RESUMO
Lowe Syndrome (LS) is a rare X-linked disorder characterized by renal dysfunction, cataracts, and several central nervous system (CNS) anomalies. The mechanisms underlying the neurological dysfunction in LS remain unclear, albeit they share some phenotypic characteristics similar to the deficiency or dysfunction of the Reelin signaling, a relevant pathway with roles in CNS development and neuronal functions. In this study, we investigated the role of OCRL1, an inositol polyphosphate 5-phosphatase encoded by the OCRL gene, mutated in LS, focusing on its impact on endosomal trafficking and receptor recycling in human neuronal cells. Specifically, we tested the effects of OCRL1 deficiency in the trafficking and signaling of ApoER2/LRP8, a receptor for the ligand Reelin. We found that loss of OCRL1 impairs ApoER2 intracellular trafficking, leading to reduced receptor expression and decreased levels at the plasma membrane. Additionally, human neurons deficient in OCRL1 showed impairments in ApoER2/Reelin-induced responses. Our findings highlight the critical role of OCRL1 in regulating ApoER2 endosomal recycling and its impact on the ApoER2/Reelin signaling pathway, providing insights into potential mechanisms underlying the neurological manifestations of LS.
Assuntos
Moléculas de Adesão Celular Neuronais , Endossomos , Proteínas da Matriz Extracelular , Proteínas Relacionadas a Receptor de LDL , Proteínas do Tecido Nervoso , Neurônios , Monoéster Fosfórico Hidrolases , Transporte Proteico , Proteína Reelina , Serina Endopeptidases , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/deficiência , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/deficiência , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/deficiência , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/deficiência , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/deficiência , Endossomos/metabolismo , Neurônios/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Transdução de Sinais , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismoRESUMO
Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.
Assuntos
COVID-19 , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Albuminas/metabolismo , Albuminas/farmacologia , Albuminúria/metabolismo , Enzima de Conversão de Angiotensina 2 , Células Cultivadas , Dextranos/farmacologia , Endocitose/fisiologia , Células Epiteliais/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacologia , Humanos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as "albumin-induced albumin endocytosis". Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.
Assuntos
Células Epiteliais/metabolismo , Retroalimentação Fisiológica , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Albuminas/metabolismo , Animais , Biomarcadores , Endocitose , Células Epiteliais/efeitos dos fármacos , Imunofluorescência , Expressão Gênica , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Transdução de Sinais/efeitos dos fármacosRESUMO
Megalin/LRP2 is a receptor that plays important roles in the physiology of several organs, such as kidney, lung, intestine, and gallbladder and also in the physiology of the nervous system. Megalin expression is reduced in diseases associated with fibrosis, including diabetic nephropathy, hepatic fibrosis and cholelithiasis, as well as in some breast and prostate cancers. One of the hallmarks of these conditions is the presence of the cytokine transforming growth factor beta (TGF-ß). Although TGF-ß has been implicated in the reduction of megalin levels, the molecular mechanism underlying this regulation is not well understood. Here, we show that treatment of two epithelial cell lines (from kidney and gallbladder) with TGF-ß1 is associated with decreased megalin mRNA and protein levels, and that these effects are reversed by inhibiting the TGF-ß1 type I receptor (TGF-ßRI). Based on in silico analyses, the two SMAD-binding elements (SBEs) in the megalin promoter are located at positions -57 and -605. Site-directed mutagenesis of the SBEs and chromatin immunoprecipitation (ChIP) experiments revealed that SMAD2/3 transcription factors interact with SBEs. Both the presence of SMAD2/3 and intact SBEs were associated with repression of the megalin promoter, in the absence as well in the presence of TGF-ß1. Also, reduced megalin expression and promoter activation triggered by high concentration of albumin are dependent on the expression of SMAD2/3. Interestingly, the histone deacetylase inhibitor Trichostatin A (TSA), which induces megalin expression, reduced the effects of TGF-ß1 on megalin mRNA levels. These data show the significance of TGF-ß and the SMAD2/3 signalling pathway in the regulation of megalin and explain the decreased megalin levels observed under conditions in which TGF-ß is upregulated, including fibrosis-associated diseases and cancer.
