Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C.
The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.
Denda
S
S
Neuroscience Program, Department of Physiology, University of California San Francisco 94143-0724, USA.
Müller
U
U
Crossin
K L
KL
Erickson
H P
HP
Reichardt
L F
LF
D000469:Alkaline Phosphatase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D002645:Chickens; D003001:Cloning, Molecular; D019281:Dimerization; D006801:Humans; D039001:Integrin alpha Chains; D016023:Integrins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012995:Solubility; D019063:Tenascin / Q000235:genetics / Q000378:metabolism*
D039001:Integrin alpha Chains; D016023:Integrins; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019063:Tenascin; C093856:integrin alpha8; C094866:integrin alpha8beta1; C047981:arginyl-glycyl-aspartic acid; D000469:Alkaline Phosphatase