Abnormal Phosphorylation Of MGluR5 In A Mouse Model Of Fragile X Syndrome Fmr1-/-Affects Homer1c-mGluR5 Interaction

Fragile X syndrome(FXS)is a common intellectual disability disease caused by single gene mutation and its incidence in patients with hereditary of disabled intelligentual is just after Ehlers-danlos syndrome.According to data released by the world health organization,the incidence of male FXS is 1/3000,but female is 1/6000.Mental disorder caused by FXS give patients huge pain,and family and society suffering heavy burden during the long process that from early disease development until end of life of FXS children.Now,there are researches show that mGluR5 signaling pathways play a crucial role in pathogenic process of FXS;and there are inhibitors of mGluR5 fenobam and AFQ056 that have come into clinical trials and have certain treatment effects,but soon to develop drug tolerance.And,we know that phosphorylation of mGluR5 regulates by its function.Therefore it is necessary to study what phosphorlation changes of mGluR5 have effects on the its function by studying Fmr1-/-mice neuron.And the research will give new insight for drug resistance mechanisms ofmGluR5 receptor protein inhibitors and pathogenesis of FXS.Our experiments are divided into two main parts:1.We detect the change of phosphorylation of mGluR5 protein in Fmrl-/-mice brain cortex by mass spectrum and confirm this result by a antibody that specifically recegnise phosphatic mGluR5 protein and phosphoproteomics.We found that phosphorylation level of 1014 and 1016 serine in mGluR5 receptor protein in Fmr1-/-mouse cortex increase.And then 389 threonine of S6K protein which is downstream of mGluR5-ERK1/2 signaling pathways is upregulated.This means that protein synthesis in Fmr1-/-mouse cortex increase.2.We investigate what effects phosphorylation changes of mGluR5 receptor 1014 and 1016 serine have on the function of mGluR5 in cells by IP.The results show that:(1)Non-phosphorylation of 1014 and 1016 serine in mGluR5 protein inhibits interaction between mGluR5 and Homer1c(Homer1c is an important neuron scaffold protein.It regulates mGluR5 downstream signaling pathways by interacting with mGluR5 and the interaction in Fmr1-/-mice decreases.).The mutantwhich mimics phosphorylated serine of mGluR5 on 1014 and 1016 has no effect on interaction between mGluR5 and Homer1c.(2)Non-phosphorylation mutant of mGluR5 protein inhibits location of mGluR5 protein and co-localization with Homer1c in cytoplasmic membrane area where mGluR5 exert itself function,compared with wildtype mGluR5 and mutant of mimic phosphorylated mGluR5.(3)Mutants of non-phosphorylation and mimic phosphorylated 1014 and 1016 serine in mGluR5 protein all inhibit differentiation of neural cells N2a.Above all,phosphorylation level of 1014 and 1016 serine in mGluR5 receptor proteins in cerebral cortex of Fmrl gene knockout mouse is higher than in wild type.And this change promotes the interaction between mGluR5 and Homer1c and inhibits differentiation of N2a in cells and tissue and co-localization of mGluR5 and Homer1c in cells.So far,no one reported what effect the phosphoarylation change of mGluR56 receptor protein had on function of mGluR5 in Fmr1-/-mouse.Therefore,our study may provide important insight on pathogenesis of FXS and theoretical basis for solve the resistance of mGIuR5 inhibitors in treatment of FXS disease.