Presentation on theme: "Winston Sanders. Mu( μ ) Opioid Receptor (MOR) Stimulus Trafficking What if the unwanted side effects of a drug could be diminished while its medicinal."— Presentation transcript:
Mu( μ ) Opioid Receptor (MOR) Stimulus Trafficking What if the unwanted side effects of a drug could be diminished while its medicinal effects amplified?
7-transmembrane protein G-protein: Heterotrimeric G-protein Coupled Receptors (GPCR) The Pathway To Activation
Bordetela pertussis toxin (PTX) -rendered PTX-sensitive Ga (subunit) proteins unresponsive to GPCR activation -allowing focused aim towards specific PTX-insensitive mutant Ga proteins -No difference in relative order of agonist efficacy or potency -Interferes with natural communication b/w GPCR and G-protein -Ineffective Mechanism Examining the Pathways PTX
This proposal addresses an alternative methodological approach to investigate the ability of GPCRs, activated by distinct ligands, to activate specific Ga- protein subtypes. The Proposal
How could desired pathway be identified? Yu 1998* ( Interaction of the Xanthine Nucleotide Binding Goa Mutant with G Protein-coupled Receptors) Double Mutant GoaX The Experiment GoaX X XTP GoaX
What were results of GoaX when compared to wild-type a-subunit? Activates in same manner! GoaX conserves natural responses The Experiment
Prepare membrane incubation using Sf9 cells with XDP in TEDM buffer Co-transfect cells with X-mutant Ga protein subtypes and with our receptor of interest (MOR rather than m2 MAChR) Instead of carabachol, measure binding affinity and ligand efficacy agonist of ligand of interest (Morphine) The Methods
Use xanthine binding Ga subunits (GoaX) as efficient indicators for study of activation of cellular pathways Successfully study individual activation pathways Experiment with cells other than Sf9 like human neuroblastom- a SHSY5Y cells, which are known to express MOR Results
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