Down-Regulation of the Met Receptor Tyrosine Kinase by Presenilin-dependent Regulated Intramembrane Proteolysis

Hepatocyte growth factor/scatter factor (HGF/SF) acts through the membrane-anchored Met receptor tyrosine kinase to induce invasive growth. Deregulation of this signaling is associated with tumorigenesis and involves, in most cases, overexpression of the receptor. We demonstrate that Met is processed in epithelial cells by presenilin-dependent regulated intramembrane proteolysis (PS-RIP) independently of ligand stimulation. The proteolytic process involves sequential cleavage by metalloproteases and the γ-secretase complex, leading to generation of labile fragments. In normal epithelial cells, although expression of cleavable Met by PS-RIP is down-regulated, uncleavable Met displayed membrane accumulation and induced ligand-independent motility and morphogenesis. Inversely, in transformed cells, the Met inhibitory antibody DN30 is able to promote Met PS-RIP, resulting in down-regulation of the receptor and inhibition of the Met-dependent invasive growth. This demonstrates the original involvement of a proteolytic process in degradation of the Met receptor implicated in negative regulation of invasive growth.     

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.     

Proteolytic Shedding of Human Colony-Stimulating Factor 1 Receptor and its implication

Both Colony-stimulating factor 1 receptor (CSF1R) and triggering receptor expressed on myeloid cells-2 (TREM2) are trans-membrane receptors and are expressed in the brain primarily by microglia. Mutations in these two microglia-expressed genes associated with neurodegenerative disease have recently been grouped under the term “microgliopathy”. Several literatures have indicated that CSF1R and TREM2 encounters a stepwise shedding and TREM2 variants impair or accelerate the processing. However, whether CSF1R variant affects the shedding of CSF1R remains elusive. Here, plasmids containing human CSF1R or TREM2 were transiently transfected into the human embryonic kidney (HEK) 293T cells. Using Western Blot and/or ELISA assay, we demonstrated that, similar to those of TREM2, an N-terminal fragment (NTF) shedding of CSF1R ectodomain and a subsequent C-terminal fragment (CTF) of CSF1R intra-membrane were generated by a disintegrin and metalloprotease (ADAM) family member and by γ-secretase, respectively. And the shedding was inhibited by treatment with Batimastat, an ADAM inhibitor, or DAPT or compound E, a γ-secretase inhibitor. Importantly, we show that the cleaved fragments, both extracellular domain and intracellular domain of a common disease associated I794T variant, were decreased significantly. Together, our studies demonstrate a stepwise approach of human CSF1R cleavage and contribute to understand the pathogenicity of CSF1R I794T variant in adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). These studies also suggest that the cleaved ectodomain fragment released from CSF1R may be proposed as a diagnostic biomarker for ALSP.     

The cytosolic domain of protein-tyrosine kinase 7 (PTK7), generated from sequential cleavage by a disintegrin and metalloprotease 17 (ADAM17) and γ-secretase, enhances cell proliferation and migration in colon cancer cells

Protein-tyrosine kinase 7 (PTK7) is a member of the defective receptor protein-tyrosine kinases and is known to function as a regulator of planar cell polarity during development. Its expression is up-regulated in some cancers including colon carcinomas. A 100-kDa fragment of PTK7 was detected in the culture media from colon cancer cells and HEK293 cells. The shed fragment was named sPTK7-Ig1-7 because its molecular mass was very similar to that of the entire extracellular domain of PTK7 that contains immunoglobulin-like loops 1 to 7 (Ig1-7). The shedding of sPTK7-Ig1-7 was enhanced by treatment with phorbol 12-myristate 13-acetate. In addition to the sPTK7-Ig1-7 found in the culture medium, two C-terminal fragments of PTK7 were detected in the cell lysates: PTK7-CTF1, which includes a transmembrane segment and a cytoplasmic domain, and PTK7-CTF2, which lacks most of the transmembrane segment from PTK7-CTF1. Analysis of PTK7 processing in the presence of various protease inhibitors or after knockdown of potential proteases suggests that shedding of PTK7 into sPTK7-Ig1-7 and PTK7-CTF1 is catalyzed by ADAM17, and further cleavage of PTK7-CTF1 into PTK7-CTF2 is mediated by the γ-secretase complex. PTK7-CTF2 localizes to the nucleus and enhances proliferation, migration, and anchorage-independent colony formation. Our findings demonstrate a novel role for PTK7 in the tumorigenesis via generation of PTK7-CTF2 by sequential cleavage of ADAM17 and γ-secretase.     

Neuronal brain-derived neurotrophic factor is synthesized in excess, with levels regulated by sortilin-mediated trafficking and lysosomal degradation

Brain-derived neurotrophic factor (BDNF) regulates neuronal differentiation, synaptic plasticity, and morphology, and modest changes in BDNF levels results in complex behavioral phenotypes. BDNF levels and intracellular localization in neurons are regulated by multiple mechanisms, including use of distinct promoters, mRNA and protein transport, and regulated cleavage of proBDNF to mature BDNF. Sortilin is an intracellular chaperone that binds to the prodomain of BDNF to traffic it to the regulated secretory pathway. However, sortilin binds to numerous ligands and plays a major role in mannose 6-phosphate receptor-independent transport of lysosomal hydrolases utilizing motifs in the intracellular domain that mediate trafficking from the Golgi and late endosomes. Sortilin is modified by ectodomain shedding, although the biological implications of this are not known. Here we demonstrate that ADAM10 is the preferred protease to cleave sortilin in the extracellular stalk region, to release the ligand binding sortilin ectodomain from the transmembrane and cytoplasmic domains. We identify sortilin shedding at the cell surface and in an intracellular compartment. Both sortilin and BDNF are trafficked to and degraded by the lysosome in neurons, and this is dependent upon the sortilin cytoplasmic tail. Indeed, expression of the sortilin ectodomain, which corresponds to the domain released after shedding, impairs lysosomal targeting and degradation of BDNF. These findings characterize the regulation of sortilin shedding and identify a novel mechanism by which sortilin ectodomain shedding acts as a regulatory switch for delivery of BDNF to the secretory pathway or to the lysosome, thus modulating the bioavailability of endogenous BDNF.     

Nuclear translocation of intracellular domain of Protogenin by proteolytic cleavage

Protogenin (PRTG) is a transmembrane protein of immunoglobulin superfamily, which has multiple roles in embryogenesis as a receptor or an adhesion molecule. In this study, we present sequential proteolytic cleavage of PRTG. The cleavage first occurs at the extracellular domain, then at the interface of the transmembrane and the intracellular domain by γ-secretase, which results in the release of the intracellular domain of PRTG (PRTG-ICD). PRTG-ICD contains putative nuclear localization signal (NLS) at its N-terminal, and translocates to the nucleus in cultured cells and in the neuroepithelial cells of chick embryos. We propose that the PRTG-ICD is cleaved by γ-secretase and translocates to the nucleus, which is potentially implicated in signaling for neural differentiation and in cell adhesion mediated by PRTG.     

Involvement of lysosomal degradation in VEGF-C-induced down-regulation of VEGFR-3

The vascular endothelial growth factor (VEGF)-C-induced down-regulation of VEGF receptor (VEGFR)-3 is important in lymphangiogenesis. Here, we demonstrate that VEGF-C, -D, and -C156S, but not VEGF-A, down-regulate VEGFR-3. VEGF-C stimulates VEGFR-3 tyrosyl phosphorylation and transient phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinases in lymphatic endothelial cells. VEGF-C-induced down-regulation of VEGFR-3 was blocked by a VEGF-C trap, tyrosine kinase inhibitor, and leupeptin, pepstatin, and E64 (LPE), but was unaffected by Notch 1 activator and γ-secretase inhibitors. Our findings indicate that VEGF-C down-regulates VEGFR-3 in lymphatic endothelial cells through VEGFR-3 kinase activation and, in part, via lysosomal degradation.     

The dyslexia-associated KIAA0319 protein undergoes proteolytic processing with {gamma}-secretase-independent intramembrane cleavage

The KIAA0319 gene has been associated with reading disability in several studies. It encodes a plasma membrane protein with a large, highly glycosylated, extracellular domain. This protein is proposed to function in adhesion and attachment and thought to play an important role during neuronal migration in the developing brain. We have previously proposed that endocytosis of this protein could constitute an important mechanism to regulate its function. Here we show that KIAA0319 undergoes ectodomain shedding and intramembrane cleavage. At least five different cleavage events occur, four in the extracellular domain and one within the transmembrane domain. The ectodomain shedding processing cleaves the extracellular domain, generating several small fragments, including the N-terminal region with the Cys-rich MANEC domain. It is possible that these fragments are released to the extracellular medium and trigger cellular responses. The intramembrane cleavage releases the intracellular domain from its membrane attachment. Our results suggest that this cleavage event is not carried out by γ-secretase, the enzyme complex involved in similar processing in many other type I proteins. The soluble cytoplasmic domain of KIAA0319 is able to translocate to the nucleus, accumulating in nucleoli after overexpression. This fragment has an unknown role, although it could be involved in regulation of gene expression. The absence of DNA-interacting motifs indicates that such a function would most probably be mediated through interaction with other proteins, not by direct DNA binding. These results suggest that KIAA0319 not only has a direct role in neuronal migration but may also have additional signaling functions.     

Characterization of nectin processing mediated by presenilin-dependent γ-secretase

Nectins play an important role in forming various intercellular junctions including synapses. This role is regulated by several secretases present at intercellular junctions. We have investigated presenilin (PS)-dependent secretase-mediated processing of nectins in PS1 KO cells and primary hippocampal neurons. The loss of PS1/γ-secretase activity delayed the processing of nectin-1 and caused the accumulation of its full-length and C-terminal fragments. Over-expression of PS2 in PS1 KO cells compensated for the loss of PS1, suggesting that PS2 also has the ability to regulate nectin-1 processing. In mouse brain slices, a pronounced increase in levels of 30 and 24 kDa C-terminal fragments in response to chemical long-term potentiation was observed. The mouse brain synaptosomal fractionation study indicated that nectin-1 localized to post-synaptic and preferentially pre-synaptic membranes and that shedding occurs in both compartments. These data suggest that nectin-1 shedding and PS-dependent intramembrane cleavage occur at synapses, and is a regulated event during conditions of synaptic plasticity in the brain. Point mutation analysis identified several residues within the transmembrane domain that play a critical role in the positioning of cleavage sites by ectodomain sheddases. Nectin-3, which forms hetero-trans-dimers with nectin-1, also undergoes intramembrane cleavage mediated by PS1/γ-secretase, suggesting that PS1/γ-secreatse activity regulates synapse formation and remodeling by nectin processing.     

The type III TGF-β receptor betaglycan transmembrane-cytoplasmic domain fragment is stable after ectodomain cleavage and is a substrate of the intramembrane protease γ-secretase

The Type III TGF-β receptor, betaglycan, is a widely expressed proteoglycan co-receptor for TGF-β superfamily ligands. The full-length protein undergoes ectodomain cleavage with release of a soluble ectodomain fragment. The fate of the resulting transmembrane-cytoplasmic fragment, however, has never been explored. We demonstrate here that the transmembrane-cytoplasmic fragment is stable in transfected cells and in cell lines expressing endogenous betaglycan. Production of this fragment is inhibited by the ectodomain shedding inhibitor TAPI-2. Treatment of cells with inhibitors of the intramembrane protease γ-secretase stabilizes this fragment, suggesting that it is a substrate of γ-secretase. Expression of the transmembrane-cytoplasmic fragment as well as γ-secretase inhibitor stabilization are independent of TGF-β1 or -β2 and are unaffected by mutation of the cytoplasmic domain serines that undergo phosphorylation. γ-Secretase inhibition or the expression of a transmembrane-cytoplasmic fragment in HepG2 cells blunted TGF-β2 signaling. Our findings thus suggest that the transmembrane-cytoplasmic fragment remaining after betaglycan ectodomain cleavage is stable and a substrate of γ-secretase, which may have significant implications for the TGF-β signaling response.     

Met degradation: more than one stone to shoot a receptor down

The receptor tyrosine kinase Met and its high-affinity ligand, the hepatocyte growth factor/scatter factor (HGF/SF), are essential to embryonic development. Deregulation of their signaling is associated with tumorigenesis and metastasis, notably through receptor overexpression. It is thus important to understand the mechanisms controlling Met expression. The ligand-dependent internalization of Met and its subsequent degradation in the lysosomal compartment are well described. This process is known to attenuate downstream Met signaling pathways. Yet internalized Met takes part directly in intracellular signaling by chaperoning signaling factors in the course of its trafficking. Furthermore, recent studies describe various new degradation mechanisms of membrane-anchored Met, involving proteolytic cleavages or association with novel partners. Although all these degradations are ligand-independent, they share, to different extents, some common features with canonical HGF/SF-dependent degradation. Interestingly, activated Met variants display resistance to degradation, suggesting defective degradation is involved in tumorigenesis. Conversely, forced degradation of Met through reinduction of one or more degradation pathways is a promising therapeutic strategy.     

Regulated proteolytic processing of Tie1 modulates ligand responsiveness of the receptor-tyrosine kinase Tie2

Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor-tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane-tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. After Tie1 ectodomain cleavage the newly formed 45-kDa endodomain undergoes additional proteolytic processing mediated by gamma-secretase to generate an amino-terminal-truncated 42-kDa fragment that is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing, we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of cartilage oligomeric protein angiopoietin 1. A key function of gamma-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases cartilage oligomeric protein angiopoietin 1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand after shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggest that regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2.     

Necrosis- and apoptosis-related Met cleavages have divergent functional consequences

Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged.Met is a receptor tyrosine kinase expressed predominantly by epithelial cells and activated by its stromal ligand, hepatocyte growth factor/scatter factor (HGF/SF). Met activation stimulates a biological program called invasive growth,¹ involving survival, proliferation, invasion, and morphogenesis of epithelial cells. Ligand-stimulated Met acts, furthermore, as an angiogenic and neurotrophic factor.^{2, 3} HGF/SF and Met are essential to several steps of embryogenesis, experiments on transgenic mice having shown that they are necessary for formation of the placenta, liver, limb muscle, neurons, and lung airspace.^{4, 5, 6, 7, 8} In adults, HGF/SF and Met promote regeneration of several organs, including the liver, kidneys, and thymus.^{9, 10, 11, 12, 13}Aberrant Met and HGF/SF signaling contributes to promoting tumorigenesis and metastasis (for review see Furlan et al.).¹⁴ A direct link between Met and cancer has been evidenced by observation of Met germinal mutations linked to hereditary papillary renal carcinoma.¹⁵ Met and/or HGF/SF are/is also overexpressed in several human cancers.¹⁶ Given its important oncogenic activity, Met is the target of many therapeutic agents currently under clinical investigation.¹⁷Downregulation of Met following its activation by HGF/SF is an important negative regulatory mechanism preventing receptor overactivation. We have previously shown that Met expression and activity are also controlled by proteolytic cleavages. Under steady-state conditions, Met is processed by PS-RIP (presenilin-regulated intramembrane proteolysis).^{18, 19} This process involves cleavage of Met within its extracellular juxtamembrane domain by A disintegrin and metalloproteinase (ADAM)-10,²⁰ generating a soluble N-terminal fragment (Met-NTF), which is released into the extracellular space, and a membrane-anchored C-terminal Met fragment (Met-CTF). The latter is in turn efficiently degraded by the lysosome and by further γ-secretase cleavages. Constitutive degradation of the Met receptor by PS-RIP contributes to regulating its half-life.Under apoptotic conditions, Met is cleaved by caspases²¹ within its C-terminal tail and its intracellular juxtamembrane domain. These cleavages remove the C-terminal tail of Met and separate the extracellular ligand-binding domain from the intracellular kinase domain. The generated 40-kDa intracellular fragment, previously called ‘p40Met'' and here called p40Met^caspase, can increase cell death by promoting mitochondrial permeabilization.^{22, 23} Removal of the C-terminal tail of Met is required for the efficient pro-apoptotic action of the fragment. This pro-apoptotic function of Met makes it a member of the dependence receptor family.²⁴ Met cleavages are illustrated in Figure 6a.Although the mechanisms underlying apoptosis have been studied extensively, necrosis has only recently been described as a regulated cell death mechanism.²⁵ Necrosis is an adenosine triphosphate (ATP)-independent cell death mechanism featuring early plasma membrane and organelle disruption. Many pathways can lead to cell necrosis, including calcium overload. This type of cell stress has been amply described in the nervous system, where an increase in intracellular calcium results in neuronal injury and neurodegenerative diseases. In many other cell types, calcium ionophores such as ionomycin can induce rapid necrosis. An increase in intracellular calcium triggers activation of several proteases, including calpains and cathepsins.^{26, 27, 28} Calpains are calcium-dependent proteases capable of cleaving multiple substrates and involved in regulating various cellular processes, including migration, autophagy, apoptosis, and necrosis. Interestingly, the effector role of calpains during necrosis is reminiscent of the function of caspases during apoptosis. Caspases are directly involved in morphological changes observed during apoptosis, while calpains can cleave cytoskeletal proteins such as spectrin and tubulin, thus favoring dismantling of cell structure during necrosis.^{29, 30, 31}Although apoptosis resistance is a hallmark of many cancer cells,³² some such cells remain sensitive to other cell death processes, including necrosis.³³ Thus, a better understanding of the mechanisms underlying necrosis is important, as it could help to elaborate novel therapeutic strategies. Here we show that calcium stress induced by calcium ionophores triggers Met degradation during necrotic cell death. This loss of Met receptor occurs early during the process and is mediated by Met cleavages: by calpains in its intracellular part and by metalloproteases in its extracellular part. These cleavages generate an extracellular fragment and an intracellular fragment with a molecular weight close to that of p40Met^caspase.     

Rac1 changes the substrate specificity of gamma-secretase between amyloid precursor protein and Notch1

Beta amyloid peptide is generated from amyloid precursor protein (APP) by proteolytic cleavage of β- and γ-secretases, and plays a critical role in the pathogenesis of Alzheimer’s disease. Since γ-secretase cleaves several proteins including APP and Notch in a number of cell types, it is important to understand the conditions determining γ-secretase substrate specificity. In the present study, inhibition of Rac1 attenuated γ-secretase activity for APP, resulting in decreased production of the APP intracellular domain but accumulated C-terminal fragments (APP-CTF). In contrast, Rac1 inhibitor, NSC23766 increased production of the Notch1 intracellular domain but slightly decreased the ectodomain-shed form of Notch1 (NotchΔE). To elucidate the mechanism underlying these observations, we performed co-immunoprecipitation experiments to analyze the interaction between Rac1 and presenilin1 (PS1), a component of the γ-secretase complex. Inhibition of Rac1 enhanced its interaction with PS1. Under the same condition, PS1 interacted more strongly with NotchΔE than with APP-CTF. Our results suggested that PS1 determines the preferred substrate for γ-secretase between APP and Notch1, depending on the activation status of Rac1.     

