Down-regulation of alphav/beta3 integrin via misrouting to lysosomes by overexpression of a beta3Lamp1 fusion protein

Conesa, Magali; Prat, Annik; Mort, John S.; Marvaldi, Jacques; Lissitzky, Jean‐Claude; Seidah, Nabil G.

doi:10.1042/bj20021301

Cited by 10 publications

(8 citation statements)

References 34 publications

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“…Instead, the major factor affecting what a-b combinations are present on the cell surface seems to be the cell's integrin subunit expression profile, the types, and relative abundance. For example, manipulating the amount of an a subunit (e.g., by overexpression) affects the a-b combinations expressed by a cell [57][58][59][60][61][62]. In many cell types (including eggs [17,19]), ITGB1 is a highly abundant b subunit, favoring ITGB1 dimerization with the available a subunits.…”

Section: Discussionmentioning

confidence: 99%

Reduction of Mouse Egg Surface Integrin Alpha9 Subunit (ITGA9) Reduces the Egg's Ability to Support Sperm-Egg Binding and Fusion1

Vjugina

Zhu

et al. 2009

Biology of Reproduction

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The involvement of egg integrins in mammalian sperm-egg interactions has been controversial, with data from integrin inhibitor studies contrasting with evidence from knockouts showing that specific integrin subunits are not essential for fertility. An alpha(4)/alpha(9) (ITGA4/ITGA9) integrin subfamily member has been implicated in fertilization but not extensively examined, so we tested the following three hypotheses: 1) an ITGA4/ITGA9 integrin participates in sperm-egg interactions, 2) short-term acute knockdown by RNA interference of integrin subunits would result in a fertilization phenotype differing from that of chronic depletion via knockout, and 3) detection of a fertilization phenotype is sensitive to in vitro fertilization (IVF) assay conditions. We show that mouse and human eggs express the alpha(9) integrin subunit (ITGA9). RNA interference-mediated knockdown resulted in reduced levels of Itga9 mRNA and surface protein in mouse eggs. RNA interference attempts to knockdown ITGA9's likely beta partner, beta(1) (ITGB1), resulted in reduced Itgb1 mRNA but no reduction in ITGB1 surface protein. Therefore, studies using a function-blocking anti-ITGB1 antibody tested the hypothesis that ITGB1 participates in gamete interactions. Analyses of sperm-egg interactions with Itga9-knockdown eggs and anti-ITGB1 antibody-treated eggs in IVF assays using specific sperm:egg ratios revealed the following: 1) a reduction, but not complete loss, of sperm-egg binding and fusion was observed and 2) the reduction of sperm-egg binding and fusion was not detected in inseminations with high sperm:egg ratios. These data demonstrate that ITGA9 and ITGB1 participate in sperm-egg interactions but clearly are not the only molecules involved. This also shows that careful design of IVF parameters allows detection of deficiencies in gamete interactions.

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Section: Discussionmentioning

confidence: 99%

Reduction of Mouse Egg Surface Integrin Alpha9 Subunit (ITGA9) Reduces the Egg's Ability to Support Sperm-Egg Binding and Fusion1

Vjugina

Zhu

et al. 2009

Biology of Reproduction

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“…The next question was whether TIMP-2 could recruit PC5A to the cell surface, possibly in proximity to a protein that binds the complex TIMP-2/PC5A. Accordingly, we used a cell-based missorting approach (Conesa et al, 2003) as well as coimmunoprecipitation and immunocytochemical colocalization techniques. The data demonstrated that the C-terminal segment of TIMP-2 binds the CRD of PC5A (Figure 8) and that cell surface immobilized TIMP-2 is required for the plasma membrane colocalization of PC5A with TIMP-2 (Figure 7).…”

Section: Discussionmentioning

confidence: 99%

“…To test this hypothesis, we used a cellular missorting technique previously applied to ␤3-integrin (Conesa et al, 2003), whereby the C termini of soluble TIMP-2 or the ectodomain of MT1-MMP were fused to a lysosomal/endosomal targeting sequence consisting of the Lamp1 transmembrane-cytosolic tail, herein named TIMP-2-LP ( Figure 3A) and MT1-MMP-LP. According to this dominant negative technique, partners of TIMP-2 or MT1-MMP would be expected to cycle to the cell surface and be dragged to the endosomal/lysosomal compartments for degradation (Conesa et al, 2003). The data revealed that both MT1-MMP and MT1-MMP-LP do not significantly affect the level of C-terminal cleavage of PC5A or the secretion of its CRD (our unpublished data), suggesting that PC5 may not bind robustly MT1-MMP.…”

