Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-06-09
2001-11-27
Gambel, Phillip (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C530S350000, C536S023100, C536S023500
Reexamination Certificate
active
06322990
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of integrin-mediated signaling, particularly signal transduction mediated by &bgr;3 integrins such as &agr;V&bgr;3 and &agr;IIb&bgr;3. The invention relates specifically to the identification of a novel human gene, tentatively named Bap-1. Bap-1 encodes a protein, Bap-1, that interacts with the cytoplasmic domains of &agr;IIb or &bgr;3 integrins, and Src kinase, and is involved in &bgr;3 integrin-mediated signal transduction.
BACKGROUND OF THE INVENTION
Integrins are a family of &agr;&bgr; heterodimers that mediate adhesion of cells to extracellular matrix proteins and to other cells (Clark et al., Science 268: 233-239, 1995). Integrins also participate in signal transduction, as evidenced by either an alteration in adhesive affinity of cell surface integrins in response to cellular activation (termed inside-out signal transduction) or by affecting intracellular signaling pathways following integrin-mediated adhesion (termed outside-in signal transduction). Many biological responses are dependent at least to some extent upon integrin-mediated adhesion and cell migration, including embryonic development, hemostasis, clot retraction, mitosis, angiogenesis, cell migration, inflammation, immune response, leukocyte homing and activation, phagocytosis, bone resorption, tumor growth and metastasis, atherosclerosis, restenosis, wound healing, viral infectivity, amyloid toxicity, programmed cell death and the response of cells to mechanical stress.
The integrin family consists of 15 related known &agr; subunits (&agr;1, &agr;2, &agr;3, &agr;4, &agr;5, &agr;6, &agr;7, &agr;8, &agr;9, &agr;E, &agr;V, &agr;IIb, &agr;L, &agr;M, and &agr;X) and 8 related known &bgr; subunits (&bgr;1, &bgr;2, &bgr;3, &bgr;4, &bgr;5, &bgr;6, &bgr;7, and &bgr;8). (Luscinskas et al.,
FASEB J.
8:929-938, 1994.) Integrin &agr; and &bgr; subunits are known to exist in a variety of pairings. Integrin ligand specificity is determined by the specific pairing of the &agr; and &bgr; subunits, although some redundancy exists as several of the integrins are known to bind the same ligand. Most integrins containing the &bgr;1, &bgr;2, &bgr;3, &bgr;5, &bgr;6, and &bgr;7 subunits have been found to transduce signals (reviewed by Hynes,
Cell
69:11-25, 1992). Integrins are involved in both “inside-out” and “outside-in” signaling events.
Various pathologies associated with integrin-related defects are known. For example, inherited deficiencies of GP IIb-IIIa (also termed &agr;IIb&bgr;3) content or function have been described (termed Glanzmann's thrombasthenia) and are characterized by platelets that do not bind adhesive proteins and therefore fail to aggregate, resulting in a life-long bleeding diathesis. Inhibitors of the binding of fibrinogen and von Willebrand factor to GP IIb-IIIa have been described and have been found to block platelet aggregation in vitro and to inhibit clinical thrombosis in vivo (The EPIC Investigators,
New England Journal of Med.
330:956-961, 1994, J. E. Tcheng et al.,
Circulation
91:2151-2157, 1995). Also, leukocyte adhesion deficiency (LAD) results from the absence of a &bgr;2 subunit, and is characterized by leukocytes which fail to bind &bgr;2 integrin ligands, resulting in individuals that are susceptible to infections.
The most studied platelet integrin &agr;IIb&bgr;3 (GPIIbIIIa) plays a critical role in homeostasis (platelet aggregation) and also in thrombosis. The &agr;V&bgr;3 plays a critical role in melanoma metastasis and angiogenesis, which is essential for cancer cell growth. The adhesion capacity of &agr;IIb&bgr;3 is known to be stimulated by various agonists such as thrombin, collagen, and ADP. This is termed inside-out signaling. There is accumulating evidence suggesting that integrins, in various cells and tissues including platelets, are also capable of mediating signals from the exterior to the cell interior, and that these signals can trigger cellular processes such as stimulating protein tyrosine phosphorylation, activating Na
+
/H
+
antiporter, assembly of cytoskeletal structures and regulating gene expression that is involved in cell migration and proliferation. However, the mechanisms by which these signals are transmitted remain elusive. It has been hypothesized that the cytoplasmic tails of &agr;IIb&bgr;3 and other integrins may play important roles in adhesion by modulating the ligand-binding function of the extracellular domains through responses to intracellular signals generated by agonists stimulation (inside-out), and by mediating signals triggered by integrin receptor occupancy to intracellular molecules that may play a pivotal role in cellular physiological and pathological functions (outside-in).
