Activated α<sub>2</sub>‐Macroglobulin Promotes Mitogenesis in Rat Vascular Smooth Muscle Cells by a Mechanism that is Independent of Growth‐Factor‐Carrier Activity

Webb, Donna J.; Hussaini, Isa M.; Weaver, Alissa M.; Atkins, T. L.; Chu, Charleen T.; Pizzo, Salvatore V.; Owens, Gary K.; Gonias, Steven L.

doi:10.1111/j.1432-1033.1995.714_a.x

Cited by 32 publications

(26 citation statements)

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“…1B). ␣ 2 M* binds to growth factors and cytokines, but RBF and K1370A lack the growth factor binding domain (44). Thus, these studies demonstrate that the observed effects are related directly to ␣ 2 M* cellular binding.…”

Section: Western Blotting Of C-fos C-myc Creb and Nfb Proteins Inmentioning

confidence: 58%

Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin

Misra

Pizzo

2002

Journal of Biological Chemistry

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3 H]thymidine uptake and cell proliferation was completely abolished if activation of cPLA 2 was prevented. The response of macrophages to ␣ 2 M* requires transcription factors nuclear factor B, and cAMP-responsive element-binding protein as well as expression of the proto-oncogenes c-fos and c-myc. These studies indicate that the activation of cPLA 2 plays a crucial role in ␣ 2 M*-induced mitogenesis and cell proliferation.The plasma proteinase inhibitor ␣ 2 -macroglobulin (␣ 2 M*) 1 undergoes a major conformational change when it binds proteinases (1, 2). Each ␣ 2 M subunit also contains an internal thiol ester, which can be directly attacked by small nucleophiles resulting in a similar conformational change (1). In either event, receptor recognition sites are exposed in each of the subunits (1). These receptor-recognized forms of ␣ 2 M, termed ␣ 2 M*, bind to the low density lipoprotein receptorrelated protein (LRP) present on a variety of cells including macrophages (1-3). In 1993, we demonstrated that the binding of ␣ 2 M* to macrophages activated signaling cascades characterized by an inositol 1,4,5-trisphosphate (IP 3 )-dependent increase in [Ca 2ϩ ] i (4). Subsequent studies demonstrated that the binding to macrophages activates a pertussis toxin-insensitive phospholipase C, which hydrolyzes membrane phosphoinositides generating both IP 3 and diacylglycerol (5, 6). These studies also demonstrated that ␣ 2 M*-mediated signal transduction was not blocked by addition of receptor-associated protein (RAP). Because RAP blocks the binding of all known ligands to LRP, this observation suggested the presence of a second ␣ 2 M* receptor on macrophages, which was termed the ␣ 2 M* signaling receptor (␣ 2 MSR) (1, 5, 6). In support of the identification of a distinct ␣ 2 M* receptor were several other observations. Two classes of binding sites were identified on macrophages, one of high affinity and low capacity (K d ϳ 50 pM and ϳ1600 sites/cell) and the other LRP, which demonstrated lower affinity and higher binding capacity (K d ϳ2-5 nM and ϳ70,000 sites/cell) (7-10). Binding of other ligands to LRP, moreover, initiated signaling cascades that activated a pertussis toxin-sensitive G protein and were blocked by addition of RAP (4 -12). More recent observations, however, suggest that ␣ 2 M*-mediated signal transduction requires the presence of LRP on cells (13). Thus, Backsai et al. (13) have shown that ␣ 2 M* binding to LRP on neuronal cells mediates signaling via N-methyl-D-aspartate receptors. These authors suggest that an adapter protein causes LRP to associate with this receptor, allowing ␣ 2 M* to activate signal transduction. Furthermore, Herz and colleagues (14, 15) have identified a large number of adapter proteins that can associate with LRP, and Barnes et al. (16) have demonstrated that Tyr-phosphorylated LRP associates with the adapter protein SHC in SRC-transformed cells.Based on our observations with respect to activation of the p21 ras -dependent MAPK and PI 3-kinase signaling cascades and subseq...

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Section: Western Blotting Of C-fos C-myc Creb and Nfb Proteins Inmentioning

confidence: 58%

Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin

Misra

Pizzo

2002

Journal of Biological Chemistry

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“…Glutathione-Stransferase receptor-associated protein (GST-RAP) was expressed in bacteria and purified as previously described. 28 TaqMan quantitative polymerase chain reaction (qPCR) primers, probes, and reagents were purchased from Applied Biosystems (Foster City, CA). Smart-pool siRNA targeting mouse LRP-1 and nontargeting pooled control (NTC) siRNA were from Dharmacon RNA Technologies (Lafayette, CO).…”

Section: Reagents and Proteinsmentioning

confidence: 99%

Regulation of tumor necrosis factor receptor-1 and the IKK-NF-κB pathway by LDL receptor–related protein explains the antiinflammatory activity of this receptor

Gaultier

Arandjelovic

Niessen

et al. 2008

Blood

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114

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Low-density lipoprotein receptor-related protein (LRP-1) functions in endocytosis and in cell signaling directly (by binding signaling adaptor proteins) or indirectly (by regulating levels of other cell-surface receptors). Because recent studies in rodents suggest that LRP-1 inhibits inflammation, we conducted activity-based protein profiling experiments to discover novel proteases, involved in inflammation, that are regulated by LRP-1. We found that activated complement proteases accumulate at increased levels when LRP-1 is absent. Although LRP-1 functions as an endocytic receptor for IntroductionLow-density lipoprotein (LDL) receptor-related protein (LRP-1) is a member of the LDL receptor gene family, which includes type I transmembrane proteins that function in receptor-mediated endocytosis and cell signaling. 1 LRP-1 was first recognized as a receptor for apolipoprotein E and for the protease inhibitor, ␣ 2 -macroglobulin (␣ 2 M) 2,3 ; however, it is now clear that LRP-1 binds more than 40 different ligands, well beyond the more limited scope of the LDL receptor. 1 LRP-1 ligands include proteases, such as tissuetype plasminogen activator (tPA) and matrix metalloprotease-9, growth factors, extracellular matrix proteins, and foreign toxins. LRP-1 also mediates the endocytosis of plasma membrane proteins, including the urokinase receptor (uPAR), amyloid precursor protein, and tissue factor. 4 By regulating cell-surface uPAR, LRP-1 controls the activity of cell signaling factors downstream of uPAR, including extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and Rac1. 5,6 In mice, systemic LRP-1 gene deletion is embryonic lethal. 7 LRP-1 is synthesized as a 600-kDa single-chain precursor and processed by a furin-like protease into the mature 2-chain form. 8 The 85-kDa ␤-chain includes a small ectodomain, a single transmembrane domain, and the cytoplasmic tail. The 515-kDa ␣-chain is entirely extracellular but coupled to the ␤-chain through strong noncovalent forces. 8 The ␣-chain includes 4 clusters of cysteine-rich complementtype repeats (CRs). 1 The second and fourth clusters of CRs mediate most ligand-binding interactions, with the exception of the protease inhibitor, ␣ 2 M, which binds to the first and second CR clusters. 9 In addition to its role as a regulator of cell signaling downstream of uPAR, LRP-1 directly activates cell signaling upon binding of ligands, including tPA, activated ␣ 2 M, and apolipoprotein E. [10][11][12] The exact mechanism by which ligand binding to LRP-1 initiates cell signaling is unclear; however, the LRP-1 ␤-chain contains 2 NPxY motifs, the more C-terminal of which binds signaling adaptor proteins, such as Shc, c-Jun amino-terminal kinase (JNK)-interacting protein-1/2 (JIP-1/JIP-2), Dab-1, and FE65. [13][14][15] Adaptor protein binding to the LRP-1 ␤-chain is controlled by tyrosine phosphorylation, which occurs in response to ligand binding or the activity of nonreceptor tyrosine kinases. 11,13,16 LRP-1-JIP complex sequesters JNK and thereby prev...

