Étude du rôle de la tyrosine kinase Src dans la régulation de la signalisation des récepteurs opioïdes delta (∆OR)
Opioids are the most effective analgesics available but their use is limited by  tolerance. Tolerance is related, at least in part, to receptor desensitization. Hence, the  role of the present study was to better characterize the desensitization process, in  particular concerning the role of the tyrosine kinase Src on regulation of delta opioid  receptor signalling. Our results show that pharmacological inhibition with PP2  (administered at low concentration: 20-40µM) or molecular inhibition of the kinase  with low expression levels of a dominant negative mutant of Src (0,2µg of DNA)  potentiate the magnitude and duration of agonist-dependent (DPDPE; 1µM; 5 min)  activation of the ERK pathway. We also showed that higher concentrations of Src  inhibitors (80 and 100µM of PP2 or 1µg/ml of dominant negative mutant DNA)  block the MAPK cascade following DOR stimulation by DPDPE. These  observations indicate that Src has a biphasic effect on ERK activity, respectively  potentiating or inhibiting agonist stimulation of the MAPK cascade at low and high  levels of Src inhibition. We reasoned that high levels of Src blockers were interfering  with ERK activation mechanism while low levels of inhibition were interfering with  receptor desensitization. This possibility was tested by using cAMP accumulation  assays to evaluate the effect of Src blockers (PP2, 20 µM; 1h) on agonist-induced  desensitization. DOR stimulation by DPDPE inhibited forskolin stimulated cAMP  production in a dose dependent manner with a maximal reduction of 61%. This  inhibitory response was reduced by 72% following pre-exposure to DPDPE (1 µM,  30 min), an effect that was blocked by pre-treating cells with PP2 (PP2, 20 µM; 1 h)  before desensitization. The protective effect of Src blockers did not involve changes  in DOR internalization but interfering with internalization by using an  internalization-deficient DOR mutant or hypertonic medium (0.4M sucrose) reduced  this protection, indicating the need for optimal internalization in order for the  protective effect of Src blockers to take place. Based on the latter observation it was  possible to conclude that Src contribution to DOR desensitization is post-endocytic.
Desensitization truncated mutant delta opioid receptor (DOR344T) Src PP2 DPDPE Internalization Tolerance mitogen-activated protein kinase (MAPK) Extracellular Signal-Regulated Protein Kinase (ERK) cyclic adenosine monophosphate (cAMP)