Monocyte Chemoattractant Activity of Ser195 → Ala Active Site Mutant Recombinant α-Thrombin

Crago, Aimeeé M.; Wu, Hai Feng; Hoffman, Maureane; Church, Frank C.

doi:10.1006/excr.1995.1275

Cited by 20 publications

(11 citation statements)

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“…In contrast, We were surprised that the ␣-thrombin receptor, PAR-1, failed to support chemotaxis in 300-19 cells. Thrombin mediates the chemotaxis of monocytes͞macrophages (34,35) as well as fibroblasts (36), but the studies that demonstrate this have been difficult to interpret mechanistically because catalytically inactive forms of thrombin, which fail to activate PAR-1, are also chemotactic (34,35). Although PAR-1 couples to both G ␣i and G ␣q in fibroblasts (37), we found that in 300-19 cells, it signaled almost exclusively through G ␣q .…”

Section: Discussionmentioning

confidence: 84%

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of G_αi-coupled receptors

Arai

Tsou

Charo

1997

Proc. Natl. Acad. Sci. U.S.A.

150

114

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Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to G ␣q (the m3 muscarinic receptor), G ␣i (the -opioid receptor), and G ␣s (the ␤-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to G ␣i , but not to G ␣q or G ␣s , migrated in response to a concentration gradient of the appropriate agonist. Overexpression of G ␣ transducin, which binds to and inactivates free G ␤␥ dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of G ␤␥ dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of G ␣i and the release of G ␤␥ dimers, and that G ␣i -coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.Chemotaxis is crucial for leukocytes to migrate to sites of inflammation and infection, and a large number of chemotactic peptides (1, 2) and lipids (3) have been demonstrated to be chemoattractants. The most recent addition to the list of leukocyte chemoattractants is the rapidly growing family of proteins known as the chemokines (chemotactic cytokines) (for reviews, see refs. 4-8). The chemokines can be subdivided into two major families, based on the positions of the first two of four conserved cysteines and on the quaternary structure of chemokine homodimers (9, 10). Two chemokines have also recently been described: lymphotactin (11), which lacks the first and third cysteines, and fractalkine (12), which is displayed at the tip of a mucin stalk on the cell surface. Monocyte chemoattractant protein 1 (MCP-1) is a member of the C-C or ␤-chemokine family, in which the first two cysteines are adjacent, and is a potent chemoattractant for monocytes, basophils, and memory T cells. The receptors for all known leukocyte chemoattractants, including the chemokines, are members of the seven-transmembrane domain superfamily and couple to a variety of G protein ␣ subunits, including G ␣i , G ␣q , and G ␣16 (13,14). The signal transduction pathways that lead to chemotaxis, however, have not yet been identified.Unlike freshly isolated leukocytes, immortalized cell lines do not migrate well in chemotaxis assays, even when transfected with receptors for potent chemoattractants (15), and this has hampered efforts to elucidate the molecular mechanisms of chemotaxis. To investigate this question, we used a lymphocyte cell line (300-19) and a transwell assay...

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Section: Discussionmentioning

confidence: 84%

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of G_αi-coupled receptors

Arai

Tsou

Charo

1997

Proc. Natl. Acad. Sci. U.S.A.

150

114

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“…To determine the concentration dependence of the thrombin effect, primary rat muscle cultures were exposed to various doses of thrombin for 24 h. The maximal efficacy was achieved at a concentration of 100 nM (Fig. 2B), which is the concentration required for optimal receptormediated responses to thrombin in a number of cell systems (9,(23)(24)(25).…”

