MMPs and Soluble ICAM-5 Increase Neuronal Excitability within In Vitro Networks of Hippocampal Neurons

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are released from neurons in an activity dependent manner. Published studies suggest their activity is important to varied forms of learning and memory. At least one MMP can stimulate an increase in the size of dendritic spines, structures which represent the post synaptic component for a large number of glutamatergic synapses. This change may be associated with increased synaptic glutamate receptor incorporation, and an increased amplitude and/or frequency of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) mini excitatory post-synaptic currents (EPSCs). An associated increase in the probability of action potential occurrence would be expected. While the mechanism(s) by which MMPs may influence synaptic structure and function are not completely understood, MMP dependent shedding of specific cell adhesion molecules (CAMs) could play an important role. CAMs are ideally positioned to be cleaved by synaptically released MMPs, and shed N terminal domains could potentially interact with previously unengaged integrins to stimulate dendritic actin polymerization with spine expansion. In the present study, we have used multielectrode arrays (MEAs) to investigate MMP and soluble CAM dependent changes in neuronal activity recorded from hippocampal cultures. We have focused on intercellular adhesion molecule-5 (ICAM-5) in particular, as this CAM is expressed on glutamatergic dendrites and shed in an MMP dependent manner. We show that chemical long-term potentiation (cLTP) evoked changes in recorded activity, and the dynamics of action potential bursts in particular, are altered by MMP inhibition. A blocking antibody to β(1) integrins has a similar effect. We also show that the ectodomain of ICAM-5 can stimulate β(1) integrin dependent increases in spike counts and burst number. These results support a growing body of literature suggesting that MMPs have important effects on neuronal excitability. They also support the possibility that MMP dependent shedding of specific synaptic CAMs can contribute to these effects.     

Soluble ICAM-5, a Product of Activity Dependent Proteolysis,Increases mEPSC Frequency and Dendritic Expression of GluA1

Matrix metalloproteinases (MMPs) are zinc dependent endopeptidases that can be released from neurons in an activity dependent manner to play a role in varied forms of learning and memory. MMP inhibitors impair hippocampal long term potentiation (LTP), spatial memory, and behavioral correlates of drug addiction. Since MMPs are thought to influence LTP through a β₁ integrin dependent mechanism, it has been suggested that these enzymes cleave specific substrates to generate integrin binding ligands. In previously published work, we have shown that neuronal activity stimulates rapid MMP dependent shedding of intercellular adhesion molecule-5 (ICAM-5), a synaptic adhesion molecule expressed on dendrites of the telencephalon. We have also shown that the ICAM-5 ectodomain can interact with β₁ integrins to stimulate integrin dependent phosphorylation of cofilin, an event that occurs with dendritic spine maturation and LTP. In the current study, we investigate the potential for the ICAM-5 ectodomain to stimulate changes in α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) dependent glutamatergic transmission. Single cell recordings show that the ICAM-5 ectodomain stimulates an increase in the frequency, but not the amplitude, of AMPA mini excitatory post synaptic currents (mEPSCs). With biotinylation and precipitation assays, we also show that the ICAM-5 ectodomain stimulates an increase in membrane levels of GluA1, but not GluA2, AMPAR subunits. In addition, we observe an ICAM-5 associated increase in GluA1 phosphorylation at serine 845. Concomitantly, ICAM-5 affects an increase in GluA1 surface staining along dendrites without affecting an increase in dendritic spine number. Together these data are consistent with the possibility that soluble ICAM-5 increases glutamatergic transmission and that post-synaptic changes, including increased phosphorylation and dendritic insertion of GluA1, could contribute. We suggest that future studies are warranted to determine whether ICAM-5 is one of a select group of synaptic CAMs whose shedding contributes to MMP dependent effects on learning and memory.     

Activation of NMDA receptors promotes dendritic spine development through MMP-mediated ICAM-5 cleavage

Matrix metalloproteinase (MMP)-2 and -9 are pivotal in remodeling many tissues. However, their functions and candidate substrates for brain development are poorly characterized. Intercellular adhesion molecule-5 (ICAM-5; Telencephalin) is a neuronal adhesion molecule that regulates dendritic elongation and spine maturation. We find that ICAM-5 is cleaved from hippocampal neurons when the cells are treated with N-methyl-d-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA). The cleavage is blocked by MMP-2 and -9 inhibitors and small interfering RNAs. Newborn MMP-2- and MMP-9-deficient mice brains contain more full-length ICAM-5 than wild-type mice. NMDA receptor activation disrupts the actin cytoskeletal association of ICAM-5, which promotes its cleavage. ICAM-5 is mainly located in dendritic filopodia and immature thin spines. MMP inhibitors block the NMDA-induced cleavage of ICAM-5 more efficiently in dendritic shafts than in thin spines. ICAM-5 deficiency causes retraction of thin spine heads in response to NMDA stimulation. Soluble ICAM-5 promotes elongation of dendritic filopodia from wild-type neurons, but not from ICAM-5-deficient neurons. Thus, MMPs are important for ICAM-5-mediated dendritic spine development.     

