title: Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes.
abstract: A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described.
authors.affiliation: Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Dasiel O
authors.initials: DO
authors.lastname: Borroto-Escuela
authors.forename: Marc
authors.initials: M
authors.lastname: Flajolet
authors.forename: Luigi F
authors.initials: LF
authors.lastname: Agnati
authors.forename: Paul
authors.initials: P
authors.lastname: Greengard
authors.forename: Kjell
authors.initials: K
authors.lastname: Fuxe
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists / Q000737:chemistry / Q000494:pharmacology; D001667:Binding, Competitive; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism*; D010627:Phenethylamines / Q000737:chemistry / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D058908:Adenosine A2 Receptor Agonists; D010627:Phenethylamines; D043705:Receptor, Adenosine A2A; D016222:Fibroblast Growth Factor 2; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1; D000241:Adenosine
keywords: Allosteric modulation; BRET competition assays; BRET kinetics and dose–response assays; BRET saturation assays; Bioluminescence resonance energy transfer (BRET); G protein-coupled receptors (GPCRs); GPCR–RTK heteroreceptor complexes; Heterodimerization; Heteroreceptor complexes; Homodimerization; Receptor tyrosine kinases (RTKs); Receptor–receptor interactions