Novel human beta-2 integrin alpha subunit

Abstract

Methods to inhibit inflammation and macrophage infiltration following spinal cord injury are disclosed along with methods to modulate TNFα release from cells expressing αd are disclosed.

Description

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 08 / 943,363 filed Oct. 3, 1997, which is pending, which a continuation-in-part of U.S. patent application Ser. No. 08 / 605,672, filed Feb. 22, 1996, which issued as U.S. Pat. No. 5,817,515 on Oct. 6, 1998, which is a continuation-in-part of U.S. application Ser. No. 08 / 362,652, filed Dec. 21, 1994, which issued as U.S. Pat. No. 5,766,850 on Jun. 16, 1998, which is a continuation-in-part of U.S. application Ser. No. 08 / 286,889, filed Aug. 5, 1994, which issued as U.S. Pat. No. 5,470,953 on Nov. 28, 1995, which in turn is a continuation-in-part of U.S. application Ser. No. 08 / 173,497, filed Dec. 23, 1993, which issued as U.S. Pat. No. 5,437,958 on Aug. 1, 1995.BACKGROUND OF THE INVENTION [0002] The integrins are a class of membrane-associated molecules which actively participate in cellular adhesion. Integrins are transmembrane heterodimers comprising an α subunit in noncovalent association with a β su...

AI Technical Summary

Benefits of technology

[0017] Assays to identify αd binding molecules are also provided, including in vitro assays such as immobilized ligand binding assays, solution binding assays, and scintillation proximity assays, as well as cell based assays such as di-hybrid screening assays, split hybrid screening assays, and the like. Cell based assays provide for a phenotypic change in a host cell as a result of specific binding interaction or disruption of a specific binding interaction, thereby permitting indirect quantitation or measurement of some specific binding interaction.

[0022] In a preferred embodiment utilizing the split hybrid assay, the invention provides a method to identify an inhibitor of binding between an αd protein or fragment thereof and an αd binding protein or binding fragment thereof comprising the steps of: (a) transforming or transfecting a host cell with a first DNA expression construct comprising a first selectable marker gene encoding a first selectable marker protein and a repressor gene encoding a repressor protein, said repressor gene under transcriptional control of a promoter; (b) transforming or transfecting said host cell with a second DNA expression construct comprising a second selectable marker gene encoding a second selectable marker protein and a third selectable marker gene encoding a third selectable marker protein, said third selectable marker gene under transcriptional control of an operator, said operator specifically acted upon by said repressor protein such that interaction of said repressor protein with said operator decreases expression of said third selectable marker protein; (c) transforming or transfecting said host cell with a third DNA expression construct comprising a fourth selectable marker gene, encoding a fourth selectable marker protein and an αd fusion protein gene encoding an αd protein or fragment thereof in frame with either a DNA binding domain of a transcriptional activation protein or a transactivating domain of said transcriptional activation protein; (d) transforming or transfecting said host cell with a fourth DNA expression construct comprising a fifth selectable marker gene encoding a fifth selectable marker protein and a second fusion protein gene encoding an αd binding protein or binding fragment thereof in frame with either the DNA binding domain of said transcriptional activation protein or the transactivating domain of said transcriptional activation protein, whichever is not included in first fusion protein gene; (e) growing said host cell under conditions which permit expression of said αd protein or fragment thereof and said αd binding protein or fragment thereof such that said αd protein or fragment thereof and αd binding protein or binding fragment thereof interact bringing into proximity said DNA binding domain and said transactivating domain reconstituting said transcriptional activating protein; said transcriptional activating protein acting on said promoter to increase expression of said repressor protein; said repressor protein interacting with said operator such that said third selectable marker protein is not expressed; (f) detecting absence of expression of said selectable gene; (g) growing said host cell in the presence of a test inhibitor of binding between said αd protein or fragment thereof and said αd binding protein or fragment thereof; and (h) comparing expression of said selectable marker protein in the presence and absence of said test inhibitor wherein decreased expression of said selectable marker protein is indicative of an ability of the test inhibitor to inhibit binding between said αd protein or fragment thereof and said αd binding protein or binding fragment thereof such that said transcriptional activating protein is not reconstituted, expression of said repressor protein is not increased, and said operator increases expression of said selectable marker protein.

Problems solved by technology

The absolute molecular weights of presumed homologs from other species have been shown to vary significantly [see, e.g., Danilenko et al., Tissue Antigens 40:13-21 (1992)], and in the absence of sequence information, a definitive correlation between human integrin subunits and those identified in other species has not been possible.

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