DNA molecules encoding a calcium-integrin binding protein

Details DNA molecules encoding a calcium-integrin binding protein DNA molecules encoding a calcium-integrin binding protein

1996-10-02

2001-06-05

Gambel, Phillip (Department: 1644)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S252300, C435S320100, C435S455000, C435S471000, C435S476000, C530S350000, C536S023100

Reexamination Certificate

active

06242587

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel Ca
2+
-binding protein that interacts specifically with the integrin &agr;IIb subunit cytoplasmic domain. This protein is expressed in platelets and interacts with the &agr;IIb subunit of integrin &agr;IIb&bgr;3, to activate the fibrinogen receptor.
BACKGROUND OF THE INVENTION
Integrins are a large family of homologous transmembrane linker proteins, and are the principal receptor proteins on animal cells which bind extracellular matrix proteins such as collagen, fibronectin, and laminin. Integrins consist of two noncovalently associated transmembrane glycoprotein subunits, called &agr; and &bgr;, both of which contribute to the binding of the target extracellular matrix protein. The binding of integrins to their ligands requires the presence of extracellular divalent cations (Ca
2+
or Mg
2+
, depending on the integrin); multiple divalent-cation-binding domains are present in the large extracellular part of the &agr; chain. Integrin receptors undergo reversible activation due to ligand binding or cellular stimulation; activation results in conformational changes in the integrin extracellular domains, reorganization of intracytoplasmic connections, and redistribution of integrins on the cell surface. (See Diamond and Springer,
Curr. Biol
. 4:506 (1994); Li et al.,
J. Cell Biol
. 129:1143 (1995); Yednock et al.,
j. Biol. Chem
. 270:28740 (1995)). About 20 integrin heterodimers, made from 9 types of &bgr; subunits and 14 types of &agr; subunits have been defined.
As noted above, binding of integrins to their ligands depends on extracellular divalent cations. This property can be used to purify integrins, for example, by passing detergent-solubilized plasma membrane proteins over an affinity column containing an extra-cellular matrix protein, and then eluting the bound integrins from the column by washing in a divalent-cation-free solution.
The Molecular Biology of the Cell
, 3d ed., Alberts et al. (eds.), Garland Publishing Inc., New York, 1994, p. 996-1000.
&bgr;3 integrins are found on a variety of cells and are known to bind several matrix proteins, including fibrinogen. Blood platelets contain the &agr;IIb&bgr;3 fibrinogen receptor (also known as GPIIb IIIa). Platelet integrins can be activated by contact with a damaged blood vessel or by any of a number of soluble signaling molecules. Activation alters the conformation of the extracellular domain of &bgr;3 integrins, so that the extracellular domain becomes able to bind fibrinogen with high-affinity, promoting platelet aggregation and blood clot formation. In unstimulated platelets the majority of &agr;IIb&bgr;3 is present in an inactive conformation or low affinity state, and is unable to bind soluble ligands. Platelet agonists through their respective receptors transduce a cascade of intracellular signals ultimately leading to the conversion of &agr;IIb&bgr;3 into an active, high affinity state that is capable of binding soluble ligands.
Shattil et al.
J. Cell Biol
. 131:807 (1995), using the yeast two-hybrid system, recently identified a novel protein termed &bgr;3-endonexin that interacts with the cytoplasmic domain of the &bgr;3 integrin.
U.S. Pat. No. 5,523,209 to Ginsberg and O'Toole (Jun. 4, 1996) describes a method using cell culture assays to screen compounds to identify inhibitors of integrin activation.
SUMMARY OF THE INVENTION
A first aspect of the present invention is an isolated DNA molecule encoding a calcium-integrin binding protein (CIB) that binds to the integrin &agr;IIb cytoplasmic domain, selected from the group consisting of isolated DNA of SEQ ID NO:1, isolated DNA that hybridizes to DNA of SEQ ID NO:1 under stringent conditions and which encodes a calcium-integrin binding protein that binds to the integrin &agr;IIb cytoplasmic domain, and isolated DNA that encodes a calcium-integrin binding protein and which differs from the above DNA due to the degeneracy of the genetic code.
A second aspect of the present invention is a vector containing isolated DNA as described above.
A further aspect of the present invention is a protein encoded by DNA as described above.
A further aspect of the present invention is a protein having the amino acid sequence of SEQ ID NO:2 given herein.
A further aspect of the present invention is an aqueous preparation containing a cell membrane and calcium-integrin binding protein of SEQ ID NO:2, the cell membrane containing a functional integrin with an &agr;IIb integrin subunit.
The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.


REFERENCES:
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patent: 5380646 (1995-01-01), Knight et al.
patent: 5492890 (1996-02-01), Ginsberg et al.
patent: 5523209 (1996-06-01), Ginsberg et al.
patent: 5529902 (1996-06-01), Kottke et al.
1. Naik et al. J Biol Chem. 272:4651-4654 (1997).*
Alberts et al. (Eds)., The Molecular Biology of the Cell, 3d ed., Garland Publishing Inc., New York, 1994 p. 996-1000.
Leung-Hagesteijn et al., “Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin &agr;-subunit-binding protein”,Journal of Cell Science,107, pp. 589-600 (1994).
Crabtree and Clipstone, “Signal Transmission Between The Plasma Membrane And Nucleus Of T Lymphocytes”,Annu. Rev. Biochem.,63: 1045-83 (1994).
Wera and Hemmings, “Serine/threonine protein phosphatases”,Biochem J.,311: 17 (1995), p. 17-29.
Shattil et al., “&bgr;3-Endonexin, A Novel Polypeptide That Interacts Specifically with the Cytoplasmic Tail of the Integrin &bgr;3Subunit”,J. Cell Biol.,131:807 (1995), p. 807-816.
Naik et al., (Abstract 246) “Integrins and Signalling Events in Cell Biology and Disease”, Keystone Symposia, Molecular & Cellular Biology, Keystone, Colorado, (Jan. 1996); p. 54.
Hannigan et al., “Regulation of cell adhesion and anchorage-dependent growth by a new &bgr;1-integrin-linked protein kinase”,Nature,379: 91 (1996).
Barroso et al., “A Novel Ca2+-binding Protein, p22, Is Required for Constitutive Membrane Traffic”,J. Biol. Chem.,271: 17, 10183-10187 (1996).
Barreuther et al., “The Role of Phosphorylation in Modulating &bgr;1Integrin Localization”,Experimental Cell Research,222, pp. 10-15 (1996).
Wei et al., “Regulation of Integrin Function by the Urokinase Receptor”,Science,273, pp. 1551-1555 (1996).

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