Assuntos
Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sequência de Bases , Sítios de Ligação , Biomarcadores , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The multi-ligand endocytic receptor, low-density lipoprotein-receptor-related protein 1 (LRP1), has anti-inflammatory roles in disease. Here, we reveal that pathogen-activated Toll-like receptors (TLRs) activate LRP1 in human and mouse primary macrophages, resulting in phosphorylation of LRP1 at Y4507. In turn, this allows LRP1 to activate and recruit the guanosine triphosphatase (GTPase), Rab8a, with p110γ/p101 as its phosphatidylinositol 3-kinase (PI3K) effector complex. PI3Kγ is a known regulator of TLR signaling and macrophage reprogramming. LRP1 coincides with Rab8a at signaling sites on macropinosomal membranes. In LRP1-deficient cells, TLR-induced Rab8 activation is abolished. CRISPR-mediated knockout of LRP1 in macrophages alters Akt/mTOR signaling and produces a pro-inflammatory bias in cytokine outputs, mimicking the Rab8a knockout and PI3Kγ-null phenotype. Thus, TLR-LRP1 crosstalk activates the Rab8a/PI3Kγ complex for reprogramming macrophages, revealing this as a key mechanism through which LRP1 helps to suppress inflammation.
Assuntos
Inflamação/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Receptores Toll-Like/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Humanos , Inflamação/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Receptores Toll-Like/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
BACKGROUND: ApoER2 and the neurotrophin receptors Trk and p75(NTR) are expressed in the CNS and regulate key functional aspects of neurons, including development, survival, and neuronal function. It is known that both ApoER2 and p75(NTR) are processed by metalloproteinases, followed by regulated intramembrane proteolysis. TrkA activation by nerve growth factor (NGF) increases the proteolytic processing of p75(NTR) mediated by ADAM17. Reelin induces the sheeding of ApoER2 ectodomain depending on metalloproteinase activity. However, it is not known if there is a common regulation mechanism for processing these receptors. RESULTS: We found that TrkA activation by NGF in PC12 cells induced ApoER2 processing, which was dependent on TrkA activation and metalloproteinases. NGF-induced ApoER2 proteolysis was independent of mitogen activated protein kinase activity and of phosphatidylinositol-3 kinase activity. In contrast, the basal proteolysis of ApoER2 increased when both kinases were pharmacologically inhibited. The ApoER2 ligand reelin regulated the proteolytic processing of its own receptor but not of p75(NTR). Finally, in primary cortical neurons, which express both ApoER2 and TrkB, we found that the proteolysis of ApoER2 was also regulated by brain-derived growth factor (BDNF). CONCLUSIONS: Our results highlight a novel relationship between neurotrophins and the reelin-ApoER2 system, suggesting that these two pathways might be linked to regulate brain development, neuronal survival, and some pathological conditions.
Assuntos
Proteínas Relacionadas a Receptor de LDL/metabolismo , Fatores de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Metaloproteases/metabolismo , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteólise , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento , Proteína Reelina , Serina Endopeptidases/metabolismo , Transdução de SinaisRESUMO
The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.
Assuntos
Endossomos/metabolismo , Exossomos/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Neurônios/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Microscopia de Fluorescência , Proteínas do Tecido Nervoso , Células PC12 , Interferência de RNA , Ratos , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/genéticaRESUMO
Sorting nexin 17 (SNX17) is an adaptor protein present in early endosomal antigen 1 (EEA1)-positive sorting endosomes that promotes the efficient recycling of low-density lipoprotein receptor-related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1-positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17-binding domain, we generated chimeric proteins in which the SNX17-binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non-polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized Madin-Darby canine kidney cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17-binding receptors and the restricted function of SNX17 in the BSE.
