title: Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells.
abstract: Green bioluminescence in Renilla species is generated by a approximately 100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.
authors.affiliation: Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Roma, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Molinari
authors.forename: Ida
authors.initials: I
authors.lastname: Casella
authors.forename: Tommaso
authors.initials: T
authors.lastname: Costa
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000737:chemistry; D049449:Luminescence; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D048790:Renilla; D013917:Thrombin / Q000737:chemistry
chemical_list: D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D013917:Thrombin; D007930:Leucine