Recombinant domain III of perlecan promotes cell attachment through its RGDS sequence.
Perlecan has been previously been shown to support attachment of a wide variety of cells through interactions of its core protein with the cell surface. The core protein domains involved in cell adhesion are, however, unknown. The laminin-like domain III of murine perlecan contains an RGDS sequence and is a likely candidate for supporting integrin-mediated cell attachment. We made a cDNA construct corresponding to domain III and containing an in frame signal peptide at the 5' end as well as in frame a stop codon at the 3' end by using cDNA clones to perlecan. The construct was inserted into the pRC/CMV vector and transfected into HT1080 cells, and the secreted recombinant domain III, a 130-kDa protein, was purified from the medium. The size of proteolytic fragments produced by digestion with V8 protease as well as analysis of the rotary shadowed image of the recombinant protein indicated it was produced in a native conformation. Recombinant domain III coated on tissue culture dishes, supports adhesion of an epithelial-like mouse mammary tumor cell line MMT 060562 in a dose-dependent manner. This interaction was inhibited specifically by the RGDS synthetic peptide and intact perlecan, but not laminin. This domain III RGD-dependent cell attachment activity indicates a role for perlecan in integrin-mediated signaling.
Chakravarti
S
S
Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4955.
Horchar
T
T
Jefferson
B
B
Laurie
G W
GW
Hassell
J R
JR
D000595:Amino Acid Sequence; D002448:Cell Adhesion / Q000187:drug effects*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011509:Proteoglycans / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D009842:Oligopeptides; D011509:Proteoglycans; D011994:Recombinant Proteins; C071927:perlecan; C046617:arginyl-glycyl-aspartyl-serine