Assuntos
Endossomos/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de LDL/metabolismo , Nexinas de Classificação/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cães , Células HEK293 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Células Madin Darby de Rim Canino , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Sinais Direcionadores de Proteínas , Transporte Proteico , Receptores de LDL/química , Receptores de LDL/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
In physiological conditions, both ß-amyloid precursor protein (ßAPP) and cellular prion (PrP(c)) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid ß (Aß)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated ßAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated ßAPP (APP(LDN)) that yields Aß oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APP(LDN)-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aß-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos/metabolismo , Proteínas PrPC/metabolismo , Multimerização Proteica , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Células CHO , Caspase 3/genética , Caspase 3/metabolismo , Morte Celular , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/genética , Proteínas PrPC/genética , Estaurosporina/farmacologiaRESUMO
Since the discovery of the low-density lipoprotein receptor (LDLR) and its association with familial hypercholesterolemia in the early 1980s, a family of structurally related proteins has been discovered that has apolipoprotein E as a common ligand, and the broad functions of its members have been described. LRP2, or megalin, is a member of the LDLR family and was initially called gp330. Megalin is an endocytic receptor expressed on the apical surface of several epithelial cells that internalizes a variety of ligands including nutrients, hormones and their carrier proteins, signaling molecules, morphogens, and extracellular matrix proteins. Once internalized, these ligands are directed to the lysosomal degradation pathway or transported by transcytosis from one side of the cell to the opposite membrane. The availability of megalin at the cell surface is controlled by several regulatory mechanisms, including the phosphorylation of its cytoplasmic domain by GSK3, the proteolysis of the extracellular domain at the cell surface (shedding), the subsequent intramembrane proteolysis of the transmembrane domain by the gamma-secretase complex, and exosome secretion. Based on the important roles of its ligands and its tissue expression pattern, megalin has been recognized as an important component of many pathological conditions, including diabetic nephropathy, Lowe syndrome, Dent disease, Alzheimer's disease (AD) and gallstone disease. In addition, the expression of megalin and some of its ligands in the central and peripheral nervous system suggests a role for this receptor in neural regeneration processes. Despite its obvious importance, the regulation of megalin expression is poorly understood. In this review, we describe the functions of megalin and its association with certain pathological conditions as well as the current understanding of the mechanisms that underlie the control of megalin expression.
Assuntos
Humanos , Doença de Alzheimer/metabolismo , /fisiologia , Doença de Alzheimer/fisiopatologia , Transporte Biológico/fisiologia , Colesterol/fisiologia , Cálculos Biliares/metabolismo , Cálculos Biliares/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Nefropatias/metabolismo , Nefropatias/fisiopatologia , /genética , /metabolismo , Distribuição Tecidual/fisiologiaRESUMO
Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic recycling receptor with two cytoplasmic tyrosine-based basolateral sorting signals. Here we show that during biosynthetic trafficking LRP1 uses AP1B adaptor complex to move from a post-TGN recycling endosome (RE) to the basolateral membrane. Then it recycles basolaterally from the basolateral sorting endosome (BSE) involving recognition by sorting nexin 17 (SNX17). In the biosynthetic pathway, Y(29) but not N(26) from a proximal NPXY directs LRP1 basolateral sorting from the TGN. A N(26)A mutant revealed that this NPXY motif recognized by SNX17 is required for the receptor's exit from BSE. An endocytic Y(63)ATL(66) motif also functions in basolateral recycling, in concert with an additional endocytic motif (LL(86,87)), by preventing LRP1 entry into the transcytotic apical pathway. All this sorting information operates similarly in hippocampal neurons to mediate LRP1 somatodendritic distribution regardless of the absence of AP1B in neurons. LRP1 basolateral distribution results then from spatially and temporally segregation steps mediated by recognition of distinct tyrosine-based motifs. We also demonstrate a novel function of SNX17 in basolateral/somatodendritic recycling from a different compartment than AP1B endosomes.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Animais , Linhagem Celular , Endossomos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/genética , Rede trans-Golgi/metabolismoRESUMO
Accumulation of extracellular amyloid beta peptide (Abeta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Abeta from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in Abeta production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to Abeta. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to Abeta.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica , Proteínas de Transporte Vesicular/fisiologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citoplasma/metabolismo , Endocitose , Humanos , Camundongos , Modelos Biológicos , Neurônios/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Nexinas de Classificação , Proteínas de Transporte Vesicular/metabolismoRESUMO
Decorin is a small leucine-rich proteoglycan that modulates the activity of transforming growth factor type beta and other growth factors and thereby influences the processes of proliferation and differentiation in a wide array of physiological and pathological reactions. Hence, understanding the regulatory mechanisms of decorin activity has broad implications. Here we report that the extracellular levels of decorin are controlled by receptor-mediated catabolism, involving the low density lipoprotein receptor family member, low density lipoprotein receptor-related protein (LRP). We show that decorin is endocytosed and degraded by C2C12 myoblast cells and that both processes are blocked by suppressing LRP expression using short interfering RNA. The same occurs with CHO cells, but not with CHO cells genetically deficient in LRP. Finally, we show that LRP-null CHO cells, transfected to express mini-LRP polypeptides containing either the second or fourth LRP ligand-binding domains, carry out decorin endocytosis and lysosomal degradation. These findings point to LRP-mediated catabolism as a new control pathway for the biological activities of decorin, specifically for its ability to influence extracellular matrix signaling.