Protocadherin-12 cleavage is a regulated process mediated by ADAM10 protein: evidence of shedding up-regulation in pre-eclampsia

Protocadherins are a group of transmembrane proteins with homophilic binding activity, members of the cadherin superfamily. Apart from their role in adhesion, the cellular functions of protocadherins are essentially unknown. Protocadherin (PCDH)12 was previously identified in invasive trophoblasts and endothelial and mesangial cells in the mouse. Invalidation studies revealed that the protein was required for optimal placental development. In this article, we show that its human homolog is abundantly expressed in various trophoblast subtypes of the human placenta and at lower levels in endothelial cells. We demonstrate that PCDH12 is shed at high rates in vitro. The shedding mechanism depends on ADAM10 and results in reduced cellular adhesion in a cell migration assay. PCDH12 is subsequently cleaved by the γ-secretase complex, and its cytoplasmic domain is rapidly degraded by the proteasome. PCDH12 shedding is regulated by interlinked intracellular pathways, including those involving protein kinase C, PI3K, and cAMP, that either increase or inhibit cleavage. In endothelial cells, VEGF, prostaglandin E(2), or histamine regulates PCDH12 shedding. The extracellular domain of PCDH12 was also detected in human serum and urine, thus providing evidence of PCDH12 shedding in vivo. Importantly, we observed an increase in circulating PCDH12 in pregnant women who later developed a pre-eclampsia, a frequent pregnancy syndrome and a major cause of maternal and fetal morbidity and mortality. In conclusion, we speculate that, like in mice, PCDH12 may play an important role in human placental development and that proteolytic cleavage in response to external factors, such as cytokines and pathological settings, regulates its activity.     

γ-Secretase-mediated growth hormone receptor proteolysis: mapping of the intramembranous cleavage site

GH receptor (GHR) undergoes regulated proteolysis by both metalloprotease (α-secretase) and γ-secretase activities. α-Secretase activity regulates GHR availability and sensitivity and generates circulating GH binding protein. The function of γ-secretase cleavage is yet uncertain. We investigated GHR determinants that affect inducible sequential α- and γ-secretase cleavage and thus remnant and stub generation, respectively. Purification and N-terminal sequencing of the stub revealed that γ-secretase cleavage occurs at an ε-site in GHR’s transmembrane domain four residues from the intracellular domain. Mutagenesis revealed that deletion of the proximal two transmembrane residues prevented both α- and γ-secretase-mediated proteolysis and deletion of four residues around the ε-site precluded surface GHR expression and proteolysis. However, point mutations in and around the ε-site affected neither α- or γ-secretase cleavage. We conclude that both cleavages likely occur at the cell surface and sequentially (α-secretase followed by γ-secretase) and that ε-site cleavage by γ-secretase does not require a consensus sequence.     

γ-Secretase Activity Is Required for Regulated Intramembrane Proteolysis of Tumor Necrosis Factor (TNF) Receptor 1 and TNF-mediated Pro-apoptotic Signaling

The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.     

Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein

Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca²⁺ from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.     

Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I

The multivesicular body (MVB) pathway is responsible for both the biosynthetic delivery of lysosomal hydrolases and the downregulation of numerous activated cell surface receptors which are degraded in the lysosome. We demonstrate that ubiquitination serves as a signal for sorting into the MVB pathway. In addition, we characterize a 350 kDa complex, ESCRT-I (composed of Vps23, Vps28, and Vps37), that recognizes ubiquitinated MVB cargo and whose function is required for sorting into MVB vesicles. This recognition event depends on a conserved UBC-like domain in Vps23. We propose that ESCRT-I represents a conserved component of the endosomal sorting machinery that functions in both yeast and mammalian cells to couple ubiquitin modification to protein sorting and receptor downregulation in the MVB pathway.     

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl

Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA.