Convergent Fc-gamma receptor signaling in the human neutrophil: Evidence for role of GPI-anchored proteins.
Item
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Title
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Convergent Fc-gamma receptor signaling in the human neutrophil: Evidence for role of GPI-anchored proteins.
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Identifier
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AAI9820520
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identifier
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9820520
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Creator
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Chuang, Frank Y. S.
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Contributor
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Advisers: Massimo Sassaroli | Jay Unkeless | Josef Eisinger
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Date
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1998
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Health Sciences, Immunology | Biophysics, General | Chemistry, Biochemistry
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Abstract
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The recognition of immune complexes and opsonized bacteria by polymorphonuclear leukocytes (PMNs or neutrophils) is mediated by receptors which bind to the constant (Fc) portion of immunoglobulin (IgG). Within the Fc{dollar}\gamma{dollar} receptor family, the Fc{dollar}\gamma{dollar}RIIIB isoform is unique for its attachment to the cell membrane via glycosyl-phosphatidylinositol (GPI). The molecular basis for transmembrane signaling by Fc{dollar}\gamma{dollar}RIIIB (as well as by other GPI-anchored receptors) has been unclear. Spectrofluorimetric data obtained using the calcium indicator Indo-1 demonstrate that direct antibody crosslinking of Fc{dollar}\gamma{dollar}RIIIB on isolated human PMNs triggers a rapid, transient rise in intracellular calcium concentration, comparable to that exhibited by crosslinking the membrane-spanning Fc{dollar}\gamma{dollar}RIIA isoform. Inhibition by wortmannin and by N,N-dimethylsphingosine of signaling through either Fc{dollar}\gamma{dollar}RIIA or Fc{dollar}\gamma{dollar}RIIIB shows similar dose response, suggesting that both receptors converge on a phosphatidylinositol 3-kinase and sphingosine kinase-dependent pathway. Immunofluorescence microscopy and internalization assays demonstrate that separately crosslinked Fc{dollar}\gamma{dollar}RIIIB and Fc{dollar}\gamma{dollar}RIIA show similar patterns of movement on living cells; and furthermore, without direct involvement in crosslinking, Fc{dollar}\gamma{dollar}RIIA co-localizes with aggregated Fc{dollar}\gamma{dollar}RIIIB. Fc{dollar}\gamma{dollar}RIIA is thus implicated as the essential transmembrane element required for signaling by GPI-anchored Fc{dollar}\gamma{dollar}RIIIB.;The physical nature of association between Fc{dollar}\gamma{dollar}RIIA and Fc{dollar}\gamma{dollar}RIIIB remains to be elucidated. The observation that dialkyl-indocarbocyanine (DiI) membrane probes also co-localize with clustered Fc{dollar}\gamma{dollar}RIIIB in vivo, suggests that the physical properties of the lipid bilayer in the region of the GPI-anchored receptor are significantly different from that of bulk membrane, and that other molecules (including Fc{dollar}\gamma{dollar}RIIA) may selectively partition into these regions. A physical model is proposed in which clustered GPI-anchored proteins form a lipid domain comprised of sphingolipids and cholesterol, and organized around hydrophobic interactions. The partitioning and enrichment of Fc{dollar}\gamma{dollar}RIIA within these domains lead to the subsequent initiation of intracellular signaling. This model provides a rationale for the intriguing observation that crosslinked, but immobilized Fc{dollar}\gamma{dollar}RIIIB does not stimulate neutrophil activity.;Other experiments to directly assess the functional dependence of Fc{dollar}\gamma{dollar}RIIIB on Fc{dollar}\gamma{dollar}RIIA, and to examine receptor proximity with nanometer-scale resolution, are also described.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