Assuntos
Endocitose/fisiologia , Proteínas da Matriz Extracelular/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Proteoglicanas/química , Animais , Células CHO , Diferenciação Celular , Cricetinae , Decorina , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Leucina/química , Camundongos , Músculo Esquelético/metabolismo , Peptídeos/química , Proteoglicanas/metabolismoRESUMO
Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276-1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, beta-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and beta-2 microglobulin (beta2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE-beta2m to the apical membrane. The amount of HFE-beta2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or beta2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE-beta2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Mucosa Intestinal/metabolismo , Ferro/farmacocinética , Proteínas de Membrana/metabolismo , Microglobulina beta-2/metabolismo , Células CACO-2 , Proteínas de Transporte de Cátions/metabolismo , DNA Antissenso/genética , Hemocromatose/metabolismo , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoprecipitação/métodos , Mucosa Intestinal/citologia , Ferro/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Membrana/genética , Modelos Biológicos , Transfecção/métodos , Microglobulina beta-2/genéticaRESUMO
The apolipoprotein E receptor 2 (apoER2) is a member of the low-density lipoprotein receptor family which binds ligands such as reelin, apolipoprotein E and apolipoprotein J/clusterin and has been shown to play roles in neuronal migration during development and in male fertility. The function of apoER2 mainly depends on cellular signaling triggered by ligand binding. Although the receptor is internalized, the mechanism and functional significance of its endocytic trafficking remain unclear. Apolipoprotein E receptor 2 partitions into lipid rafts and interacts with caveolin-1, a feature that could modulate its endocytic behavior. Recent evidence also suggested that apoER2 might be endocytosed by a pathway independent of clathrin. Here, we show that despite a raft association, apoER2 internalization depends on its cytoplasmic FxNPXY motif that is similar to canonical motifs for clathrin-mediated endocytosis. This motif mediates receptor binding to the adaptor protein Dab2, which can interact directly with clathrin. Several inhibitory conditions of clathrin-mediated endocytosis, including expression of the dominant negative forms of eps15 and Dab2, decreased apoER2 internalization. In contrast, treatment with the drug nystatin, which blocks the caveolar/raft internalization pathway, has no effect on the receptor's endocytosis. Neither the transmembrane nor the proline-rich insert of the cytoplasmic domain, which has been previously reported to exclude the receptor from the clathrin-mediated pathway, altered apoER2 endocytic activity. These studies indicate that apoER2 internalizes through a clathrin-mediated pathway and that its association with caveolar and noncaveolar rafts does not determine its endocytosis.
Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , Microdomínios da Membrana/metabolismo , Receptores de Lipoproteínas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adenoviridae/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Astrocitoma/patologia , Biotinilação , Western Blotting , Células CHO , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Caveolina 1 , Caveolinas/genética , Caveolinas/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células Clonais , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Proteínas Relacionadas a Receptor de LDL , Camundongos , Microscopia de Fluorescência , Neuroblastoma/patologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteína Reelina , Saco Vitelino/citologia , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin-Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand-binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC-PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand-binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts.