Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide.
Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.
Drake
S L
SL
Department of Laboratory Medicine, University of Minnesota, Minneapolis 55455.
Klein
D J
DJ
Mickelson
D J
DJ
Oegema
T R
TR
Furcht
L T
LT
McCarthy
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000276:immunology / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011509:Proteoglycans / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D000906:Antibodies; D005353:Fibronectins; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D011509:Proteoglycans