Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.
Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression.
K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.
The LDL receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins and protein-lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1-CT) can be phosphorylated by activated protein-tyrosine kinases at two NPXY motifs in LRP1-CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull-down experiments from brain lysate revealed numerous proteins binding to LRP1-CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY4507 (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY4473 (membrane proximal) bound many fewer proteins and only to the phosphorylated form.
receptor tyrosine kinase; phosphotyrosine; lipoprotein receptor; signal transduction; protein interactions
Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening “gap4” region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.
As a latent transcription factor, nuclear factor κB (NF-κB) translocates from the cytoplasm into the nucleus upon stimulation and mediates the expression of genes that are important in immunity, inflammation, and development. However, little is known about how it is regulated inside the nucleus. By a two-hybrid approach, we identify a prefoldin-like protein, ubiquitously expressed transcript (UXT), that is expressed predominantly and interacts specifically with NF-κB inside the nucleus. RNA interference knockdown of UXT leads to impaired NF-κB activity and dramatically attenuates the expression of NF-κB–dependent genes. This interference also sensitizes cells to apoptosis by tumor necrosis factor-α. Furthermore, UXT forms a dynamic complex with NF-κB and is recruited to the NF-κB enhanceosome upon stimulation. Interestingly, the UXT protein level correlates with constitutive NF-κB activity in human prostate cancer cell lines. The presence of NF-κB within the nucleus of stimulated or constitutively active cells is considerably diminished with decreased endogenous UXT levels. Our results reveal that UXT is an integral component of the NF-κB enhanceosome and is essential for its nuclear function, which uncovers a new mechanism of NF-κB regulation.
Alzheimer’s disease (AD) is associated with optic nerve degeneration yet the underlying pathophysiology of this disease and the optic nerve disorder remains poorly understood. Low density lipoprotein receptor-related protein (LRP) is implicated in the pathogenesis of AD by mediating the transport of amyloid-β (Aβ) out of the brain into the systemic circulation. As a key player in the reaction to central nervous system (CNS) injury, astrocytes associate with LRP in AD. This study investigates the role of LRP and astrocytes in the pathogenesis of AD optic neuropathy.
To investigate the role of LRP and astrocytes in the pathogenesis of AD optic neuropathy, we conducted immunohistochemical (IHC) studies on postmortem optic nerves in AD patients (n = 11) and age-matched controls (n = 10) to examine the presence of LRP. Quantitative analyses using imaging software were used to document the extent of LRP in neural tissues. Axonal integrity was assessed by performing IHC on the subjects’ optic nerves with an antibody to neurofilament (NF) protein. Double-immunofluorescence labeling was performed to investigate whether LRP colocalized with astrocytes expressing glial fibrillary acidic protein (GFAP).
LRP expression was decreased in AD optic nerves compared to controls (p < 0.001). LRP immunoreactivity was observed in the microvasculature and perivascularly in close proximity to astrocytic processes. Colocalization of LRP in astrocytes of optic nerves was also demonstrated. The presence of optic neuropathy was confirmed in the AD optic nerves by demonstrating greatly reduced immunostaining for NF protein as compared to controls.
The reduction of LRP in the AD degenerative optic nerves supports the hypothesis that LRP may play a role in the pathophysiology of AD optic neuropathy.
