Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

The L-selectin glycoprotein receptor mediates the initial steps of leukocyte migration into secondary lymphoid organs and sites of inflammation. Following cell activation through the engagement of G-protein-coupled receptors or immunoreceptors, the extracellular domains of L-selectin are rapidly shed, a process negatively controlled via the binding of the ubiquitous eukaryotic calcium-binding protein calmodulin to the cytoplasmic tail of L-selectin. Here we present the solution structure of calcium-calmodulin bound to a peptide encompassing the cytoplasmic tail and part of the transmembrane domain of L-selectin. The structure and accompanying biophysical study highlight the importance of both calcium and the transmembrane segment of L-selectin in the interaction between these two proteins, suggesting that by binding this region, calmodulin regulates in an "inside-out" fashion the ectodomain shedding of the receptor. Our structure provides the first molecular insight into the emerging new role for calmodulin as a transmembrane signaling partner.     

The transmembrane domain of TACE regulates protein ectodomain shedding

Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.     

Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated.To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.     

Actin polymerization induces shedding of FcgammaRIIIb (CD16) from human neutrophils

FcgammaRIIIb (CD16) is a glycosyl phosphatidylinositol (GPI)-anchored low-affinity IgG receptor, exclusively expressed on human neutrophils. FcgammaRIIIb associates with complement receptor 3 (CR3, Mac-1, CD11b/CD18), which may indirectly link FcgammaRIIIb to the actin cytoskeleton. Upon neutrophil activation, apoptosis, or chemotaxis, FcgammaRIIIb is shed from the cell surface. In all of these events, actin rearrangements play an important role. To establish a role for the actin cytoskeleton in the control of FcgammaRIIIb shedding, we treated human neutrophils with jasplakinolide, an actin-polymerizing peptide. We show that enhanced actin polymerization induces time- and dose-dependent shedding of FcgammaRIIIb. This effect was not restricted to FcgammaRIIIb, because the cell surface expression of CD43, CD44, and L-selectin was also downregulated after induction of actin polymerization. This actin-dependent pathway is staurosporine sensitive but does not appear to involve activation of PKC or CR3. These data show that the actin cytoskeleton can regulate protein ectodomain shedding from human neutrophils.     

Porphyromonas gingivalis lipopolysaccharide induces shedding of syndecan-1 expressed by gingival epithelial cells

Syndecans are constitutively shed from growing epithelial cells as the part of normal cell surface turnover. However, increased serum levels of the soluble syndecan ectodomain have been reported to occur during bacterial infections. The aim of this study was to evaluate the potential of lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis to induce the shedding of syndecan-1 expressed by human gingival epithelial cells. We showed that the syndecan-1 ectodomain is constitutively shed from the cell surface of human gingival epithelial cells. This constitutive shedding corresponding to the basal level of soluble syndecan-1 ectodomain was significantly increased when cells were stimulated with P. gingivalis LPS and reached a level comparable to that caused by phorbol myristic acid (PMA), an activator of protein kinase C (PKC) which is well known as a shedding agonist. The syndecan-1 shedding was paralleled by pro-inflammatory cytokine interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) release. Indeed, secretion of IL-1beta and TNF-alpha increased following stimulation by P. gingivalis LPS and PMA, respectively. When recombinant forms of these proteins were added to the cell culture, they induced a concentration-dependent increase in syndecan-1 ectodomain shedding. A treatment with IL-1beta converting enzyme (ICE) specific inhibitor prevented IL-1beta secretion by epithelial cells stimulated by P. gingivalis LPS and decreased the levels of shed syndecan-1 ectodomain. We also observed that PMA and TNF-alpha stimulated matrix metalloproteinase-9 secretion, whereas IL-1beta and P. gingivalis LPS did not. Our results demonstrated that P. gingivalis LPS stimulated syndecan-1 shedding, a phenomenon that may be mediated in part by IL-1beta, leading to an activation of intracellular signaling pathways different from those involved in PMA stimulation.     

Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.     

A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.     

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.     

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.     

The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading.

Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGF(TM)). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGF(TM) results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGF(TM) in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGF(TM)HA/Myc). HB-EGF(TM)HA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGF(TM)HA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGF(TM) shedding were identified as follows. (i) HB-EGF(TM)HA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGF(TM)HA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGF(TM) cleavage. (ii) Activation of an inducible Raf-1 kinase, DeltaRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGF(TM)HA/Myc within 20-40 min. (iii) Serum induced MAP kinase activation and HB-EGF(TM)HA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGF(TM)HA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading.     

Regulated cell surface pro-EGF ectodomain shedding is a zinc metalloprotease-dependent process

Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.     

Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti- cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L- selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.     

PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways

Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.     

Tumour necrosis factor alpha-converting enzyme mediates ectodomain shedding of Vps10p-domain receptor family members

Several transmembrane molecules are cleaved at juxtamembrane extracellular sites leading to shedding of ectodomains. We analysed shedding of members of the Vps10p-D (Vps10p domain; where Vps is vacuolar protein sorting) family of neuronal type-I receptors with partially overlapping functions, and additional proteolytic events initiated by the shedding. When transfected into CHO (Chinese-hamster ovary) cells (CHO-K1), sorCS1a-sorCS1c isoforms were shed at high rates (approximately 0.61% x min(-1)) that were increased approx. 3-fold upon stimulation with phorbol ester. sorCS1c identified in the cultured neuroblastoma cell line SH-SY5Y was shed similarly. In CHO-K1 transfectants, constitutive and stimulated shedding of sorCS3 also occurred at high rates (0.29% and 1.03% x min(-1)). By comparison, constitutive and stimulated shedding of sorLA occurred at somewhat lower rates (0.07% and 0.48% x min(-1)), whereas sorCS2 and sortilin were shed at very low rates even when stimulated (approximately 0.01% x min(-1)). Except for sorCS2, shedding of the receptors was dramatically reduced in mutant CHO cells (CHO-M2) devoid of active TACE (tumour necrosis factor alpha-converting enzyme), demonstrating that this enzyme accounts for most sheddase activity. The release of sorCS1 and sorLA ectodomains initiated rapid cleavage of the membrane-tethered C-terminal stubs that accumulated only in the presence of gamma-secretase inhibitors. Purified shed sorLA bound several ligands similarly to the entire luminal domain of the receptor, including PDGF-BB (platelet-derived growth factor-BB) and amyloid-beta precursor protein. In addition, PDGF-BB also bound to the luminal domains of sorCS1 and sorCS3. The results suggest that ectodomains shed from a subset of Vps10p-D receptors can function as carrier proteins.     

Studies with protein kinase C inhibitors presently available cannot elucidate the role of protein kinase C in the activation of NADPH oxidase

The effects of various protein kinase C (PKC) inhibitors on NADPH oxidase (NO) activation by the phorbol ester PMA and by the chemotactic peptide FMLP were studied. H-7 reduced the effects of both stimuli in human neutrophils (HN) and HL-60 cells by 13-63%. Polymyxin B did not inhibit NO activation by PMA and FMLP in HN and reduced the effects of both stimuli in HL-60 cells by 27-55%. Retinal and retinoic acid enhanced the effects of PMA and FMLP in HL-60 cells and of FMLP in HN up to 4.5-fold. In contrast, retinoic acid inhibited the effect of PMA in HN. In the presence of cytochalasin B, retinal inhibited the effect of FMLP in HN, whereas retinoic acid inhibited NO activation by FMLP in both cell types. The dual PKC/calmodulin inhibitors trifluoperazine and W-7 abolished NO activation by PMA and FMLP in HN and HL-60 cells. Thus, the effects of PKC inhibitors on NO activation exhibit (1) cell type specificity, (2) stimulus dependency and (3) no correlation with in vitro inhibition of PKC. Our results suggest that studies with PKC inhibitors presently available cannot clarify the role of PKC in NO activation.     

Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase

Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner. Insulin responsive shedding is not seen in 3T3-L1 fibroblasts. This shedding involves both Ras-MAP kinase and phosphatidylinositol 3-kinase pathways. In response to insulin, adipocytes are known to secrete active lipoprotein lipase, an enzyme that binds to heparan sulfate on the luminal surface of capillary endothelia. Lipoprotein lipase is transported as a stable enzyme from its site of synthesis to its site of action, but the transport mechanism is unknown. Our studies indicate that shed adipocyte syndecans associate with lipoprotein lipase. The shed syndecan ectodomain can stabilize active lipoprotein lipase. These data suggest that syndecan ectodomains, shed by adipocytes in response to insulin, are physiological extracellular chaperones for lipoprotein lipase as it translocates from its site of synthesis to its site of action.     

Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus alpha-toxin and beta-toxin

Exploitation of host components by microbes to promote their survival in the hostile host environment has been a recurring theme in recent years. Available data indicate that bacterial pathogens activate ectodomain shedding of host cell surface molecules to enhance their virulence. We reported previously that several major bacterial pathogens activate ectodomain shedding of syndecan-1, the major heparan sulfate proteoglycan of epithelial cells. Here we define the molecular basis of how Staphylococcus aureus activates syndecan-1 shedding. We screened mutant S. aureus strains devoid of various toxin and protease genes and found that only strains lacking both alpha-toxin and beta-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of alpha- and beta-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus alpha- and beta-toxins, but not enterotoxin A and toxic shock syndrome toxin-1, rapidly potentiated shedding in a concentration-dependent manner. These results establish that S. aureus activates syndecan-1 ectodomain shedding via its two virulence factors, alpha- and beta-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that alpha- and beta-toxins shed syndecan-1 ectodomains through stimulation of the host cell's shedding machinery. Interestingly, beta-toxin, but not alpha-toxin, also enhanced ectodomain shedding of syndecan-4 and heparin-binding epidermal growth factor. Because shedding of these ectodomains has been implicated in promoting bacterial pathogenesis, activation of ectodomain shedding by alpha-toxin and beta-toxin may be a previously unknown virulence mechanism of S. aureus.     

Mutagenesis of the ezrin-radixin-moesin binding domain of L-selectin tail affects shedding, microvillar positioning, and leukocyte tethering

L-selectin is a cell adhesion molecule that mediates the initial capture (tethering) and subsequent rolling of leukocytes along ligands expressed on endothelial cells. We have previously identified ezrin and moesin as novel binding partners of the 17-amino acid L-selectin tail, but the biological role of this interaction is not known. Here we identify two basic amino acid residues within the L-selectin tail that are required for binding to ezrin-radixinmoesin (ERM) proteins: arginine 357 and lysine 362. L-selectin mutants defective for ERM binding show reduced localization to microvilli and decreased phorbol 12-myristate 13-acetate-induced shedding of the L-selectin ectodomain. Cells expressing these L-selectin mutants have reduced tethering to the L-selectin ligand P-selectin glycoprotein ligand-1, but rolling velocity on P-selectin glycoprotein ligand-1 is not affected. These results suggest that ERM proteins are required for microvillar positioning of L-selectin and that this is important both for leukocyte tethering and L-selectin shedding.     

Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins

Translocation of conventional protein kinases C (PKCs) to the plasma membrane leads to their specific association with transmembrane-4 superfamily (TM4SF; tetraspanin) proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by reciprocal co-immunoprecipitation and covalent cross-linking experiments. Although formation and maintenance of TM4SF-PKC complexes are not dependent on integrins, TM4SF proteins can act as linker molecules, recruiting PKC into proximity with specific integrins. Previous studies showed that the extracellular large loop of TM4SF proteins determines integrin associations. In contrast, specificity for PKC association probably resides within cytoplasmic tails or the first two transmembrane domains of TM4SF proteins, as seen from studies with chimeric CD9 molecules. Consistent with a TM4SF linker function, only those integrins (alpha(3)beta(1), alpha(6)beta(1), and a chimeric "X3TC5" alpha(3) mutant) that associated strongly with tetraspanins were found in association with PKC. We propose that PKC-TM4SF-integrin structures represent a novel type of signaling complex. The simultaneous binding of TM4SF proteins to the extracellular domains of the integrin alpha(3) subunit and to intracellular PKC helps to explain why the integrin alpha3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the alpha(3) integrin cytoplasmic tail.     

The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates’ ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.