Expression of a soluble functional form of the integrin alpha4beta1 in mammalian cells.
The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.
Stephens
P E
PE
Ortlepp
S
S
Perkins
V C
VC
Robinson
M K
MK
Kirby
H
H
D000818:Animals; D019556:COS Cells; D002413:Cations, Divalent; D003001:Cloning, Molecular; D019281:Dimerization; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D039041:Integrin alpha4beta1; D016023:Integrins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008024:Ligands; D008274:Magnesium; D008345:Manganese; D016201:Receptors, Lymphocyte Homing / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism
D002413:Cations, Divalent; D007141:Immunoglobulin Fc Fragments; D039041:Integrin alpha4beta1; D016023:Integrins; D008024:Ligands; D016201:Receptors, Lymphocyte Homing; D011993:Recombinant Fusion Proteins; D019010:Vascular Cell Adhesion Molecule-1; D008345:Manganese; D008274:Magnesium