Molecular Mechanism Of Drosophila Metallophosphoesterase In Rhodopsin Endocytosis

G protein-coupled receptors(GPCRs)are a large cell surface receptor superfamily with a seven transmembrane structure and a wide range of functions,that participate in various biological processes.Rhodopsin is a classical G protein-coupled receptor which can be activated by directly sense light stimulation and mediate signal transmission in humans and other animals.Activated Rhodopsin needs to be rapidly deactivated in order to respond to subsequent light stimulation and to avoid apoptosis due to excessive activation of the visual signaling pathway.Arrestin,as a regulator,is mainly involved in the deactivation of Rhodopsin.There are two main subtypes of Arrestin in Drosophila: Arrestin1 and Arrestin2.Arrestin2 mainly binds activated Rhodopsin,blocks the interaction of receptors with G proteins in space,and avoids the continuous activation of signals to achieve rapid deactivation of signals.Arrestin1 mediates the endocytosis process of activated Rhodopsin.Arrestin1 lacks a clathrin binding domain and the C-terminal sequences that mediates the interaction with the ?-subunits of AP2.The molecular mechanism that Arrestin1 mediates Rh1 endocytosis remains unclear.Using Drosophila as a model,this paper systematically analyzed the mechanism of main Rhodopsin(Rhodopsin1,Rh1)endocytosis mediated by Arrestin1,and showed that Metallophosphoesterase(MPPE)can promote the association of Arrestin1 and AP2?,and regulate Rh1 endocytosis.Firstly,we show light-induced Rh1 endocytosis is significantly reduced in dmppe mutants.The expression of d MPPE can rescue this phenotype in the dmppe mutants using p[UAS-d MPPE] transgene.These data indicate that d MPPE is involved in light-induced rapid Rh1 endocytosis.The number of Rh1 endocytic vesicles(ERVs)per ommatidium in the ?-man-? mutant is comparable to that in wild type flies,suggesting that the oligosaccharide chain does not significantly interfere with Rh1 endocytosis.Normal Light-induced Rh1 endocytosis in dmppe;rh1>dmppe164-183 A demonstrates that the role of d MPPE in regulating lightinduced Rh1 endocytosis is independent of its phosphoesterase activity.Secondly,both mass spectrometry,in vitro and in vivo experiments show that d MPPE can directly bind to AP2? and Arrestin1 through protein-protein interactions.Through mapping,we determine that d MPPE binds to Arrestin1 through C-terminal 344 to 352 amino acid sequences.The number of Rh1 endocytic vesicles(ERVs)is significantly reduced in dmppe;rh1 > dmppe344-355 A.In the study of molecular mechanism,we reveal that the absence of d MPPE does not affect the protein expression level of Arrestin1,nor does it affect the binding of Arrestin1 to Rh1.d MPPE promotes the association of Arrestin1/AP2? to mediate the endocytosis of Rh1.In addition,we reveal that the absence of d MPPE does not completely block the binding of Arrestin1 and AP2?.Other molecules,such as phosphatidylinositol-4,5-diphosphate(Ptd Ins(4,5)P2)can also can bind to Arrrestin1 and affect the interaction of d MPPE/Arrestin1/ AP2?.This suggests that phosphoinositol molecules may play a role in the formation of this complex and in Rh1 endocytosis.Finally,although genetic dmppe deletion in norp A does not affect the formation of Arrestin2 and Rh1 complexes,the number of ERVs also significantly reduces.Electron microscopy shows that genetic dmppe deletion could largely prevent retinal degeneration in norp A.Arrestin2 and Arrrestin1 own highly homologous sequences,and Arr2 was shown to bind d MPPE in MS spectrum analysis.It suggests that d MPPE may regulate Rh1 endocytosis by promoting the binding of Arrestin2 and AP2?,and then affect retinal degeneration in norp A.In this paper,our study identifies d MPPE as a novel regulator involved in Rh1 endocytosis and retinal degeneration,clarifies the binding site of d MPPE and Arrestin1,and proves that d MPPE can promote the binding of Arrestin1 and AP2?.Our finding also fills the gap in the study of the interaction between Arrestin1 and AP2,and reveals the molecular mechanism of Arrestin1-mediated Rh1 endocytosis by d MPPE.This study provides a new perspective for the molecular mechanisms involved in the endocytosis of G-protein-coupled receptors and new pathological clues and therapeutic strategies for the study of retinitis pigmentosa.