Alzheimer’s disease; Alzheimer’s optic neuropathy; astrocytes; low density lipoprotein receptor-related protein; microvasculature; optic nerve
Developing sensory neurons require neurotrophic support for survival, neurite outgrowth and myelination. The low-density lipoprotein receptor-related protein-1 (LRP1) transactivates Trk receptors and thereby functions as a putative neurotrophin. Herein, we show that LRP1 is abundantly expressed in developing dorsal root ganglia (DRG) and that LRP1-dependent cell signaling supports survival, neurite extension and receptivity to Schwann cells even in the absence of neurotrophins. Cultured embryonic DRG neurons (E15) were treated with previously characterized LRP1 ligands, LRP1-receptor binding domain of α2-macroglobulin (RBD), hemopexin domain of MMP-9 (PEX) or controls (GST) for two weeks. These structurally diverse LRP1 ligands significantly activated and sustained extracellular signal-regulated kinases (ERK1/2) 5-fold (p<0.05), increased expression of growth-associated protein-43(GAP43) 15-fold (P<0.01), and increased neurite outgrowth 20-fold (P<0.01). Primary sensory neurons treated with LRP1 ligands survived > 2 weeks in vitro, to an extent equaling NGF, a finding associated with canonical signaling mechanisms and blockade of caspase-3 cleavage. LRP1 ligand-induced survival and sprouting were blocked by co-incubation with the LRP1 antagonist, receptor associated protein (RAP), whereas RAP had no effect on NGF-induced activity. Site directed mutagenesis of the LRP1 ligand, RBD, in which Lys1370 and Lys1374 are converted to alanine to preclude LRP1 binding, were ineffective in promoting cell signaling, survival or inducing neurite extension in primary sensory neurons, confirming LRP1 specificity. Furthermore, LRP1-induced neurite sprouting was mediated by Src-family kinase (SFK) activation, suggesting transactivation of Trk receptors. Co-cultures of primary embryonic neurons and Schwann cells showed that LRP1 agonists promoted axonal receptivity to myelination to Schwann cells. Collectively, these findings identify LRP1 as a novel and perhaps essential trophic molecule for sensory neuronal survival and development.
Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor–related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-α (PKC-α) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-α small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate–induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-α/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-α and PI3K could provide a new treatment paradigm for glioblastomas.
Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.
This study revealed that ubiquitously expressed transcript (UXT)-V1 is recruited to tumor necrosis factor (TNF) receptor complex I by interacting with TNF receptor-associated factor 2. UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study uncovers UXT-V1 as a novel regulator of TNF-induced apoptosis.
Proteins that directly regulate tumor necrosis factor (TNF) signaling have critical roles in determining cell death and survival. Previously we characterized ubiquitously expressed transcript (UXT)-V2 as a novel transcriptional cofactor to regulate nuclear factor-κB in the nucleus. Here we report that another splicing isoform of UXT, UXT-V1, localizes in cytoplasm and regulates TNF-induced apoptosis. UXT-V1 knockdown cells are hypersensitive to TNF-induced apoptosis. We demonstrated that UXT-V1 is a new component of TNF receptor signaling complex. We found that UXT-V1 binds to TNF receptor-associated factor 2 and prevents TNF receptor–associated death domain protein from recruiting Fas-associated protein with death domain. More importantly, UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study demonstrates that UXT-V1 is a novel regulator of TNF-induced apoptosis and sheds new light on the underlying molecular mechanism of this process.
Wnt/β-catenin signaling is fundamental in embryogenesis and tissue homeostasis in metazoans. Upon Wnt stimulation, cognate coreceptors LRP5 and LRP6 ([LRP5/6] low-density lipoprotein receptor-related proteins 5 and 6) are activated via phosphorylation at key residues. Although several kinases have been implicated, the LRP5/6 activation mechanism remains unclear. Here, we report that transmembrane protein 198 (TMEM198), a previously uncharacterized seven-transmembrane protein, is able to specifically activate LRP6 in transducing Wnt signaling. TMEM198 associates with LRP6 and recruits casein kinase family proteins, via the cytoplasmic domain, to phosphorylate key residues important for LRP6 activation. In mammalian cells, TMEM198 is required for Wnt signaling and casein kinase 1-induced LRP6 phosphorylation. During Xenopus embryogenesis, maternal and zygotic tmem198 mRNAs are widely distributed in the ectoderm and mesoderm. TMEM198 is required for Wnt-mediated neural crest formation, antero-posterior patterning, and particularly engrailed-2 expression in Xenopus embryos. Thus, our results identified TMEM198 as a membrane scaffold protein that promotes LRP6 phosphorylation and Wnt signaling activation.
Several studies found that FE65, a cytoplasmic adaptor protein, interacts with APP and LRP1, altering the trafficking and processing of APP. We have previously shown that FE65 interacts with the ApoE receptor, ApoER2, altering its trafficking and processing. Interestingly, it has been shown that FE65 can act as a linker between APP and LRP1 or ApoER2. In the present study, we tested whether FE65 can interact with another ApoE receptor, VLDLR, thereby altering its trafficking and processing, and whether FE65 can serve as a linker between APP and VLDLR.
We found that FE65 interacted with VLDLR using GST pull-down and co-immunoprecipitation assays in COS7 cells and in brain lysates. This interaction occurs via the PTB1 domain of FE65. Co-transfection with FE65 and full length VLDLR increased secreted VLDLR (sVLDLR); however, the levels of VLDLR C-terminal fragment (CTF) were undetectable as a result of proteasomal degradation. Additionally, FE65 increased cell surface levels of VLDLR. Moreover, we identified a novel complex between VLDLR and APP, which altered trafficking and processing of both proteins. Furthermore, immunoprecipitation results demonstrated that the presence of FE65 increased the interaction between APP and VLDLR in vitro and in vivo.
These data suggest that FE65 can regulate VLDLR trafficking and processing. Additionally, the interaction between VLDLR and APP altered both protein's trafficking and processing. Finally, our data suggest that FE65 serves as a link between VLDLR and APP. This novel interaction adds to a growing body of literature indicating trimeric complexes with various ApoE Receptors and APP.
FE65; VLDLR; APP trafficking
The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin–proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway.
The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.
Our group has previously reported genetic studies associating polymorphisms in the low density lipoprotein receptor related protein 8 (LRP8) gene with myocardial infarction. The aim of this study was to define the role of platelet surface LRP8 in thrombosis.
Materials and Methods
Flow cytometry, aggregometry, intravital microscopy and tail bleeding assays were used to examine platelet function and hemostasis in LRP8-deficient mice and littermate controls.
We demonstrated that activation of platelets from both LRP8+/- and LRP8-/- mice was reduced in vitro in response to either ADP or thrombin. In vivo, LRP8-hemizygous and LRP8-/- mice demonstrated 200% and 68% increased time for carotid occlusion in response to FeCl3 injury, respectively. Moreover, lipidated apoE3, a ligand for LRP8, inhibited platelet activation in a dose-dependent fashion. This inhibition was markedly attenuated in LRP8-/- but not LRP8+/- mice and did not result from membrane cholesterol efflux or a nitric oxide dependent pathway. Tail bleeding times were unaffected in both genotypes.
Our results suggest that LRP8 is capable of altering thrombosis without affecting normal hemostasis through mechanisms both dependent on and independent of apoE. This suggests a means whereby clot formation could be affected in humans with LRP8 gene variants.
ApoER2; hemostasis; LRP8; platelet; thrombosis
The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor that belongs to the LDL receptor family. Recently, studies have revealed new roles of LDL receptor family members as transducers of extracellular signals. Our previous studies have demonstrated LRP phosphorylation within its cytoplasmic tail, but the nature of LRP phosphorylation and its potential function was unknown. In the present study using both in vivo and in vitro analysis, we found that LRP phosphorylation is mediated by the cAMP-dependent protein kinase A (PKA). Using site-directed mutagenesis and LRP minireceptor constructs, we further identified the predominant LRP phosphorylation site at serine 76 of its cytoplasmic tail. Finally, we demonstrated that mutations of serine 76, which abolish LRP phosphorylation by PKA, result in a decrease in the initial endocytosis rate of LRP and a lower efficiency in delivery of ligand for degradation. Thus, the role of PKA phosphorylation of LRP in receptor-mediated endocytosis may provide a mechanism by which the endocytic function of LRP can be regulated by external signals.
The LDL receptor-related protein 1B (LRP1B) is a putative tumor suppressor homologous to LRP1. Both LRP1 and LRP1B contain cytoplasmic tails with several potential endocytosis motifs. Although the positions of these endocytic motifs are similar in both receptors, LRP1B is internalized at a 15-fold slower rate than LRP1. To determine whether the slow endocytosis of LRP1B is due to the utilization of an endocytosis motif other than the YATL motif used by LRP1, we tested minireceptors with mutations in each of the five potential motifs in the LRP1B tail. Only mutation of both NPXY motifs together abolished LRP1B endocytosis, suggesting LRP1B can use either of these motifs for internalization. LRP1B contains a unique insertion of 33 amino acids not present in LRP1 that could lead to altered recognition of trafficking motifs. Surprisingly, deletion of this insertion had no effect on the endocytosis rate of LRP1B. However, replacing either half of the LRP1B tail with the corresponding LRP1 sequence markedly accelerated LRP1B endocytosis. From these data, we propose that both halves of the LRP1B cytoplasmic tail contribute to a unique global conformation, which results in less efficient recognition by endocytic adaptors and a slow endocytosis rate.
