We show that after washing out the unbound antibodies, the time-resolved FRET signal can be measured together with the expression level of both partners via the quantification of the donor and the acceptor fluorophores bound to the cells. Thanks to the high sensitivity of this method and to the low concentration of antibodies required, we show that the signal can also be measured directly after the incubation period without washing out the unbound antibody (homogeneous time-resolved FRET). As such, this method is highly sensitive, reproducible, and compatible with the development of high-throughput screening protocols.
authors.affiliation: UPR 2580 CNRS, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Maurel
authors.forename: Julie
authors.initials: J
authors.lastname: Kniazeff
authors.forename: Gérard
authors.initials: G
authors.lastname: Mathis
authors.forename: Eric
authors.initials: E
authors.lastname: Trinquet
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pin
authors.forename: Hervé
authors.initials: H
authors.lastname: Ansanay
mesh_terms: D000906:Antibodies / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods*; D018080:Receptors, GABA-B / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000906:Antibodies; D018080:Receptors, GABA-B