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“…The 18-kDa receptor-binding domain (RBD) of ␣ 2 M was generated by treating ␣ 2 M-MA with papain and purified by chromatography, as previously described (4). Receptor-associated protein was expressed as a GST fusion protein (GST-RAP) in bacteria and purified as previously described (37). GST-RAP binds to LRP-1 and blocks the binding of all other LRP-1 ligands (38).…”

Section: Methodsmentioning

confidence: 99%

Molecular Dissection of the Human α2-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling

Mantuano

Mukandala

et al. 2008

Journal of Biological Chemistry

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␣ 2 -Macroglobulin (␣ 2 M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in ␣ 2 M are responsible for its various functions, we hypothesized that the overall effects of ␣ 2 M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). 2 is a 718-kDa homotetrameric glycoprotein, found in the plasma and extracellular spaces, which was first recognized as a broad spectrum protease inhibitor (1). Reaction with proteases induces a major conformational change in ␣ 2 M so that the protease is physically trapped (2, 3). The same conformational change reveals a cryptic recognition site for low density lipoprotein receptor-related protein (LRP-1) (4, 5). Because of the function of LRP-1 as an endocytic receptor, ␣ 2 M-protease complexes are rapidly cleared from the bloodstream and probably other sites of generation (6).In addition to its role as a protease inhibitor, ␣ 2 M is an important carrier of specific growth factors, including transforming growth factor-␤ (TGF-␤), platelet-derived growth factor-BB (PDGF-BB), nerve growth factor-␤ (NGF-␤), and neurotrophin-4 (7, 8). ␣ 2 M-carrier interactions are principally reversible in nature (7). As a result, ␣ 2 M may inhibit growth factor activity (9, 10) or stabilize the growth factor for potential delivery to cell signaling receptors (11). There also is evidence that ␣ 2 M, which is "activated" by reaction with proteases, initiates cell signaling by binding to LRP-1 (12-15) or other receptors, such as glucose-regulated protein-78 (Grp 78) (16,17). However, in studies with intact ␣ 2 M, the possibility that cell signaling results from growth factors that are carried by ␣ 2 M must be considered (11,18).A structural model of ␣ 2 M has been developed, based on the crystal structure of complement component C3, which is homologous to ␣ 2 M (19). This model describes ␣ 2 M as a modular structure, consisting of multiple independently folded domains. To localize ␣ 2 M domains that are responsible for various activities, our laboratory generated a library of glutathione S-transferase (GST) fusion proteins, containing overlapping segments of the ␣ 2 M subunit (20 -22). Fusion protein-3 (FP3) includes residues 591-774 and contains the sequence in ␣ 2 M responsible for binding growth factors. FP6 contains amino acids 1242-1451 and the LRP-1-binding site, in which a single ␣-helix that includes Lys 1370 and Lys 1374 plays a central role (23,24). Assignment of ␣ 2 M activities to specific fusion proteins, such as FP3 and FP6, has been validated by mutagenesis of full-length recombinant ␣ 2 M (25, 26). Thus, the ␣ 2 M fusion proteins provide an opportunity to assess activities assigne...

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Activated α₂‐Macroglobulin Promotes Mitogenesis in Rat Vascular Smooth Muscle Cells by a Mechanism that is Independent of Growth‐Factor‐Carrier Activity

Cited by 32 publications

References 61 publications

Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin

Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin

Regulation of tumor necrosis factor receptor-1 and the IKK-NF-κB pathway by LDL receptor–related protein explains the antiinflammatory activity of this receptor

Molecular Dissection of the Human α2-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling

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Activated α2‐Macroglobulin Promotes Mitogenesis in Rat Vascular Smooth Muscle Cells by a Mechanism that is Independent of Growth‐Factor‐Carrier Activity

Cited by 32 publications

References 61 publications

Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin

Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin

Regulation of tumor necrosis factor receptor-1 and the IKK-NF-κB pathway by LDL receptor–related protein explains the antiinflammatory activity of this receptor

Molecular Dissection of the Human α2-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling

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Product

Resources

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Activated α₂‐Macroglobulin Promotes Mitogenesis in Rat Vascular Smooth Muscle Cells by a Mechanism that is Independent of Growth‐Factor‐Carrier Activity