Effects Of Peptide Intervention In μ-opioid Receptor Phosphorylation On Receptor Downstream Signaling Pathway

Objective:Morphine is one of the opioid analgesics,which is widely used in clinical practice with good analgesic effect.However,morphine can produce drug tolerance,respiratory depression,constipation,addiction and other side effects,which greatly limits its clinical application.Therefore,how to minimize the accompanying side effects of morphine while exerting its analgesic effect has become the focus of research.The purpose of this study was to find out the relationship between different key phosphorylation sites at carboxyl terminal ofμopioid receptor and the downstream signaling pathway of the receptor.By designing and synthesizing peptides which cover various sites and competitively inhibit the phosphorylation of receptor,the influence of peptides interfering with the phosphorylation ofμopioid receptor on the downstream signaling pathway of morphine-mediated receptor was explored,thus providing a new idea for enhancing morphine analgesia and improving morphine tolerance.Methods:G_i/Oinduced c AMP inhibition experiment:HEK-293 cells co-transfected with HA-MOR plasmid and p Glo Sensor TM-22F were divided into blank group,DAMGO group（10μmol/L）,morphine group,and different concentrations of peptide TAT-Seq I,II,Ⅲ,IV,V（5,10,20μmol/L）combined with morphine group.The Glo Sensor c AMP biosensor was used to measure the change of c AMP content in each group.β-arrestin2 recruitment experiment:HEK-293 cells co-transfection with the MOR-C-terminal labeled Nanoluc plasmid and theβ-arrestin2-N-terminal labeled EYFP plasmid were divided into blank group,DAMGO group（10μmol/L）,morphine group,peptide TAT-Seq I,II,Ⅲ,IV,V（10μmol/L）combined with morphine group,Cmpd101（30μmol/L）combined with morphine group.Protein interaction analysis assay was used to determine the recruitment ofβ-arrestin2 after MOR activation in each group.Confocal imaging experiment of MOR endocytosis:HEK-293 cells transfected with HA-MOR plasmid were divided into blank group,morphine（10μmol/L）group,peptide TAT-Seq Ⅲ（10μmol/L）combined with morphine（10μmol/L）group,Cmpd101（30μmol/L）and morphine（10μmol/L）group.The effect of peptide TAT-Seq Ⅲ and Cmpd101on morphine-induced MOR endocytosis was investigated by observing the distribution of fluorescent receptors in the cells.Cellular immunofluorescence assay was used to observe the endocytosis of receptors in each group.Protein Western Blotting:HEK-293 cells transfected with HA-MOR plasmid were divided into blank group,morphine（10μmol/L）group,peptide TAT-Seq Ⅲ（10μmol/L）combined with morphine（10μmol/L）group,Cmpd101（30μmol/L）and morphine（10μmol/L）group.Western Blot assay was used to detect the Ser375 phosphorylation degree of MOR in each group.HEK-293 cells transfected with HA-MOR plasmid were divided into blank group,morphine（10μmol/L）group,peptide TAT-Seq I,II,Ⅲ,IV,V（10μmol/L）combined with morphine（10μmol/L）group,Cmpd101（30μmol/L）and morphine（10μmol/L）group.Western Blot assay was used to detect the ERK1/2 phosphorylation degree of MOR in each group.Results:The results of G_i/O-induced c AMP inhibition experiment showed that peptide TAT-Seq I,II,Ⅲ,IV,V alone did not activate c AMP signaling pathways downstream of MOR（P<0.05）.The EC₅₀ value of peptide TAT-Seq I combined with morphine was statistically significant among groups（F=7.89,P<0.05）,and the EC₅₀ values of different concentrations of peptide TAT-Seq I（5,10,20μmol/L）combined with morphine group were significantly reduced compared with morphine group（P<0.01）.The EC₅₀ value of peptide TAT-Seq II combined with morphine was statistically significant among groups（F=3.87,P<0.05）,and the EC₅₀ values of different concentrations of peptide TAT-Seq II（10,20μmol/L）combined with morphine group were significantly reduced compared with morphine group（P<0.05）.The EC₅₀ value of peptide TAT-Seq Ⅲ combined with morphine was statistically significant among groups（F=7.77,P<0.05）,and the EC₅₀ values of different concentrations of peptide TAT-Seq Ⅲ（5,10,20μmol/L）combined with morphine group were significantly reduced compared with morphine group（P<0.01）.The EC₅₀value of peptide TAT-Seq IV combined with morphine was not statistically significant among groups（F=1.83,P<0.05）,and the EC₅₀ value of peptide TAT-Seq IV（20μmol/L）combined with morphine group was significantly reduced compared with morphine group（P<0.05）.The EC₅₀ value of peptide TAT-Seq V combined with morphine was statistically significant among groups（F=4.83,P<0.05）,and the EC₅₀ values of different concentrations of peptide TAT-Seq V（5,10,20μmol/L）combined with morphine group were significantly reduced compared with morphine group（P<0.05）.The results ofβ-arrestin2 recruitment experiment showed that compared with the blank group,peptide TAT-Seq I,II,Ⅲ,IV,V alone can increase the basal BERT value in different degrees（P<0.05）.There was no statistical significance in the E_max values of peptide TAT-Seq I and II peptides combined with morphine（F=2.03,P<0.05）.The E_max values of peptide TAT-Seq Ⅲ,Cmpd101 combined with morphine were statistically significant among groups（F=13.14,P<0.05）,and the E_max values of peptide TAT-Seq Ⅲ,Cmpd101combined with morphine group were significantly reduced compared with morphine group（P<0.05）.The E_max values of peptide TAT-Seq IV and V combined with morphine were statistically significant among groups（F=6.34,P<0.05）,and the E_max value of peptide TAT-Seq V combined with morphine group was significantly l reduced compared with morphine group（P<0.05）.The results of confocal imaging of MOR endocytosis showed that the number of MOR on the cell membrane of peptide TAT-Seq Ⅲ,Cmpd101 combined with morphine was significantly increased compared with morphine group.The results of Western blot experiment showed that the phosphorylation level of MOR Ser375 of peptide TAT-Seq Ⅲ,Cmpd101 combined with morphine were statistically significant among groups（F=59.87,P<0.05）.The phosphorylation level of MOR Ser375in the peptide TAT-Seq Ⅲ,Cmpd101 combined with morphine group was significantly lower than that in the morphine group（P<0.05）.When peptide TAT-Seq I,II,Ⅲ,IV,V and Cmpd101 were used in combination with morphine,there was no significant difference in MOR ERK1/2 phosphorylation among the groups（F=1.20,P>0.05）.Conclusion:The carboxy-terminal phosphorylation site ofμopioid receptor is closely related to the downstream signaling pathway of the receptor.Different phosphorylation sites play different roles in the downstream G-protein-dependent signaling pathway andβ-arrestin2-dependent signaling pathway of MOR.Phosphorylation at ³⁵⁴TSST³⁵⁷,³⁷⁵STANT³⁷⁹ and Thr394 plays an important role in the morphine-mediated downstream G-protein signaling pathway of MOR.Phosphorylation at ³⁷⁵STANT³⁷⁹ and Thr394 plays an important role in the downstreamβ-arrestin2 signaling pathway of morphine-mediated MOR,and phosphorylation at ³⁷⁵STANT³⁷⁹is closely related to morphine-induced MOR endocytosis.By designing peptides combined with morphine to competitively inhibit phosphorylation of key sites,the activation of G-protein-dependent signaling pathway downstream of MOR can be effectively enhanced,and the recruitment ofβ-arrestin2 can be reduced,providing a new idea for the treatment of morphine and pain.