Ectodomain shedding of nectin-1 regulates the maintenance of dendritic spine density

Synaptic remodeling has been postulated as a mechanism underlying synaptic plasticity and cell adhesion molecules are thought to contribute to this process. We examined the role of nectin-1 ectodomain shedding on synaptogenesis in cultured rat hippocampal neurons. Nectins are Ca(2+) -independent immunoglobulin-like adhesion molecules, involved in cell-cell adherens junctions. Herein, we show that the processing of nectin-1 occurs by multiple endoproteolytic steps both in vivo and in vitro. We identified regions containing two distinct cleavage sites within the ectodomain of nectin-1. By alanine scanning mutagenesis, two point mutations that disrupt nectin-1 ectodomain cleavage events were identified. Expression of these mutants significantly alters the density of dendritic spines. These findings suggest that ectodomain shedding of nectin-1 regulates dendritic spine density and related synaptic functions.     

Synaptically Released Matrix Metalloproteinase Activity in Control of Structural Plasticity and the Cell Surface Distribution of GluA1-AMPA Receptors

Synapses are particularly prone to dynamic alterations and thus play a major role in neuronal plasticity. Dynamic excitatory synapses are located at the membranous neuronal protrusions called dendritic spines. The ability to change synaptic connections involves both alterations at the morphological level and changes in postsynaptic receptor composition. We report that endogenous matrix metalloproteinase (MMP) activity promotes the structural and functional plasticity of local synapses by its effect on glutamate receptor mobility and content. We used live imaging of cultured hippocampal neurons and quantitative morphological analysis to show that chemical long-term potentiation (cLTP) induces the permanent enlargement of a subset of small dendritic spines in an MMP-dependent manner. We also used a superresolution microscopy approach and found that spine expansion induced by cLTP was accompanied by MMP-dependent immobilization and synaptic accumulation as well as the clustering of GluA1-containing AMPA receptors. Altogether, our results reveal novel molecular and cellular mechanisms of synaptic plasticity.     

Signals mediating cleavage of intercellular adhesion molecule-1

ICAM-1, a membrane-bound receptor, is released as soluble ICAM-1 in inflammatory diseases. To delineate mechanisms regulating ICAM-1 cleavage, studies were performed in endothelial cells (EC), human embryonic kidney (HEK)-293 cells transfected with wild-type (WT) ICAM-1, and ICAM-1 containing single tyrosine-to-alanine substitutions (Y474A, Y476A, and Y485A) in the cytoplasmic region. Tyrosine residues at 474 and 485 become phosphorylated upon ICAM-1 ligation and associate with signaling modules. Cleavage was assessed by using an antibody against the cytoplasmic tail of ICAM-1, which recognizes intact ICAM-1 and the 7-kDa membrane-bound fragment remaining after cleavage. Cleavage in HEK-293 WT cells was accelerated by phorbol ester PMA, whereas in EC it was induced by tumor necrosis factor-. In both cell types, a 7-kDa ICAM-1 remnant was detected. Tyrosine phosphatase inhibitors dephostatin and sodium orthovanadate augmented cleavage. PD-98059 (MEK kinase inhibitor), geldanamycin and PP2 (Src kinase inhibitors), and wortmannin (phosphatidylinositol 3-kinase inhibitor) dose-dependently inhibited cleavage in both cell types. SB-203580 (p38 inhibitor) was more effective in EC, and D609 (PLC inhibitor) mostly affected cleavage in HEK-293 cells. Cleavage was drastically decreased in Y474A and Y485A, whereas it was marginally reduced in Y476A. Surprisingly, phosphorylation was not detectable on the 7-kDa fragment of ICAM-1. These results implicate distinct pathways in the cleavage process and suggest a preferred signal transmission route for ICAM-1 shedding in the two cell systems tested. Tyrosine residues Y474 and Y485 within the cytoplasmic sequence of ICAM-1 regulate the cleavage process. ectodomain shedding; signaling; tyrosine phosphorylation     