Section: Resultsmentioning

confidence: 99%

Thrombin, a Survival Factor for Cultured Myoblasts

Chinni¹,

Niese²,

Tew³

et al. 1999

Journal of Biological Chemistry

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Three members of the family of protease-activated receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin receptors. PAR-1 is expressed by primary myoblast cultures, and expression is repressed once myoblasts fuse to form myotubes. The current study was undertaken to investigate the hypothesis that thrombin inhibits myoblast fusion. Primary rodent myoblast cultures were deprived of serum to promote myoblast fusion and then cultured in the presence or absence of thrombin. Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells were apoptotic within 24 h of serum deprivation, whereas less than 15% of thrombin-treated cells showed signs of apoptosis. Proteolysis was required for the effect of thrombin, but no other serine protease tested mimicked the action of thrombin. Neither a PAR-1-nor a PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast cultures were negative for PAR-3 expression. Myoblasts exposed to thrombin for 1 h and then changed to medium without thrombin accumulated apoptosis inhibitory activity in their medium over the subsequent 20 h. Thus the protective action of thrombin appears to be effected through cleavage of an unidentified thrombin receptor, leading to secretion of a downstream apoptosis inhibitory factor. These results demonstrate that thrombin functions as a survival factor for myoblasts and is likely to play an important role in muscle development and repair.

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“…38,40 Human ␣-thrombin is a chemoattractant for monocytes. 41 Fibrin-derived polypeptides are chemoattractants for neutrophils. 42 It would thus appear that many chemotactic factors may be present in tumors; the particular mix of factors present in any individual tumor may dictate the type of leukocytes that infiltrate.…”

Section: (Am J Pathol 2000 156:509 -518)mentioning

confidence: 99%

Neutrophils, Nitric Oxide Synthase, and Mutations in the Mutatect Murine Tumor Model

Sandhu

Privora

Wenckebach

et al. 2000

The American Journal of Pathology

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Mutatect MN-11 is a tumor line that can be grown subcutaneously in syngeneic C57BL/6 mice. The frequency of spontaneously arising mutants at the hypoxanthine phosphoribosyltransferase (Hprt) locus was observed to be elevated as a result of in vivo growth. The objective of the present study was to identify factors in the tumor microenvironment that might explain this increase in mutant frequency (MF). When tumors were examined histologically, neutrophils were found to be the predominant infiltrating cell type. Quantitative estimates of the number of neutrophils and MF of tumors in different animals revealed a statistically significant correlation (r ‫؍‬ 0.63, P < 0.0001). Immunohistochemical analysis for inducible nitric oxide synthase (iNOS) demonstrated its presence, mainly in neutrophils. Biochemical analysis of tumor homogenates for nitric oxide synthase (NOS) activity indicated a statistically significant correlation with MF (r ‫؍‬ 0.77, P < 0.0001). Nitrotyrosine was detected throughout the tumor immunohistochemically; both cytoplasmic and nuclear staining was seen. To increase the number of infiltrating neutrophils, tumors were injected with chemoattractant interleukin-8 and prostaglandin E2. This produced a statistically significant increase in neutrophil content (P ‫؍‬ 0.005) and MF (P ‫؍‬ 0.0002). As in control MN-11 tumors, neutrophil content and MF were strongly correlated (r ‫؍‬ 0.63, P ‫؍‬ 0.003). Because neutrophils are a potential source of genotoxic reactive oxygen and/or nitrogen species, our results support the notion that these tumor-infiltrating cells may be mutagenic and contribute to the burden of genetic abnormalities associated with tumor progression.

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Monocyte Chemoattractant Activity of Ser195 → Ala Active Site Mutant Recombinant α-Thrombin

Cited by 20 publications

References 0 publications

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of G_αi-coupled receptors

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of G_αi-coupled receptors

Thrombin, a Survival Factor for Cultured Myoblasts

Neutrophils, Nitric Oxide Synthase, and Mutations in the Mutatect Murine Tumor Model

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Monocyte Chemoattractant Activity of Ser195 → Ala Active Site Mutant Recombinant α-Thrombin

Cited by 20 publications

References 0 publications

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of Gαi-coupled receptors

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of Gαi-coupled receptors

Thrombin, a Survival Factor for Cultured Myoblasts

Neutrophils, Nitric Oxide Synthase, and Mutations in the Mutatect Murine Tumor Model

Contact Info

Product

Resources

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Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of G_αi-coupled receptors

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of G_αi-coupled receptors