STAT5A interacts with and is phosphorylated upon activation of the mu-opioid receptor.
Signal Transducers and Activators of Transcription (STATs) are transcription factors shown to be activated by G protein-coupled receptors. In the present study, we demonstrate that acute morphine or [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) exposure of COS-7 cells transiently transfected with the micro-opioid receptor and STAT5A, leads to receptor-dependent tyrosine phosphorylation of STAT5A. Activation of HEK293 cells, stably expressing the micro-opioid receptor with micro-opioid agonists results in the transcriptional activation of a STAT-responsive reporter gene. Pertussis toxin has no effect on the level of STAT5A phosphorylation, while the Src inhibitor PP1 abolishes opioid-dependent STAT5A phosphorylation. All three opioid receptor subtypes -micro, delta and kappa- share the conserved motif YXXL (amino-acids 336-339 for the micro-opioid receptor), known to be critical for STAT5A/5B binding. Co-immunoprecipitation and pull-down experiments using a GST-carboxyl-terminal tail of the micro-opioid receptor and rat brain, or COS-7 cell cytosolic extracts, demonstrate the direct binding of STAT5A to this region. Mutation of the Y336 to alanine does not prevent STAT5A binding, whereas deletion of the entire putative STAT5A binding site YXXL abolishes STAT5A interaction to the carboxyl-terminal tail of the micro-opioid receptor. Collectively, our results demonstrate the association of STAT5A with the micro-opioid receptor and reveal novel signalling pathways in the regulation of transcription by the micro-opioid receptor.
Mazarakou
Georgia
G
Laboratory of Cellular Signalling and Molecular Pharmacology, Institute of Biology, National Center for Scientific Research Demokritos, Athens, Greece.
Georgoussi
Zafiroula
Z
D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution / Q000502:physiology; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D004789:Enzyme Activation; D004921:Erythropoietin / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D053614:Janus Kinase 2; D008894:Milk Proteins / Q000378:metabolism*; D008959:Models, Neurological; D009020:Morphine / Q000494:pharmacology; D016296:Mutagenesis / Q000502:physiology; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D051381:Rats; D017450:Receptors, Opioid, mu / Q000378:metabolism*; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D025521:Tumor Suppressor Proteins
D000701:Analgesics, Opioid; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D017450:Receptors, Opioid, mu; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494094:Stat5a protein, rat; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004921:Erythropoietin; D009020:Morphine; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507927:Jak2 protein, rat; D053614:Janus Kinase 2; D016392:Proto-Oncogene Proteins pp60(c-src)