title: Translocation of signalling proteins to the plasma membrane revealed by a new bioluminescent procedure.
abstract: BACKGROUND
  Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors.High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.
  
  
  RESULTS
  Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²⁺)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²⁺concentration [Ca²⁺] difference between bulk cytosolic and the sub-plasma membrane rim.
  
  
  CONCLUSIONS
  This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane.Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation.Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.
authors.affiliation: Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI) and LTTA center, University of Ferrara, Ferrara, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Giorgi
authors.forename: Anna
authors.initials: A
authors.lastname: Romagnoli
authors.forename: Chiara
authors.initials: C
authors.lastname: Agnoletto
authors.forename: Leda
authors.initials: L
authors.lastname: Bergamelli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Sorrentino
authors.forename: Marisa
authors.initials: M
authors.lastname: Brini
authors.forename: Tullio
authors.initials: T
authors.lastname: Pozzan
authors.forename: Jacopo
authors.initials: J
authors.lastname: Meldolesi
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D064546:Protein Kinase C beta; D021381:Protein Transport; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D000331:Aequorin; D020794:Receptor Protein-Tyrosine Kinases; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D002118:Calcium