Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins.
The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.
Vandenberg
P
P
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Kern
A
A
Ries
A
A
Luckenbill-Edds
L
L
Mann
K
K
Kühn
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D002448:Cell Adhesion; D002460:Cell Line; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism*; D003488:Cyanogen Bromide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010920:Placenta / Q000737:chemistry; D051381:Rats; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D038981:Receptors, Collagen; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016023:Integrins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D038981:Receptors, Collagen; D003094:Collagen; D014357:Trypsin; D003488:Cyanogen Bromide