Molecular imaging: into in vivo interaction of HIF-1alpha and HIF-2alpha with ARNT.
Fluorescence resonance energy transfer (FRET) combined with confocal laser microscopy is a powerful tool to analyze protein-protein interaction in vivo. We have applied this combination to study the assembly of the hypoxia-inducible factor (HIF) complex in living cells under hypoxic conditions. In hypoxia, the basic helix-loop-helix/Period/ARNT/Single-minded (PAS) proteins HIF-1alpha and HIF-2alpha accumulate and are translocated into the nucleus. Here, HIF-1alpha and HIF-2alpha dimerize with HIF-1beta, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), to form HIF-1/HIF-2 complexes, which control the expression of specific target genes. Therefore, a new Java-based analyzing program was developed at our institute to calculate the nanometer distance between alpha and beta subunits of the transcriptionally active HIF-1/-2 complex bound to DNA. Fusion proteins of HIF subunits and variants of green fluorescent proteins (cyan and yellow fluorescent proteins) were expressed in living cells and protein-protein interactions were imaged in vivo by means of FRET.
Institute for Physiology, University of Duisburg-Essen, Essen, Germany.
Rebecca
R
Konietzny
Anna
A
König
Christoph
C
Wotzlaw
André
A
Bernadini
Utta
U
Berchner-Pfannschmidt
Joachim
J
Fandrey
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D011485:Protein Binding; D055503:Protein Multimerization; D000099162:Endothelial PAS Domain-Containing Protein 1
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000099162:Endothelial PAS Domain-Containing Protein 1