SummaryThe mechanisms controlling the steady-state cell surface levels of cytokine receptors, and consequently the cellular response to cytokines, remain poorly understood. The number of surface-exposed receptors is a dynamic balance of de novo synthesis, transport to the plasma membrane, internalization, recycling, degradation and ectodomain shedding. We previously reported that the E3 ubiquitin ligase RING finger protein 41 (RNF41) inhibits basal lysosomal degradation and enhances ectodomain shedding of JAK2-associated cytokine receptors. Ubiquitin-specific protease 8 (USP8), an RNF41-interacting deubiquitylating enzyme (DUB) stabilizes RNF41 and is involved in trafficking of various transmembrane proteins. The present study identifies USP8 as a substrate of RNF41 and reveals that loss of USP8 explains the aforementioned RNF41 effects. RNF41 redistributes and ubiquitylates USP8, and reduces USP8 levels. In addition, USP8 knockdown functionally matches the effects of RNF41 ectopic expression on the model leptin and leukemia inhibitory factor (LIF) receptors. Moreover, RNF41 indirectly destabilizes the ESCRT-0 complex through suppression of USP8. Collectively, our findings demonstrate that RNF41 controls JAK2-associated cytokine receptor trafficking by acting as a key regulator of USP8 and ESCRT-0 stability. Balanced reciprocal cross-regulation of RNF41 and USP8 thus determines whether receptors are sorted for lysosomal degradation or recycling, this way regulating basal cytokine receptor levels.

DSCAM and DSCAML1 are immunoglobulin and cell adhesion‐type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self‐avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high‐throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ‐secretase‐dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain‐of‐function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full‐length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane‐to‐nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS.