The Lin12-notch repeats of pregnancy-associated plasma protein-A bind calcium and determine its proteolytic specificity

The Lin12-Notch repeat (LNR) module of about 35 residues is a hallmark of the Notch receptor family. Three copies, arranged in tandem, are invariably present in the extracellular portion of the Notch receptors. Although their function is unknown, genetic and biochemical data indicate that the LNR modules participate in the regulation of ligand-induced proteolytic cleavage of the Notch receptor, a prerequisite to intramembrane cleavage and Notch signaling. Outside the Notch receptor family, the LNR module is present only in the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2, which also contain three copies. Curiously, LNR modules 1 and 2 are present within the proteolytic domain of PAPP-A/A2, but LNR3 is separated from LNR2 by more than 1000 amino acids. The growth factor antagonists insulin-like growth factor-binding protein (IGFBP)-4 and -5 are both substrates of PAPP-A. We provide here evidence that the PAPP-A LNR modules function together to determine the proteolytic specificity of PAPP-A. Analysis of C-terminally truncated PAPP-A mutants followed by the analysis of LNR deletion mutants demonstrated that each of the three PAPP-A LNR modules is strictly required for proteolytic activity against IGFBP-4 but not for proteolytic activity against IGFBP-5. Individual substitution of conserved LNR residues predicted to participate in calcium coordination caused elimination (D341A, D356A, D389A, D1484A, D1499A, and D1502A) or a significant reduction (D359A and E392A) of IGFBP-4 proteolysis, whereas IGFBP-5 proteolysis was unaffected. The activity of the latter mutants against IGFBP-4 could be partially rescued by calcium, and the addition of the calcium-binding protein calbindin D9k to wild-type PAPP-A eliminated activity against IGFBP-4 but not against IGFBP-5, demonstrating that the PAPP-A LNR modules bind calcium ions. We propose a model in which LNR3 is spatially localized in proximity to LNR1 and -2, forming a single functional unit.     

A substrate specificity-determining unit of three Lin12-Notch repeat modules is formed in trans within the pappalysin-1 dimer and requires a sequence stretch C-terminal to the third module

Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1-3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1-2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore activity against IGFBP-4, cleaves IGFBP-4 when co-expressed with a PAPP-A variant, which is mutated in the active site. This suggests that LNR3 from the inactive subunit interacts in trans with LNR1-2 of the truncated PAPP-A subunit to form a functional trimeric LNR unit. We also show that formation of such a functional LNR unit depends on dimerization, as dissociation of a mutated non-covalent PAPP-A dimer results in reduced activity against IGFBP-4, but not IGFBP-5. Using PAPP-A/PAPP-A2 chimeras, we demonstrate that PAPP-A2 LNR1-2, but not LNR3, are functionally conserved with respect to IGFBP proteolysis. Additionally, we find that a sequence stretch C-terminal to LNR3 and single residues (Asp1521, Arg1529, and Asp1530) within this are required for LNR functionality.     

Force-induced unfolding simulations of the human Notch1 negative regulatory region: possible roles of the heterodimerization domain in mechanosensing

Notch receptors are core components of the Notch signaling pathway and play a central role in cell fate decisions during development as well as tissue homeostasis. Upon ligand binding, Notch is sequentially cleaved at the S2 site by an ADAM protease and at the S3 site by the γ-secretase complex. Recent X-ray structures of the negative regulatory region (NRR) of the Notch receptor reveal an auto-inhibited fold where three protective Lin12/Notch repeats (LNR) of the NRR shield the S2 cleavage site housed in the heterodimerization (HD) domain. One of the models explaining how ligand binding drives the NRR conformation from a protease-resistant state to a protease-sensitive one invokes a mechanical force exerted on the NRR upon ligand endocytosis. Here, we combined physics-based atomistic simulations and topology-based coarse-grained modeling to investigate the intrinsic and force-induced folding and unfolding mechanisms of the human Notch1 NRR. The simulations support that external force applied to the termini of the NRR disengages the LNR modules from the heterodimerization (HD) domain in a well-defined, largely sequential manner. Importantly, the mechanical force can further drive local unfolding of the HD domain in a functionally relevant fashion that would provide full proteolytic access to the S2 site prior to heterodimer disassociation. We further analyzed local structural features, intrinsic folding free energy surfaces, and correlated motions of the HD domain. The results are consistent with a model in which the HD domain possesses inherent mechanosensing characteristics that could be utilized during Notch activation. This potential role of the HD domain in ligand-dependent Notch activation may have implications for understanding normal and aberrant Notch signaling.     

Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.     

Ligand-induced signaling in the absence of furin processing of Notch1

Notch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells. The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is thought to be the only form of the receptor which is both present on the cell surface and able to generate an intracellular signal in response to ligand. Consistent with previous reports, we found that disruption of furin processing of Notch1, either by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbed ligand-dependent signaling through the well-characterized mediator of Notch signal transduction, CSL (CBF1, Su(H), and LAG-1). Yet contrary to these reports, we could detect the full-length p300 Notch1 product on the cell surface. Moreover, this uncleaved form of Notch1 could suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together, these data support our previous studies characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. Our results suggest a novel paradigm in signal transduction, one in which two isoforms of the same cell-surface receptor could mediate two distinct signaling pathways in response to ligand.     

Auxilin is essential for Delta signaling

Endocytosis regulates Notch signaling in both signaling and receiving cells. A puzzling observation is that endocytosis of transmembrane ligand by the signaling cells is required for Notch activation in adjacent receiving cells. A key to understanding why signaling depends on ligand endocytosis lies in identifying and understanding the functions of crucial endocytic proteins. One such protein is Epsin, an endocytic factor first identified in vertebrate cells. Here, we show in Drosophila that Auxilin, an endocytic factor that regulates Clathrin dynamics, is also essential for Notch signaling. Auxilin, a co-factor for the ATPase Hsc70, brings Hsc70 to Clathrin cages. Hsc70/Auxilin functions in vesicle scission and also in uncoating Clathrin-coated vesicles. We find that like Epsin, Auxilin is required in Notch signaling cells for ligand internalization and signaling. Results of several experiments suggest that the crucial role of Auxilin in signaling is, at least in part, the generation of free Clathrin. We discuss these observations in the light of current models for the role of Epsin in ligand endocytosis and the role of ligand endocytosis in Notch signaling.     

Calcium depletion dissociates and activates heterodimeric notch receptors

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.     

Highly conserved O-fucose sites have distinct effects on Notch1 function

The extracellular domain of mouse Notch1 contains 36 tandem epidermal growth factor-like (EGF) repeats, many of which are modified with O-fucose. Previous work from several laboratories has indicated that O-fucosylation plays an important role in ligand mediated Notch activation. Nonetheless, it is not clear whether all, or a subset, of the EGF repeats need to be O-fucosylated. Three O-fucose sites are invariantly conserved in all Notch homologues with 36 EGF repeats (within EGF repeats 12, 26, and 27). To investigate which O-fucose sites on Notch1 are important for ligand-mediated signaling, we mutated the three invariant O-fucose sites in mouse Notch1, along with several less highly conserved sites, and evaluated their ability to transduce Jagged1- and Delta1-mediated signaling in a cell-based assay. Our analysis revealed that mutation of any of the three invariant O-fucose sites resulted in significant changes in both Delta1 and Jagged1 mediated signaling, but mutations in less highly conserved sites had no detectable effect. Interestingly, mutation of each invariant site gave a distinct effect on Notch function. Mutation of the O-fucose site in EGF repeat 12 resulted in loss of Delta1 and Jagged1 signaling, while mutation of the O-fucose site in EGF repeat 26 resulted in hyperactivation of both Delta1 and Jagged1 signaling. Mutation of the O-fucose site in EGF repeat 27 resulted in faulty trafficking of the Notch receptor to the cell surface and a decreased S1 processing of the receptor. These results indicate that the most highly conserved O-fucose sites in Notch1 are important for both processing and ligand-mediated signaling in the context of a cell-based signaling assay.     