LRP1B; LRP1; endocytosis; tumor suppressor
Low-density lipoprotein receptor-related protein 1 (LRP1) functions in endocytosis and intracellular signaling for a variety of structurally diverse ligands. Although LRP1 has been implicated in several aspects of neuronal function, molecular mechanisms underlying the activity of neuronal LRP1 remain unclear. Here, we describe a signaling pathway whereby LRP1 transactivates Trk receptors. Binding of tissue-type plasminogen activator or α2-macroglobulin (α2M) to LRP1 resulted in Src family kinase (SFK) activation and SFK-dependent Trk receptor transactivation in PC12 cells and neurons. Trk receptor transactivation was necessary for activation of Akt and extracellular signal-regulated kinase, and for neurite outgrowth downstream of LRP1. Injection of the LRP1-binding domain of α2M into rat dorsal root ganglia induced Trk receptor phosphorylation, which was blocked by receptor-associated protein, an antagonist of ligand binding to LRP1. Trk receptor transactivation provides a mechanism by which diverse LRP1 ligands may demonstrate neurotrophic activity.
The major defining pathological hallmark of Alzheimer’s disease (AD) is the accumulation of amyloid β protein (Aβ), a small peptide derived from β- and γ-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that the Low-density lipoprotein receptor-related protein (LRP) plays a pivotal role in the trafficking of APP and generation of Aβ. In particular, we recently showed that the soluble cytoplasmic tail of LRP (LRP-ST) without a membrane tether was sufficient to promote Aβ generation. In this study, we demonstrate that the last 37 residues of LRP cytoplasmic tail (LRP-C37) lacking the NPxY motifs and FE65 binding mediate the core pro-amyloidogenic activity of LRP-ST. Moreover, we show that the conserved dileucine motif within the LRP-C37 region is a key determinant of its Aβ promoting activity. Finally, results from a yeast two-hybrid screen using LRP-C37 region as bait reveal four new LRP-binding proteins implicated in intracellular signalling and membrane protein trafficking. Our findings indicate that the LRP-C37 sequence represents a new protein-binding domain that may be useful as a therapeutic target and tool to lower Aβ generation in AD.
LRP; APP; FE65; amyloid; Alzheimer; Snapin; RanBPM; SH3; Filamin
The leucine-responsive regulatory protein (Lrp) regulates the expression of more than 40 genes and proteins in Escherichia coli. Among the operons that are positively regulated by Lrp are operons involved in amino acid biosynthesis (ilvIH, serA)), in the biosynthesis of pili (pap, fan, fim), and in the assimilation of ammonia (glnA, gltBD). Negatively regulated operons include operons involved in amino acid catabolism (sdaA, tdh) and peptide transport (opp) and the operon coding for Lrp itself (lrp). Detailed studies of a few members of the regulon have shown that Lrp can act directly to activate or repress transcription of target operons. A substantial fraction of operons regulated by Lrp are also regulated by leucine, and the effect of leucine on expression of these operons requires a functional Lrp protein. The patterns of regulation are surprising and interesting: in some cases activation or repression mediated by Lrp is antagonized by leucine, in other cases Lrp-mediated activation or repression is potentiated by leucine, and in still other cases leucine has no effect on Lrp-mediated regulation. Current research is just beginning to elucidate the detailed mechanisms by which Lrp can mediate such a broad spectrum of regulatory effects. Our view of the role of Lrp in metabolism may change as more members of the regulon are identified and their regulation characterized, but at this point Lrp seems to be important in regulating nitrogen metabolism and one-carbon metabolism, permitting adaptations to feast and to famine.
The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's disease (AD) but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1.
We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP), which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b). Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression.
These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered early in disease. Genetic and functional studies have implicated both LRP1 and NYGGF4 in obesity and cardiovascular disease and the physical association of these proteins may reflect a common mechanism. This is particularly interesting in light of the dual role of ApoE in both cardiovascular risk and AD. The results support further studies on the functional relationship between NYGGF4 and LRP1.
