Abstract
Differentiated human neuroblastoma LA-N1 cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days (primed cells) showed increased adhesion to laminin-, fibronectin-, and collagen type I-coated plates as compared to unprimed cells. Moreover, primed cells seemed to adhere best to laminin. The binding site in laminin, mediating cell attachment, was identified as containing the YIGSR sequence, a known cell binding motif, located in the short arm of the B1 chain of laminin. The synthetic peptide amide, C(YIGSR)3-NH2, containing a repeat of this binding motif, inhibited the attachment of neuroblastoma cells to laminin in a competitive manner, and its inhibitory activity was inversely dependent on laminin concentrations. Affinity chromatography of membrane-extracted proteins over an Affi-Gel 10 column conjugated to C(YIGSR)3-NH2, revealed a major YIGSR-binding protein with an apparent molecular mass of 67 kDa. The 67-kDa surface membrane protein was specifically eluted from the column with the soluble C(YIGSR)3-NH2 peptide, but not with an unrelated peptide. Furthermore, no 67-kDa laminin-binding protein was recovered from an unrelated peptide matrix with the free C(YIGSR)3-NH2 peptide. Ligand blot overlay assays with biotin-labeled C(YIGSR)3-NH2 peptide demonstrated that the 67-kDa receptor is indeed a YIGSR-binding protein. This 67-kDa laminin-binding protein appeared to be down-regulated upon differentiation of LA-N1 cells, as indicated by the level of this protein and its mRNA.
Highlights
Differentiated human neuroblastoma LA-Nl cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days showed increased adhesion to laminin, fibronectin, and collagen type 1coated plates as compared to unprimed cells
The synthetic peptide amide, C(YIGSR)3-NH2' containing a repeat of this binding motif, inhibited the attachment of neuroblastoma cells to laminin in a competitive manner, and its inhibitory activity was inversely dependent on laminin concentrations
Adhesion of Human Neuroblastoma Cells to Extracellular Matrix Components-Adhesion of adrenergic LA-N1 cells was measured after 2 h of incubation on dishes precoated with extracellular matrix (ECM) components
Summary
Differentiated human neuroblastoma LA-Nl cells that were exposed to dibutyryl adenosine 3',5'-cyclic monophosphate for 5 days (primed cells) showed increased adhesion to laminin-, fibronectin-, and collagen type 1coated plates as compared to unprimed cells. Certain clones of human and mouse neuroblastoma (NB) cell lines acquire differentiated properties when treated with a variety of inducing agents such as cAMP analogs, retinoic acid, nerve growth factor, interferon-v, tumor necrosis factor, dimethyl sulfoxide, and hexamethylene bisacetamide. These compounds cause NB cells to extend neurites, increase the expression of sodium channels and plasminogen activator, enhance the activity of neurotransmitter-synthesizing enzymes, and change the spatial organization of cytoskeletal elements. One of the binding sites of the 67-kDa protein is defined by the peptide sequence YIGSR located on the B1 chain oflaminin [16]. One more LBP is a 35-kDa protein that has very high homology to murine galactose-binding lectin, the macrophage antigen Mac-2, suggesting the importance of ga-
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