Proteolytic cleavage of platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31) is regulated by a calmodulin‐binding motif

Wong, Margaret G.; Harbour, Stacey N.; Wee, Janet L.; Lau, Lai Man; Andrews, Robert K.; Jackson, Denise E.

doi:10.1016/j.febslet.2004.04.094

Cited by 31 publications

(32 citation statements)

References 57 publications

(65 reference statements)

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“…The underlying molecular and structural basis remains elusive, but the association of the cytoplasmic domain with intracellular proteins clearly plays a role. CaM is known to be associated with the cytoplasmic domain of a number of shed proteins (11,(13)(14)(15)(16)(17)(18)(19)(20)(21). However, our study showed that the R152E/L153E double mutation in the GPIb␤ cytoplasmic domain disrupts GPIb␤ association with CaM but exerts little effect on GPIb␣ shedding (Fig.…”

Section: Discussioncontrasting

confidence: 48%

“…Induction of ectodomain shedding by membrane-permeable CaM inhibitors is well documented, implicating CaM as a regulatory factor in ectodomain shedding (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). However, if CaM association with the GPIb␤ cytoplasmic domain is not directly involved in regulating shedding, how could the effect of CaM inhibitors on GPIb␣ shedding be explained?…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, point mutations in the L-selectin cytoplasmic domain, such as L320E, disrupt CaM association and significantly increase L-selectin shedding (11,12), providing additional evidence for regulation at the level of substrate. Since the report on L-selectin, treatment of CaM inhibitors, such as trifluoperazine and W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), has been found to induce shedding of a number of membrane receptors (13)(14)(15)(16)(17)(18)(19)(20)(21). These membrane receptors contain a cluster of basic residues in the membraneproximal region of their cytoplasmic domain, many of which have been shown to associate with CaM.…”

mentioning

confidence: 99%

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Transmembrane and Trans-subunit Regulation of Ectodomain Shedding of Platelet Glycoprotein Ibα

Mo¹,

Nguyen²,

et al. 2010

Journal of Biological Chemistry

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Ectodomain shedding of transmembrane proteins may be regulated by their cytoplasmic domains. To date, the effecting cytoplasmic domain and the shed extracellular domain have been in the same polypeptide. In this study, shedding of GPIb␣, the ligand-binding subunit of the platelet GPIb-IX complex and a marker for platelet senescence and storage lesion, was assessed in Chinese hamster ovary cells with/without functional GPIb␣ sheddase ADAM17. Mutagenesis of the GPIb-IX complex, which contains GPIb␣, GPIb␤, and GPIX subunits, revealed that the intracellular membrane-proximal calmodulin-binding region of GPIb␤ is critical for ADAM17-dependent shedding of GPIb␣ induced by the calmodulin inhibitor, W7. Perturbing the interaction between GPIb␣ and GPIb␤ subunits further lessened the restraint of GPIb␤ on GPIb␣ shedding. However, contrary to the widely accepted model of calmodulin regulation of ectodomain shedding, the R152E/L153E mutation in the GPIb␤ cytoplasmic domain disrupted calmodulin binding to GPIb␤ but had little effect on GPIb␣ shedding. Analysis of induction of GPIb␣ shedding by membrane-permeable GPIb␤-derived peptides implicated the association of GPIb␤ with an unidentified intracellular protein in mediating regulation of GPIb␣ shedding. Overall, these results provide evidence for a novel trans-subunit mechanism for regulating ectodomain shedding.A large number of transmembrane proteins expressed on the cell surface undergo ectodomain shedding, a process in which the protein is cleaved at a site close to the outer surface of the cell membrane and subsequently its ectodomain released from the cell (1). Because ectodomain shedding plays a vital role in various cellular processes, including cell growth and proliferation, mammalian development, cell migration, and inflammation, its aberration often leads to disease states including cancer (2-5). Ectodomain shedding is in essence an enzyme-driven proteolytic reaction; therefore, in general there are two ways to regulate it. One way is to modulate the activity of the enzyme that is often referred to as sheddase; the other is to modulate the accessibility of the shedding substrate to the sheddase. Most sheddases belong to the ADAM (a disintegrin and metalloprotease) 2 protease family within the metzincin superfamily (1). Although the structure, function, and regulation of ADAMs have been extensively studied (6 -8), mechanisms modulating the accessibility of shedding substrates have not been well explored.In addition to extracellular signals such as ligand binding to the shedding substrate (9, 10), intracellular signals can also induce shedding of a protein without affecting the catalytic activity of the sheddase. The first reported example of this mechanism is the induction of shedding of L-selectin by calmodulin (CaM) inhibitors (11). CaM co-immunoprecipitates with L-selectin from cell lysates and binds directly to the isolated cytoplasmic domain of L-selectin. Treating L-selectin-expressing cells with trifluoperazine, a CaM inhibitor, causes dissociation of CaM...

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Section: Discussioncontrasting

confidence: 48%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Transmembrane and Trans-subunit Regulation of Ectodomain Shedding of Platelet Glycoprotein Ibα

Mo¹,

Nguyen²,

et al. 2010

Journal of Biological Chemistry

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“…Error bars, SD. Wong et al, 2004); membrane-bound L-selectin (Kahn et al, 1998); growth factor precursors; the receptor tyrosine kinase TrkA; and the ␤-amyloid precursor (Diaz-Rodriguez et al, 2000). If calmodulin binding influenced conformation or stability of the VTC complex in the membrane, it might be possible to distinguish between these different conformations by limited proteolysis.…”

Section: Molecular Biology Of the Cell 168mentioning

confidence: 99%

The Vacuolar Transporter Chaperone (VTC) Complex Is Required for Microautophagy

Uttenweiler

Schwarz

Neumann

et al. 2007

MBoC

106

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Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation.This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes. INTRODUCTIONAutophagy occurs in all eukaryotic cells (Reggiori and Klionsky, 2002). In yeast it has mainly been characterized as an adaptation to nutrient stress such as nitrogen limitation: during autophagy large portions of cytosolic and membraneous material are delivered to the lysosome (in yeast called vacuole) for degradation and recycling (Takeshige et al., 1992). This phenomenon enables cells to survive long periods of starvation. However, in higher eukaryotes autophagy also plays an important role in developmental changes (Levine and Klionsky, 2004), regulation of lifespan (Bergamini et al., 2003;Longo and Finch, 2003;Melendez et al., 2003;Vellai et al., 2003), cancer (Qu et al., 2003;Yue et al., 2003;Gozuacik and Kimchi, 2004), and in neurodegenerative disorders like Huntington's, Parkinson's, or Alzheimer's disease (Yuan et al., 2003). It is also part of the innate immune system assisting in eliminating intracellular pathogens after infection (Gutierrez et al., 2004;Nakagawa et al., 2004;Ogawa et al., 2005).Macroautophagy in yeast is defined as the uptake of cytosolic contents by fusion of double-layered vesicles (autophagosomes) with vacuoles (Baba et al., 1994). Autophagosomes originate from a preautophagosomal structure (PAS) in the vicinity of the vacuole and, during their formation, enwrap portions of cytosol. Fusion of the outer autophagosomal layer with the vacuolar membrane liberates autophagic bodies (single-layered intravacuolar vesicles) into the vacuolar lumen for degradation (Takeshige et al., 1992). Relevant actors of macroautophagy (Atg proteins; Klionsky et al., 2003) have been identified, mainly by genetic screens (Tsukada and Ohsumi, 1993;Thumm et al., 1994;Harding et al., 1995Harding et al., , 1996Titorenko et al., 1995) and have been studied intensively over the last decade.Little is known about microautophagy, a process consisting of a di...

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“…on May 11, 2018. by guest www.bloodjournal.org From Shedding of both proteins involves the association of their intracellular domains with calmodulin, [32][33][34][35] as does the shedding of PECAM-1 (CD31). 36 It is not clear, however, that this is a general mechanism for regulating shedding in platelets.Cleavage not only eliminates proteins from the surface of cells, it can also generate bioactive fragments. Early studies showed that soluble Sema4D is generated by a spontaneous shedding from lymphocytes.…”

mentioning

confidence: 99%

Identification of a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets

Mou

Zeng

Qiang

et al. 2013

Blood

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• This study identifies a calmodulin-binding sequence in Sema4D and shows that calmodulin binds to Sema4D in resting platelets.• Dissociation of the Sema4D:calmodulin complex is sufficient to trigger Sema4D cleavage and shedding of the extracellular domain.Semaphorin 4D (Sema4D) is a transmembrane protein that supports contact-dependent amplification of platelet activation by collagen before being gradually cleaved by the metalloprotease ADAM17, as we have previously shown. Cleavage releases a soluble 120-kDa exodomain fragment for which receptors exist on platelets and endothelial cells. Here we have examined the mechanism that regulates Sema4D exodomain cleavage. The results show that the membrane-proximal cytoplasmic domain of Sema4D contains a binding site for calmodulin within the polybasic region Arg762-Lys779. Coprecipitation studies show that Sema4D and calmodulin are associated in resting platelets, forming a complex that dissociates upon platelet activation by the agonists that trigger Sema4D cleavage. Inhibiting calmodulin with W7 or introducing a membrane-permeable peptide corresponding to the calmodulin-binding site is sufficient to trigger the dissociation of Sema4D from calmodulin and initiate cleavage. Conversely, deletion of the calmodulin-binding site causes constitutive shedding of Sema4D. These results show that (1) Sema4D is a calmodulin-binding protein with a site of interaction in its membrane-proximal cytoplasmic domain, (2) platelet agonists cause dissociation of the calmodulin-Sema4D complex, and (3) dissociation of the complex is sufficient to trigger ADAM17-dependent cleavage of Sema4D, releasing a bioactive fragment. (Blood. 2013;121(20):4221-4230) Introduction Sema4D (CD100), a class IV semaphorin family member, is a 150-kDa type I membrane glycoprotein. The mature protein is a disulfidelinked homodimer that includes an NH 2 -terminal (N-terminal) sema domain and a cytoplasmic domain containing sites for tyrosine and serine and/or threonine phosphorylation.1-5 Sema4D was first described on T cells where it supports B-cell differentiation by binding to the low-affinity receptor CD72. 3,[6][7][8] Subsequent studies have identified roles for Sema4D in axon guidance, 9-12 angiogenesis, [13][14][15][16][17] and tumor progression [18][19][20] and showed that some of these effects are mediated by the high-affinity receptor plexin-B1 20 and the related lower-affinity receptor plexin-B2. 21,22 In previously published studies, we have shown that Sema4D is expressed by platelets and that it participates in the hemostatic response to injury by reinforcing collagen-initiated platelet activation in a contact-dependent manner.23,24 Deletion of Sema4D protects mice against the development of atherosclerosis by attenuating platelet hyperactivity in the setting of hyperlipidemia 25 and reducing intimal neovascularization, 26 which suggests that platelet Sema4D has an impact on the vessel wall as well as on platelets. Our previous studies also showed that the extracellular domain of Sema4D is gradu...

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Proteolytic cleavage of platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31) is regulated by a calmodulin‐binding motif

Cited by 31 publications

References 57 publications

Transmembrane and Trans-subunit Regulation of Ectodomain Shedding of Platelet Glycoprotein Ibα

Transmembrane and Trans-subunit Regulation of Ectodomain Shedding of Platelet Glycoprotein Ibα

The Vacuolar Transporter Chaperone (VTC) Complex Is Required for Microautophagy

Identification of a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets

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