Matrix metalloproteinase-7 disrupts dendritic spines in hippocampal neurons through NMDA receptor activation

Dendritic spines are protrusions from the dendritic shaft that host most excitatory synapses in the brain. Although they first emerge during neuronal maturation, dendritic spines remain plastic through adulthood, and recent advances in the molecular mechanisms governing spine morphology have shown them to be exquisitely sensitive to changes in the micro-environment. Among the many factors affecting spine morphology are components and regulators of the extracellular matrix (ECM). Modification of the ECM is critical to the repair of injuries throughout the body, including the CNS. Matrix metalloproteinase (MMP)-7/matrilysin is a key regulator of the ECM during pathogen infection, after nerve crush and in encephalitogenic disorders. We have investigated the effects of MMP-7 on dendritic spines in hippocampal neuron cultures and found that it induces the transformation of mature, short mushroom-shaped spines into long, thin filopodia reminiscent of immature spines. These changes were accompanied by a dramatic redistribution of F-actin from spine heads into thick, rope-like structures in the dendritic shaft. Strikingly, MMP-7 effects on dendritic spines were similar to those of NMDA treatment, and both could be blocked by channel-specific antagonists. These findings are the first direct evidence that MMPs can influence the morphology of mature dendritic spines, and hence synaptic stability.     

Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1)

Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.     

Ectodomain shedding of TGF-alpha and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades

A variety of transmembrane proteins, such as transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and L-selectin, undergo shedding, i.e. cleavage of the ectodomain, resulting in release of a soluble protein. Although the physiological relevance of ectodomain shedding is well recognized, little is known about the signaling mechanisms activating this process. We show that growth factor activation of cell surface tyrosine kinase receptors induces ectodomain cleavage of transmembrane TGF-alpha through activation of the Erk MAP kinase signaling cascade without the need for new protein synthesis. In addition, expression of constitutively activated MEK1 or its downstream target Erk2 MAP kinase was sufficient to stimulate TGF-alpha shedding. The basal cleavage level in the absence of exogenous growth factor stimulation was due to p38 MAP kinase signaling. Accordingly, a constitutively activated MKK6, a p38 activator, activated TGF-alpha shedding in the absence of exogenous stimuli. In addition to TGF-alpha shedding, these mechanisms also mediate L-selectin and TNF-alpha cleavage. Thus, L-selectin shedding by neutrophils, induced by N-formylmethionyl-leucyl-phenylalanine, was strongly inhibited by inhibitors of Erk MAP kinase or p38 MAP kinase signaling. Our results indicate that activation of Erk and p38 signaling pathways may represent a general physiological mechanism to induce shedding of a variety of transmembrane proteins.     

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.     

GSK-3β and MMP-9 Cooperate in the Control of Dendritic Spine Morphology

Changes in the morphology of dendritic spines are prominent during learning and in different neurological and neuropsychiatric diseases, including those in which glycogen synthase kinase-3β (GSK-3β) has been implicated. Despite much evidence of the involvement of GSK-3β in functional synaptic plasticity, it is unclear how GSK-3β controls structural synaptic plasticity (i.e., the number and shape of dendritic spines). In the present study, we used two mouse models overexpressing and lacking GSK-3β in neurons to investigate how GSK-3β affects the structural plasticity of dendritic spines. Following visualization of dendritic spines with DiI dye, we found that increasing GSK-3β activity increased the number of thin spines, whereas lacking GSK-3β increased the number of stubby spines in the dentate gyrus. Under conditions of neuronal excitation, increasing GSK-3β activity caused higher activity of extracellularly acting matrix metalloproteinase-9 (MMP-9), and MMP inhibition normalized thin spines in GSK-3β overexpressing mice. Administration of the nonspecific GSK-3β inhibitor lithium in animals with active MMP-9 and animals lacking MMP-9 revealed that GSK-3β and MMP-9 act in concert to control dendritic spine morphology. Altogether, our data demonstrate that the dysregulation of GSK-3β activity has dramatic consequences on dendritic spine morphology, implicating MMP-9 as a mediator of GSK-3β-induced synaptic alterations.     

Small,smaller… dendritic spine

Spines are highly motile protrusions emerging from the dendritic shafts of neurons. The dynamics of these post‐synaptic structures are ruled by actin filament turnover. However, our understanding of the mechanisms of actin polymerization in dendritic spines is quite ambiguous. A recent study by the Giannone laboratory (Chazeau et al, 2014 ) is now shedding some light on the peculiar features of actin polymerization in dendritic spines, which are distinct from the known canonical mechanisms.     

