| Opioid alkaloids and related drugs are the most efficacious analgesics for the treatment of acute and chronic pain.The main active opioid alkaloids are morphine and codeine,induce analgesia,sedation,respiratory depression,constipation and euphoria by acting on the μ opioid receptor(MOR)of the central nervous system.It is generally reported that two parallel signaling pathways downstream of the G protein-coupled receptor mediated the function of MOR through its activation,one is G protein-dependent signaling which mediates analgesia,sedation and the other is β-arrestin-dependent signaling which mediates respiratory depression,tolerance,gastrointestinal dysfunction.Different ligands interact with MOR,which cause different conformational changes of MOR,and it will form some different stable conformations showing different degrees of activation of downstream signal pathways.It is found that certain motifs or amino acid sites in the structure will affect the signal pathway by analyzing of the crystal structure of MOR.Thus,if we can find the key amino acid sites that affect the binding of ligands and receptors,we will study the specific mechanism of μ opioid receptor agonists affecting the signal pathway,providing ideas for guiding ligand synthesis.Therefore,we use molecular docking to predict the amino acid sites that affect the binding of μ opioid receptor agonists and MOR,and mutate the corresponding sites to determine the binding of agonists with MOR using fluorescence labeled ligand competition binding experiment.Then,we analyzed the effect of site mutation on G-protein-dependent signaling pathway and β-arrestin-dependent signaling pathway,and explored the specific mechanism of site influence on signaling pathway by molecular dynamics simulation.The results showed that H6.52 mutations could not bind with fluorescent ligand,and reduced the activation of G-protein-dependent signaling pathway stimulated by DAMGO,morphine and fentanyl.It was speculated that H6.52 affected the activity of G protein pathway by the binding of tested compounds with MOR.When Y7.43 was mutated,only Y7.43S mutation could reduce the affinity between fentanyl and MOR,while the other mutants did not change the affinity between the three tested compounds and MOR.Y7.43 mutations all reduced the activation of G protein pathway stimulated by DAMGO and fentanyl,Y7.43A and Y7.43S mutants more significantly reduced the activation of MOR-mediated G protein-dependent signaling stimulated by DAMGO than Y7.43F mutant MOR.The Y7.43 mutations did not change the activation state of the G protein pathway stimulated by morphine.For the β-arrestin dependent-signaling pathway,the Y7.43 mutation MOR blocked or significantly reduced its activation stimulated by DAMGO and fentanyl,but did not change the action state stimulated by morphine.That is,it was still in the same state as the wild-type MOR,wild-type and mutant MOR were not activated β-arrestin dependent-signaling pathway.Finally,the molecular dynamics simulation of morphine,fentanyl with wild-type and Y7.43A mutant MOR suggested that morphine reduced the degree of the sixth transmembrane helical(TM6)migration of Y7.43A mutantion compared with wild-type MOR,which was beneficial to maintain the states of G-protein-dependent signaling pathway and β-arrestin-dependent signaling pathway.In addition,fentanyl increased the degree of TM6 migration of Y7.43 A mutantion compared with wild-type MOR,which weakened the activation degree of G-protein-dependent signaling pathway and β-arrestin-dependent signaling pathway. |