A. Inside-Out Signaling
Inside-out signal transduction has been observed for &bgr;1, &bgr;2, and &bgr;3 integrins. (R. O. Hynes,
Cell
69:11-25, 1992; D. R. Phillips, et al.
Cell
65:359-362, 1991, S. S. Smyth et al.,
Blood
81:2827-2843, 1993; M. H. Ginsberg, et al.
Thromb. Haemostasis
70:87-93, 1993, R. L. Juliano and S. Haskill,
J. Cell Biol.
120:577-585, 1993; E. Rouslahti,
J. Clin. Invest.
87:1-5. 1991; Weber et al.,
J. Cell Biol.
134:1063-1073, 1996.)
Perhaps the most widely studied integrin that is involved in inside-out signaling is GP IIb-IIIa, the receptor for four adhesive proteins, fibrinogen, von Willebrand factor, vitronectin and fibronectin that bind to stimulated platelets (D. R. Phillips, et al.,
Blood
71:831-43, 1988). The binding of adhesive proteins to GP IIb-IIIa is required for platelet aggregation and normal hemostasis and is also responsible for occlusive thrombosis in high shear arteries.
GP IIb-IIIa is known to be involved in inside-out signal transduction because GP IIb-IIIa on the surface of unstimulated platelets is capable of recognizing only immobilized fibrinogen. In response to platelet stimulation by agents such as thrombin, collagen and ADP, GP IIb-IIIa becomes a receptor for the four adhesive proteins identified in the previous paragraph, and the binding of fibrinogen and von Willebrand factor causes platelets to aggregate. A monoclonal antibody has been described which detects the activated, receptor competent state of GP IIb-IIIa, suggesting that the conformation of the receptor competent form of GP IIb-IIIa differs from that of GP IIb-IIIa which does not bind soluble fibrinogen or von Willebrand factor (S. J. Shattil, et al.,
J. Biol. Chem.
260:11107-11114, 1985). It has been postulated that inside-out GP IIb-IIIa signal transduction is dependent on cellular proteins that act to repress or stimulate GP IIb-IIIa activation (M. H. Ginsberg, et al.,
Curr. Opin. Cell Biol.
4:766-771, 1992).
&bgr;2 integrins on leukocytes also respond to inside-out signal transduction which accounts, for example, for the increased binding activity of LFA-1 (&agr;L&bgr;
2
) on stimulated lymphocytes and the increased binding activity of MAC-1 (&agr;m&bgr;
2
) on stimulated neutrophils (reviewed by T. Springer, Curr. Biol. 4:506-517, 1994).
B. Outside-In Signaling
Most integrins can be involved in outside-in signal transduction as evidenced by observations showing that binding of adhesive proteins or antibodies to integrins affects the activities of many cells, for example cellular differentiation, various markers of cell activation, gene expression, and cell proliferation (R. O. Hynes,
Cell
69:11-25, 1992). The involvement of GP IIb-IIIa in outside-in signaling is apparent because the binding of unstimulated platelets to immobilized fibrinogen, a process mediated by GP IIb-IIIa, leads to platelet activation and platelet spreading (N. Kieffer and D. R. Phillips,
J. Cell Biol.
113:451-461, 1991, Haimovich et al.,
J. Biol. Chem.
268:15868-15877, 1993).
Outside-in signaling through GP IIb-IIIa also occurs during platelet aggregation. Signaling occurs because fibrinogen or von Willebrand factor bound to the activated form of GP IIb-IIIa on the surface of stimulated platelets, coupled with the formation of platelet-platelet contacts, causes further pla
Li Shengfeng
Phillips David R.
Gambel Phillip
Morgan & Lewis & Bockius, LLP
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