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COIMMUNOPRECIPITATION Bélocine Musolo Archana Koneswaran M1 BBC. The oxytocin (OT) is a circulating hormone. It is involved in several physiological functions, including uterine contraction, milk ejection and in maternal behaviour. The action of OT is mediated by the OT receptor (OTR). This receptor is coupled to a G Protein (GPCR). GPCRS have the feature to be desensitized under a sustained stimuation. The internalisation is induced by phophorylation of the receptor by GRK2, which is a GPCR specific kinase. We want to know if this desensitization is caused by the internalisation of OTR. Thereby we search if there is an interaction between OTR and GRK2. BRET assay (Bioluminescence Resonance Energy Transfer) Introduction Highlighting the interaction between GRK2 and OTR -After 1 min: interaction -After 5 min: decrease of the interaction Time dependant - Increase of Bret ratio upon agonist stimulation - Decrease by 65% after 8 mins • Rapid and transcient association of GRK2 and OTR can explain the failure of previous attempts. • GRK6 is involved in the desensitization of human myometrial cells OTR oxytocin during labour may increase the risk of postpartum haemorrhage. •Try to understand the receptor, allows a better use of it. - OTA blocks interactions Advantages Drawbacks •Specifity of Ab •Strong affinity •Quantity of protein •Ab can block a interaction site of protein Advantages Drawbacks •Living cells •Real time •Dim signals can’t be amplified. Reference: 1. Ahmed Hasbi, Dominic Devost, Stéphane A. Laporte and Hans H.Zingg, « Real-time detection of interactions between the huan oxytocin receptor and G-protein-coupled receptor kinase-2 » 2.Freedman NJ and al « Desensitization of G protein –coupled receptors » 3.Jonathon M.Willets and al, « Regulation of oxytocin receptor responsiveness by G protein coupled receptor kinase 6 in human myometrial smooth muscle. 4 Belghiti et coll « Oxytocin during labour and risk of severe postpartum haemorrhage: a population-based; cohort nested case-control studyt Thanks to Mrs.Sabrina Kellouche-Gaillard and to thedepartment of Biology. Conclusion and perspectives
