STAT5A interacts with and is phosphorylated upon activation of the µ‐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 µ‐opioid receptor and STAT5A, leads to receptor‐dependent tyrosine phosphorylation of STAT5A. Activation of HEK293 cells, stably expressing the µ‐opioid receptor with µ‐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 –µ, δ and κ– share the conserved motif YXXL (amino‐acids 336–339 for the µ‐opioid receptor), known to be critical for STAT5A/5B binding. Co‐immunoprecipitation and pull‐down experiments using a GST‐carboxyl‐terminal tail of the µ‐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 µ‐opioid receptor. Collectively, our results demonstrate the association of STAT5A with the µ‐opioid receptor and reveal novel signalling pathways in the regulation of transcription by the µ‐opioid receptor.