Interleukin-1 receptor type 1 is a substrate for gamma-secretase-dependent regulated intramembrane proteolysis

Biochemical and genetic studies have revealed that the presenilins interact with several proteins and are involved in the regulated intramembrane proteolysis of numerous type 1 membrane proteins, thereby linking presenilins to a range of cellular processes. In this study, we report the characterization of a highly conserved tumor necrosis factor receptor-associated factor-6 (TRAF6) consensus-binding site within the hydrophilic loop domain of presenilin-1 (PS-1). In coimmunoprecipitation studies we indicate that presenilin-1 interacts with TRAF6 and interleukin-1 receptor-associated kinase 2. Substitution of presenilin-1 residues Pro-374 and Glu-376 by site-directed mutagenesis greatly reduces the ability of PS1 to associate with TRAF6. By studying these interactions, we also demonstrate that the interleukin-1 receptor type 1 (IL-1R1) undergoes intramembrane proteolytic processing, mediated by presenilin-dependent gamma-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble IL-1R1 ectodomain and produces an approximately 32-kDa C-terminal domain. This IL-1R1 C-terminal domain is a substrate for subsequent gamma-secretase cleavage, which generates an approximately 26-kDa intracellular domain. Specific pharmacological gamma-secretase inhibitors, expression of dominant negative presenilin-1, or presenilin deficiency independently inhibit generation of the IL-1R1 intracellular domain. Attenuation of gamma-secretase activity also impairs responsiveness to IL-1beta-stimulated activation of the MAPKs and cytokine secretion. Thus, TRAF6 and interleukin receptor-associated kinase 2 are novel binding partners for PS1, and IL-1R1 is a new substrate for presenilin-dependent gamma-secretase cleavage. These findings also suggest that regulated intramembrane proteolysis may be a control mechanism for IL-1R1-mediated signaling.     

Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor

The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent gamma-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including beta-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for gamma-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75NTR) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75NTR is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that gamma-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid beta-peptide 40 (Abeta40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkA-containing high molecular weight complex, indicating that gamma-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that gamma-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction.     

The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis

Several type-1 membrane proteins undergo regulated intramembrane proteolysis resulting in the generation of biologically active protein fragments. Presenilin-dependant gamma-secretase activity is central to this event and includes amyloid precursor protein (APP), Notch and ErbB4 as substrates. Here we show that the insulin-like growth factor 1 receptor (IGF-IR) undergoes regulated intramembrane proteolysis. A metalloprotease-dependant ectodomain-shedding event generates a approximately 52 kDa IGF-IR-carboxyl terminal domain (CTD). The IGF-IR-CTD is consequentially a substrate for gamma-secretase cleavage, liberating a approximately 50 kDa intracellular domain (ICD) that can be inhibited by a specific gamma-secretase inhibitor. This study suggests that the IGF-IR is a substrate for gamma-secretase and may mediate a function independent of its role as a receptor tyrosine kinase.     

TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain

Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.     

Metalloproteases and γ‐secretase: new membrane partners regulating p75 neurotrophin receptor signaling?

Signaling by the p75 neurotrophin receptor (p75) has been implicated in diverse neuronal responses, including the control of neuronal survival versus death and axonal regeneration and growth cone collapse, involving p75 in different neuropathological conditions. There are different levels of complexity regulating p75-mediated signaling. First, p75 can interact with different ligands and co-receptors in the plasma membrane, forming tripartite complexes, whose activation result in different cellular outcomes. Moreover, it was recently described that trafficking capacities of p75 in neurons are regulating, in addition to p75 downstream interactions, also the sequential cleavage of p75. The proteolytical processing of p75 involves, first, a shedding event that releases a membrane-bound carboxiterminal fragment (p75-CTF), followed by a gamma-secretase mediated cleavage, generating a soluble intracellular domain (p75-ICD) with signaling capabilities. The first shedding event, generating a p75-CTF, is the key step to regulating the production of p75-ICD, and although the generation of p75-ICD is important for both p75-mediated control of neuronal survival and the control of neurite outgrowth, little is known how both cleavage events are regulated. In this review, we argue that both sheddases and gamma-secretase are key membrane components regulating p75-mediated signaling transduction; therefore, further attention should be paid to their roles as p75 signaling regulators.     

{gamma}-Protocadherins, presenilin-mediated release of C-terminal fragment promotes locus expression

gamma-Protocadherins (gamma-pcdhs) are type I membrane-spanning glycoproteins, widely expressed in the mammal and required for survival. These cell adhesion molecules are expressed from a complex locus comprising 22 functional variable exons arranged in tandem, each encoding extracellular, transmembrane and intracellular sequence, and three exons for an invariant C-terminal domain (gamma-ICD). However, the signaling mechanisms that lie downstream of gamma-pcdhs have not been elucidated. Here we report that gamma-pcdhs are subject to presenilin-dependent intramembrane cleavage (PS-IP), accompanied by shedding of the extracellular domain. The cleaved intracellular domain (gamma-ICD) translocates to the cell nucleus and was detected in subsets of cortical neurons. Notably, gene-targeted mice lacking functional gamma-ICD sequence showed severely reduced gamma-pcdh mRNA levels and neonatal lethality. Most importantly, inhibition of gamma-secretase decreased gamma-pcdh locus expression. Luciferase reporter assays demonstrated that gamma-pcdh promoter activity is increased by gamma-ICD. These results reveal an intracellular signaling mechanism for gamma-pcdhs and identify a novel vital target for the gamma-secretase complex.     

Generation of intracellular domain of insulin receptor tyrosine kinase by gamma-secretase

The proteolytic cleavage of a precursor protein into alpha- and beta-subunits by furin is required to form functional insulin receptor (IR). In this study, we examined if IR undergoes the additional presenilin (PS)/gamma-secretase-dependent processing. In cells treated with gamma-secretase inhibitors or expressing the dominant-negative PS1 variant led to the accumulation of an endogenous IR C-terminal fragment. In the presence of proteasome inhibitors, we detected a PS/gamma-secretase cleavage product of the IR, termed the IR intracellular domain (ICD). Cellular fractionation and confocal microscopy analyses showed that the IR-ICD is predominantly detected in the nucleus. These data indicate that IR is a tyrosine kinase receptor, which undergoes PS/gamma-secretase-dependent processing. We also show that the autophosphorylation levels of the IR beta-subunit upon insulin stimulation were decreased by the inactivation of PS/gamma-secretase, raising the possibility that the PS/gamma-secretase proteolysis of IR may play a modulatory role in insulin signaling.     

Localization of presenilin-nicastrin complexes and gamma-secretase activity to the trans-Golgi network

Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of gamma-secretase, the protease responsible for generating the Alzheimer amyloid beta-protein. However, the differential localization of presenilins to early secretory compartments and gamma-secretase substrates to late secretory compartments and the plasma membrane (the "spatial paradox") argues against this view. We investigated this issue by studying the localization of nicastrin, another putative gamma-secretase component, and its association with presenilin-1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with the trans-Golgi network. Trafficking of nicastrin to the trans-Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin-deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin in trans-Golgi network fractions was complexed quantitatively with N- and C-terminal fragments of presenilin-1, whereas immature nicastrin present in early secretory compartments was not. Additionally, trans-Golgi network fractions contained the gamma-secretase substrate beta-amyloid precursor protein C83 and were enriched in presenilin-dependent gamma-secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin-nicastrin complexes and presenilin-dependent gamma-secretase activity are co-localized to a late secretory compartment. The findings provide further evidence that presenilin-containing complexes are the gamma-secretase, and indicate that presenilins also regulate gamma-secretase assembly.