title: A FRET-based approach for quantitative evaluation of forskolin-induced pendrin trafficking at the plasma membrane in bronchial NCI H292 cells.
abstract: BACKGROUND
  Human pendrin (SLC26A4, PDS) is an integral membrane protein acting as an electroneutral anion exchanger. Loss of function mutations in pendrin protein cause Pendred syndrome, a disorder characterized by sensorineural deafness and a partial iodide organification defect that may lead to thyroid goiter. Additionally, pendrin up-regulation could play a role in the pathogenesis of several diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD). Therefore, monitoring the plasma membrane abundance and trafficking of pendrin in the context of a living cell is crucially important.
  
  
  METHODS
  Trafficking of pendrin to the plasma membrane was monitored by fluorescence resonance energy transfer (FRET), a physical phenomenon occurring between two fluorophores (the FRET donor and acceptor) located in close spatial proximity. Because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, FRET is extremely sensitive to small changes in distance between the donor and acceptor and is therefore a powerful tool to determine protein-protein interactions.
  
  
  RESULTS
  FRET studies revealed that forskolin-induced cAMP production is associated with a significant increase of pendrin expression at plasma membrane, which is paralleled by a decrease in intracellular pH. Pendrin transposition to the membrane is accompanied with a partial depolymerization of actin cytoskeleton via Rho-GTPase inhibition.
  
  
  CONCLUSIONS
  Trafficking to the plasma membrane is critical in the regulation of pendrin activity. Therefore, reliable tools for monitoring and quantifying this phenomenon are highly desirable.
authors.affiliation: Dept. of Biosciences Biotechnologies and Biopharmaceutics, University of Bari, Italy.
authors.forename: Grazia
authors.initials: G
authors.lastname: Tamma
authors.forename: Marianna
authors.initials: M
authors.lastname: Ranieri
authors.forename: Silvia
authors.initials: S
authors.lastname: Dossena
authors.forename: Annarita
authors.initials: A
authors.lastname: Di Mise
authors.forename: Charity
authors.initials: C
authors.lastname: Nofziger
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
authors.forename: Markus
authors.initials: M
authors.lastname: Paulmichl
authors.forename: Giovanna
authors.initials: G
authors.lastname: Valenti
mesh_terms: D000199:Actins / Q000378:metabolism; D001980:Bronchi / Q000166:cytology / Q000187:drug effects*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006044:Goiter, Nodular / Q000235:genetics* / Q000473:pathology; D006319:Hearing Loss, Sensorineural / Q000235:genetics* / Q000473:pathology; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D021381:Protein Transport / Q000187:drug effects; D000076988:Sulfate Transporters
chemical_list: D000199:Actins; D026901:Membrane Transport Proteins; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D005576:Colforsin