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.