RECOMBINANT DOMAIN-III OF PERLECAN PROMOTES CELL ATTACHMENT THROUGH ITS RGDS SEQUENCE

Perlecan has been previously been shown to support attachment of a wid e variety of cells through interactions of its core protein with the c ell surface. The core protein domains involved in cell adhesion are, h owever, unknown. The laminin-like domain III of murine perlecan can co ntains an RGDS sequence and is a likely candidate for supporting integ rin-mediated cell attachment. We made a cDNA construct corresponding t o domain III and containing an in frame signal peptide at the 5' end a s 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 trans fected into HT1080 cells, and the secreted recombinant domain III, a 1 30-kDa protein, was purified from the medium. The size of proteolytic fragments produced by digestion with V8 protease as well as analysis o f the rotary shadowed image of the recombinant protein indicated it wa s produced in a native conformation. Recombinant domain III coated on tissue culture dishes, supports adhesion of an epithelial-like mouse m ammary tumor cell line MMT 060562 in a dose-dependent manner. This int eraction was inhibited specifically by the RGDS synthetic peptide and intact perlecan, but not laminin. This domain III RGD-dependent cell a ttachment activity indicates a role for perlecan in integrin-mediated signaling.