A METHOD FOR SCREENING SUBSTANCES FOR EFFECT ON CAMP LEVELS BASED ON INTRACELLULAR TRANSLOCATION OF PKA

摘要

Cells are genetically modified to express a luminophore, e.g., a modified (F64L, S65T, Y66H) Green Fluorescent Protein (GFP, EGFP) coupled to a component of an intracellular signalling pathway such as a transcription factor, a cGMP-or cAMP-dependent protein kinase, a cyclin-, calmodulin- or phospholipid-dependent or mitogen-activated serine-threonin protein kinase, a tyrosine protein kinase, or a protein phosphatase (e.g. PK A, PKC, Erk, Smad, VASP, actin, p38, Jnk1, PKG, IkappaB, CDK2, Grk5, Zap70 , p85, protein-tyrosine phosphatase 1C, Stat5, NFAT, NFkappaB, RhoA, PKB). An influence modulates the intracellular signalling pathway in such a way th at the luminophore is being redistributed or translocated with the component in living cells in a manner experimentally determined to be correlated to the degree of the influence. Measurement of redistribution is performed by recording of light intensity, fluorescence lifetime, polarization, wavelengt h shift, resonance energy transfer, or other properties by an apparatus consisting of e.g. a fluorescence microscope and a CCD camera. Data stored as digital images are processed to numbers representing the degree of redistribution. The method can be used as a screening program for identifying a compound that modulates a component and is capable of treating adisease related to the function of the component.