Section: Timp-2 Interacts With Pc5a Via the Crdmentioning

confidence: 99%

“…Human TIMP-1-4 were cloned into the digested Ecor1/Sma1 phCMV3 vector. The TIMP-[1-4]-Lamp1 proteins (TIMP[1-4]-LP) were obtained by fusion of C terminus of each human TIMP to the transmembrane-cytosolic tail of human Lamp1 (TM-CT-Lamp1; Figure 3A, 9), as described previously (Conesa et al, 2003). The myc-tagged cDNA (pcDNA3.1-Myc-His vector; Invitrogen, Carlsbad, CA) of human endothelial lipase-myc was a generous gift of Drs.…”

Section: Vectors Constructsmentioning

confidence: 99%

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The Cysteine-rich Domain of the Secreted Proprotein Convertases PC5A and PACE4 Functions as a Cell Surface Anchor and Interacts with Tissue Inhibitors of Metalloproteinases

Nour

Mayer

Mort

et al. 2005

MBoC

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The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.

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“…Antibody to LRP1 (C-terminal Ab377) was a generous gift of J. Herz (University of Texas, Dallas). Stable transfectants of empty bicistronic pIRES2-EGFP vector, V5-tagged wild type NARC-1 and its mutants H226A, S127R, and D374Y were obtained in the human hepatocyte-derived cell line HepG2 following G418 selection and FACS purification of fluorescent cells (25).…”

Section: Methodsmentioning

confidence: 99%

NARC-1/PCSK9 and Its Natural Mutants

Benjannet

Rhainds

Essalmani

et al. 2004

Journal of Biological Chemistry

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230

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The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca 2؉ -independent and that within the zymogen autocatalytic processing site SSVFAQ2SIP Val at P4 and Pro at P3 are critical. The S127R and D374Y mutations result in ϳ50 -60% and >98% decrease in zymogen processing, respectively. In contrast, the double [D374Y ؉ N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mM ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal ϳ9-fold increase in circulating LDL cholesterol, while in LDLR(؊/؊) mice a delayed ϳ2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.The mammalian proprotein convertases constitute a family of 9 serine proteinases related to bacterial subtilisin. These include the 7 basic amino acid-specific convertases known as PC1/PC3, PC2, furin, PC4, PACE4, PC5/PC6, PC7/LPS (1, 2) and the two enzymes cleaving at nonbasic residues SKI-1/S1P (3, 4) and NARC-1/PCSK9 (5). These proteases are implicated in the limited proteolysis of precursors of secretory proteins that regulate a variety of cellular functions, including cellular growth, adhesion, differentiation, cell to cell communications, and endocrine/paracrine functions (6, 7). Published gene knockout analyses (reviewed in Ref. 8) revealed that only furin (9) and SKI-1/S1P (10) are embryonic lethal. So far, nothing is known about the phenotype consequences of NARC-1 1 knockout in mice. The cDNA of the enzyme NARC-1 was cloned during pharmaceutical screening of mRNAs up-regulated following induction of neural apoptosis by serum withdrawal, and the encoded protein was called "neural apoptosis regulated convertase 1" (NARC-1) (11). We characterized this enzyme, and we showed that it is highly expressed in liver and small intestine and that specific mutations in the prosegment of NARC-1 completely abrogated its autocatalytic processing (5). We further showed that overexpression of NARC-1 enhances neurogenesis of progenitor brain telencephalic cells. The sustained expression of NARC-1 in liver and small intestine and its transient expression in telencephalon, kidney, and cerebellum beg for the identification of its physiological substrates, which are still unknown.Human genetic point mutations resulting in pathology have been rep...

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Down-regulation of alphav/beta3 integrin via misrouting to lysosomes by overexpression of a beta3Lamp1 fusion protein

Cited by 10 publications

References 34 publications

Reduction of Mouse Egg Surface Integrin Alpha9 Subunit (ITGA9) Reduces the Egg's Ability to Support Sperm-Egg Binding and Fusion1

Reduction of Mouse Egg Surface Integrin Alpha9 Subunit (ITGA9) Reduces the Egg's Ability to Support Sperm-Egg Binding and Fusion1

The Cysteine-rich Domain of the Secreted Proprotein Convertases PC5A and PACE4 Functions as a Cell Surface Anchor and Interacts with Tissue Inhibitors of Metalloproteinases

NARC-1/PCSK9 and Its Natural Mutants

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