Notch信号通路与血液系统和白血病形成

Notch基因编码着一类进化上高度保守的跨膜受体蛋白家族,其信号通路是由Notch受体、Notch配体、CSL DNA结合蛋白组成。该信号通路能够调节淋巴细胞的发育和分化过程,介导心血管系统的形成,参与肿瘤的发生和发展。近年来有研究表明,Notch信号通路在造血作用及白血病的发生过程中起关键作用。本文综述了Notch信号通路在淋巴细胞及造血干细胞的发育和分化中的作用,进一步探讨了其对造血作用和白血病发生发展的调节,以期能够对临床造血疾病的治疗提供帮助。     

Low Density Lipoprotein Receptor-related Protein-1 (LRP1) Regulates Thrombospondin-2 (TSP2) Enhancement of Notch3 Signaling

Intracellular trafficking of Notch and Notch ligands modulates signaling, suggesting that choreography of ligand and receptor translocation is essential for optimal Notch activity. Indeed, a major model for Notch signaling posits that Notch trans-endocytosis into the ligand-expressing (signal sending) cell is a key driving force for Notch signal transduction. The extracellular protein thrombospondin-2 (TSP2) enhances Notch signaling and binds to both Jagged1 and Notch3 ectodomains, potentially bridging two essential extracellular components of Notch signaling. We investigated the role of low density lipoprotein receptor-related protein-1 (LRP1), a TSP2 receptor, in the regulation of Notch3 signaling. TSP2 potentiation of Notch is blocked by the receptor-associated protein (an inhibitor of low density lipoprotein receptor-related protein function) and requires LRP1 expression in the signal-sending cell. TSP2 stimulates Notch3 endocytosis into wild type fibroblasts but not LRP1-deficient fibroblasts. Finally, recombinant Notch3 and Jagged1 interact with the LRP1 85-kDa B-chain, a subunit that lacks known ligand binding function. Our data suggest that LRP1 and TSP2 stimulate Notch activity by driving trans-endocytosis of the Notch ectodomain into the signal-sending cell and demonstrate a novel, non-cell autonomous function of LRP1 in cell-cell signaling.     

Tissue-specific regulation of the LIM homeobox gene lin-11 during development of the Caenorhabditis elegans egg-laying system

The egg-laying system of Caenorhabditis elegans hermaphrodites requires development of the vulva and its precise connection with the uterus. This process is regulated by LET-23-mediated epidermal growth factor signaling and LIN-12-mediated lateral signaling pathways. Among the nuclear factors that act downstream of these pathways, the LIM homeobox gene lin-11 plays a major role. lin-11 mutant animals are egg-laying defective because of the abnormalities in vulval lineage and uterine seam-cell formation. However, the mechanisms providing specificity to lin-11 function are not understood. Here, we examine the regulation of lin-11 during development of the egg-laying system. Our results demonstrate that the tissue-specific expression of lin-11 is controlled by two distinct regulatory elements that function as independent modules and together specify a wild-type egg-laying system. A uterine pi lineage module depends on the LIN-12/Notch signaling, while a vulval module depends on the LIN-17-mediated Wnt signaling. These results provide a unique example of the tissue-specific regulation of a LIM homeobox gene by two evolutionarily conserved signaling pathways. Finally, we provide evidence that the regulation of lin-11 by LIN-12/Notch signaling is directly mediated by the Su(H)/CBF1 family member LAG-1.     

Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the Notch ligand Delta1

The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis. Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells. Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell. Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1). We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo. We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains. In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds. Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos.     

Down-regulation of Delta by proteolytic processing

Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, we examined the biological activity of purified soluble Delta forms and conclude that Delta cleavage is an important down-regulating event in Notch signaling. The data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner.