RESUMO
Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.
Assuntos
Complexo 4 de Proteínas Adaptadoras , Proteínas Relacionadas a Receptor de LDL , Paraplegia Espástica Hereditária , Animais , Humanos , Camundongos , Complexo 4 de Proteínas Adaptadoras/genética , Complexo 4 de Proteínas Adaptadoras/metabolismo , Células HeLa , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Receptores de Superfície Celular , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismoRESUMO
Adaptor protein (AP) complexes facilitate protein trafficking by playing key roles in the selection of cargo molecules to be sorted in post-Golgi compartments. Four AP complexes (AP-1 to AP-4) contain a medium-sized subunit (µ1-µ4) that recognizes YXXØ-sequences (Ø is a bulky hydrophobic residue), which are sorting signals in transmembrane proteins. A conserved, canonical region in µ subunits mediates recognition of YXXØ-signals by means of a critical aspartic acid. Recently we found that a non-canonical YXXØ-signal on the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP) binds to a distinct region of the µ4 subunit of the AP-4 complex. In this study we aimed to determine the functionality of both binding sites of µ4 on the recognition of the non-canonical YXXØ-signal of APP. We found that substitutions in either binding site abrogated the interaction with the APP-tail in yeast-two hybrid experiments. Further characterization by isothermal titration calorimetry showed instead loss of binding to the APP signal with only the substitution R283D at the non-canonical site, in contrast to a decrease in binding affinity with the substitution D190A at the canonical site. We solved the crystal structure of the C-terminal domain of the D190A mutant bound to this non-canonical YXXØ-signal. This structure showed no significant difference compared to that of wild-type µ4. Both differential scanning fluorimetry and limited proteolysis analyses demonstrated that the D190A substitution rendered µ4 less stable, suggesting an explanation for its lower binding affinity to the APP signal. Finally, in contrast to overexpression of the D190A mutant, and acting in a dominant-negative manner, overexpression of µ4 with either a F255A or a R283D substitution at the non-canonical site halted APP transport at the Golgi apparatus. Together, our analyses support that the functional recognition of the non-canonical YXXØ-signal of APP is limited to the non-canonical site of µ4.