Lrp (leucine-responsive regulatory protein) activates some Escherichia coli operons that function in anabolism and represses others involved in catabolism (for a review, see J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). This overall pattern suggests that Lrp may help cells adapt to changes in the nutritional environment. Here, we tested the idea that the nutritional richness of the medium determines the amount of Lrp in cells. Lrp was measured directly by Western blotting (immunoblotting) in cells grown in a chemically defined rich medium or in a minimal medium. In addition, transcription from the lrp promoter was assessed with a lacZ reporter gene. The results with these two different measurements were nearly the same, indicating that under the conditions employed, beta-galactosidase measurements can accurately reflect Lrp levels. For cells in a minimal medium, Lrp levels were consistently lowest during the logarithmic phase of growth, but overall, there was not much variation in levels as a function of growth phase (1.3-fold difference between highest and lowest values). However, for cells in a rich medium, Lrp levels dropped 3- to 4-fold during the lag phase, remained constant during the log phase, and then rose to starting levels upon entry into the stationary phase. When cells in the log phase were compared, Lrp levels were 3- to 4-fold higher in cells growing in a minimal medium than those in a rich medium. The levels of lrp expression were the same or slightly higher in strains containing mutations in rpoS, cya, or crp compared with wild-type strains, suggesting that neither RpoS nor the cyclic AMP (cAMP) receptor protein-cAMP complex is required for expression. On the other hand, lrp expression was severely restricted in cells that could not make ppGpp because of mutations in relA and spoT. The reduced expression of lrp during logarithmic growth in a rich medium may be due to low ppGpp levels under these conditions. The repressive effects of rich medium and the stimulatory effects of ppGpp were also observed with a construct having only a minimal lrp promoter (-57 to +21). The results of other experiments suggest that Lrp levels vary inversely with the growth rate of cells instead of being determined by some component of the medium.
Inflammation is a potentially self-destructive process that needs tight control. We have identified a novel nuclear signaling mechanism through which the low-density lipoprotein receptor-related protein 1 (LRP1) limits transcription of lipopolysaccharide (LPS)-inducible inflammatory genes in vitro and in vivo. Stimulation with LPS increases proteolytic processing of the LRP1 ectodomain, resulting in the γ-secretase-dependent release of the LRP1 intracellular domain (ICD) from the plasma membrane and its translocation to the nucleus, where it binds to and represses the interferon-γ promoter. Basal transcription of LPS target genes and LPS-induced secretion of proinflammatory cytokines are increased in the absence of LRP1. Physical interaction of the LRP1 ICD with IRF-3 promotes its nuclear export and proteasomal degradation. Feedback inhibition of the inflammatory response through intramembranous processing of LRP1 thus defines a novel physiological role for γ-secretase.
γ-secretase; inflammation; lipoprotein receptor; LPS; transcriptional regulation
The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-α nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of importin-α with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-α, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-α to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131–141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-α and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-α as well as the PI3-kinase–dependent phosphorylation of Sgk. Our results define a new role importin-α in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.
Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor’s silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.
LRP1b and the closely related LRP1 are large members of the low-density lipoprotein receptor family. At the protein level LRP1b is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cargo transport and cellular signaling. Somatic LRP1b mutations frequently occur in non-small cell lung cancer and urothelial cancers, suggesting a role in the modulation of cellular growth. In contrast to LRP1, LRP1b-deficient mice develop normally, most likely due to its restricted expression pattern and functional compensation by LRP1 or other receptors. LRP1b is expressed predominantly in the brain, and a differentially spliced form is present in the adrenal gland and in the testis. Despite the presence of a potential furin cleavage site and in contrast to LRP1, immunoblotting for LRP1b reveals the presence of a single 600-kDa polypeptide species. Using a yeast two-hybrid approach, we have identified two intracellular proteins, the postsynaptic density protein 95 and the aryl hydrocarbon receptor-interacting protein, that bind to the intracellular domain of LRP1b. In addition, we have found several potential ligands that bind to the extracellular domain. Analysis of LRP1b knockout mice may provide further insights into the role of LRP1b as a tumor suppressor and into the mechanisms of cancer development.