Microtubule-associated protein 1B (MAP1B) is required for dendritic spine development and synaptic maturation

Microtubule-associated protein 1B (MAP1B) is prominently expressed during early stages of neuronal development, and it has been implicated in axonal growth and guidance. MAP1B expression is also found in the adult brain in areas of significant synaptic plasticity. Here, we demonstrate that MAP1B is present in dendritic spines, and we describe a decrease in the density of mature dendritic spines in neurons of MAP1B-deficient mice that was accompanied by an increase in the number of immature filopodia-like protrusions. Although these neurons exhibited normal passive membrane properties and action potential firing, AMPA receptor-mediated synaptic currents were significantly diminished. Moreover, we observed a significant decrease in Rac1 activity and an increase in RhoA activity in the post-synaptic densities of adult MAP1B(+/-) mice when compared with wild type controls. MAP1B(+/-) fractions also exhibited a decrease in phosphorylated cofilin. Taken together, these results indicate a new and important role for MAP1B in the formation and maturation of dendritic spines, possibly through the regulation of the actin cytoskeleton. This activity of MAP1B could contribute to the regulation of synaptic activity and plasticity in the adult brain.     

ROCK, PAK, and Toll of synapses in Alzheimer's disease

Alzheimer’s disease is a neurodegenerative disorder where the cognitive deficit is the hallmark symptom reflecting the progression of the disease. Synaptic dysfunction is a sensitive parameter of the AD pathology. Rho GTPases and the Rho kinases, ROCK1/2, and PAK1-3, are important regulators of synaptic plasticity, especially in maintaining the actin cytoskeleton of dendritic spines. Recent studies have revealed that β-amyloid oligomers can inhibit PAK and stimulate ROCK-mediated signaling. Both of these effects enhance the disassembly of synaptic actin filaments and ultimately evoke synaptic loss. Brain tissue in AD recognizes the β-amyloid peptide oligomers as foreign protein particles and mounts an inflammatory defense via Toll-like receptor (TLR) signaling which causes synaptic impairment. We will review here the dysfunction of ROCK, PAK, and Toll signaling associated with AD pathology. The protection of synapses in AD may provide new therapeutic approaches to combatting the cognitive impairment in AD.     

Extracellular signal-regulated kinase phosphorylates tumor necrosis factor alpha-converting enzyme at threonine 735: a potential role in regulated shedding

The ectodomain of certain transmembrane proteins can be released by the action of cell surface proteases, termed secretases. Here we have investigated how mitogen-activated protein kinases (MAPKs) control the shedding of membrane proteins. We show that extracellular signal-regulated kinase (Erk) acts as an intermediate in protein kinase C-regulated TrkA cleavage. We report that the cytosolic tail of the tumor necrosis factor alpha-converting enzyme (TACE) is phosphorylated by Erk at threonine 735. In addition, we show that Erk and TACE associate. This association is favored by Erk activation and by the presence of threonine 735. In contrast to the Erk route, the p38 MAPK was able to stimulate TrkA cleavage in cells devoid of TACE activity, indicating that other proteases are also involved in TrkA shedding. These results demonstrate that secretases are able to discriminate between the different stimuli that trigger membrane protein ectodomain cleavage and indicate that phosphorylation by MAPKs may regulate the proteolytic function of membrane secretases.     

Actin in dendritic spines: connecting dynamics to function

Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses and are major sites of information processing and storage in the brain. Changes in the shape and size of dendritic spines are correlated with the strength of excitatory synaptic connections and heavily depend on remodeling of its underlying actin cytoskeleton. Emerging evidence suggests that most signaling pathways linking synaptic activity to spine morphology influence local actin dynamics. Therefore, specific mechanisms of actin regulation are integral to the formation, maturation, and plasticity of dendritic spines and to learning and memory.     

Neurabin/protein phosphatase-1 complex regulates dendritic spine morphogenesis and maturation

The majority of excitatory synapses in the mammalian brain form on filopodia and spines, actin-rich membrane protrusions present on neuronal dendrites. The biochemical events that induce filopodia and remodel these structures into dendritic spines remain poorly understood. Here, we show that the neuronal actin- and protein phosphatase-1-binding protein, neurabin-I, promotes filopodia in neurons and nonneuronal cells. Neurabin-I actin-binding domain bundled F-actin, promoted filopodia, and delayed the maturation of dendritic spines in cultured hippocampal neurons. In contrast, dimerization of neurabin-I via C-terminal coiled-coil domains and association of protein phosphatase-1 (PP1) with neurabin-I through a canonical KIXF motif inhibited filopodia. Furthermore, the expression of a neurabin-I polypeptide unable to bind PP1 delayed the maturation of neuronal filopodia into spines, reduced the synaptic targeting of AMPA-type glutamate (GluR1) receptors, and decreased AMPA receptor-mediated synaptic transmission. Reduction of endogenous neurabin levels by interference RNA (RNAi)-mediated knockdown also inhibited the surface expression of GluR1 receptors. Together, our studies suggested that disrupting the functions of a cytoskeletal neurabin/PP1 complex enhanced filopodia and impaired surface GluR1 expression in hippocampal neurons, thereby hindering the morphological and functional maturation of dendritic spines.     

Mitochondria-targeted antioxidant SkQR1 reduces TNF-induced endothelial permeability <Emphasis Type="Italic">in vitro</Emphasis>

Prolonged or excessive increase in the circulatory level of proinflammatory tumor necrosis factor (TNF) leads to abnormal activation and subsequent damage to endothelium. TNF at high concentrations causes apoptosis of endothelial cells. Previously, using mitochondria-targeted antioxidants of SkQ family, we have shown that apoptosis of endothelial cells is dependent on the production of reactive oxygen species (ROS) in mitochondria (mito-ROS). Now we have found that TNF at low concentrations does not cause cell death but activates caspase-3 and caspase-dependent increase in endothelial permeability in vitro. This effect is probably due to the cleavage of β-catenin–an adherent junction protein localized in the cytoplasm. We have also shown that extracellular matrix metalloprotease 9 (MMP9) VE-cadherin shedding plays a major role in the TNF-induced endothelial permeability. The mechanisms of the caspase-3 and MMP9 activation are probably not related to each other since caspase inhibition did not affect VE-cadherin cleavage and MMP9 inhibition had no effect on the caspase-3 activation. Mitochondria-targeted antioxidant SkQR1 inhibited TNF-induced increase in endothelial permeability. SkQR1 also inhibited caspase-3 activation, β-catenin cleavage, and MMP9-dependent VE-cadherin shedding. The data suggest that mito-ROS are involved in the increase in endothelial permeability due to the activation of both caspase-dependent cleavage of intracellular proteins and of MMP9-dependent cleavage of the transmembrane cell-to-cell contact proteins.     

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware,concepts, and recent developments

Proteolytic removal of membrane protein ectodomains (ectodomain shedding) is a post‐translational modification that controls levels and function of hundreds of membrane proteins. The contributing proteases, referred to as sheddases, act as important molecular switches in processes ranging from signaling to cell adhesion. When deregulated, ectodomain shedding is linked to pathologies such as inflammation and Alzheimer's disease. While proteases of the “a disintegrin and metalloprotease” (ADAM) and “beta‐site APP cleaving enzyme” (BACE) families are widely considered as sheddases, in recent years a much broader range of proteases, including intramembrane and soluble proteases, were shown to catalyze similar cleavage reactions. This review demonstrates that shedding is a fundamental process in cell biology and discusses the current understanding of sheddases and their substrates, molecular mechanisms and cellular localizations, as well as physiological functions of protein ectodomain shedding. Moreover, we provide an operational definition of shedding and highlight recent conceptual advances in the field. While new developments in proteomics facilitate substrate discovery, we expect that shedding is not a rare exception, but rather the rule for many membrane proteins, and that many more interesting shedding functions await discovery.     

Coiled Coils Ensure the Physiological Ectodomain Shedding of Collagen XVII

α-Helical coiled coils, frequent protein oligomerization motifs, are commonly observed in vital proteins. Here, using collagen XVII as an example, we provide evidence for a novel function of coiled coils in the regulation of ectodomain shedding. Transmembrane collagen XVII, an epithelial cell surface receptor, mediates dermal-epidermal adhesion in the skin, and its dysfunction is linked to human skin blistering diseases. The ectodomain of this collagen is constitutively shed from the cell surface by proteinases of a disintegrin and metalloprotease family; however, the mechanisms regulating shedding remain elusive. Here, we used site-specific mutagenesis to target the coiled-coil heptad repeats within the juxtamembranous, extracellular noncollagenous 16th A (NC16A) domain of collagen XVII. This resulted in a substantial increase of ectodomain shedding, which was not mediated by disintegrin and metalloproteases. Instead, conformational changes induced by the mutation(s) unmasked a furin recognition sequence that was used for cleavage. This study shows that apart from their functions in protein oligomerization, coiled coils can also act as regulators of ectodomain shedding depending on the biological context.