EVIDENCE THAT A NON-RGD DOMAIN IN RAT OSTEOPONTIN IS INVOLVED IN CELLATTACHMENT

The bone sialoprotein osteopontin (OPN) promotes cell attachment and s preading through its RGD (Arg-Gly-Asp) sequence. To study additional r egions of OPN involved in cell attachment, peptides of rat OPN were ev aluated for their capacity to mediate cell binding to wells in vitro. Human gingival fibroblasts were incubated on microtiter plates coated with either OPN or OPN peptides. A peptide of M(r) 28 kD, obtained aft er digestion with endoproteinase Arg-C and isolated by reversed-phase HPLC, enhanced cell attachment. to a similar degree as OPN. Sequence a nalysis showed that the amino terminus of the 28 kD peptide starts at Ser142 and therefore does not contain the RGD cell attachment sequence (residues 128-130). Cell attachment mediated through both OPN and the 28 kD peptide was blocked by the addition of GRGDSPA peptides or LM-6 09, a monoclonal antibody to the integrin alpha(V)beta3, a receptor fo r vitronectin. A variant peptide, GRGESPA, did not alter cell attachme nt. Based on these observations, we conclude that (1) binding of OPN a nd the 28 kD peptide to fibroblasts involves binding to alpha(V)beta3, (2) a site other than the RGD sequence on OPN is also involved in bin ding to integrins, and (3) the binding of this second site to alpha(V) beta3 is inhibited by RGD-containing peptides.