Abstract
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.
Highlights
IntroductionThe primary structure deduced from sequencing murine and human cDNA clones indicates that the core protein has five distinct domains (Noonan et al, 1988, 1991; Murdoch et al, 1992; Kallunki and Tryggvason, 1992)
400,000) core protein, is a major component of all basement membranes
Construction of Domain III-All cloning procedures were derived from Sambrook et at. (1987).A cDNAencoding all of domain IIIwas prepared from restriction enzyme digests of cDNA clones 54, 5, and 72 (Noonan et al, 1991), in pBIISK+ vector (Stratagene), by using sites in the multicloning region and within inserts
Summary
The primary structure deduced from sequencing murine and human cDNA clones indicates that the core protein has five distinct domains (Noonan et al, 1988, 1991; Murdoch et al, 1992; Kallunki and Tryggvason, 1992). The N-terminal end of the core protein, domain I, consists of amino acid sequences unique to perlecan and has three potential heparan sulfate attachment sites. Domain III consists of three cysteine-free laminin-like globules that alternate with cysteine-rich EGF1-like units and is most homologous to the N-terminal third oflaminin A chain. GM45380, FY09747, and P30 EY08098 and by Research to Prevent Blindness, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. That alternate with EGF-like units and is most homologous to the C-terminal third of agrin (Patthy and Nikolics, 1994)
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