Evidence that a non-RGD domain in rat osteopontin is involved in cell attachment.
The bone sialoprotein osteopontin (OPN) promotes cell attachment and spreading through its RGD (Arg-Gly-Asp) sequence. To study additional regions of OPN involved in cell attachment, peptides of rat OPN were evaluated 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 after digestion with endoproteinase Arg-C and isolated by reversed-phase HPLC, enhanced cell attachment to a similar degree as OPN. Sequence analysis 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-609, a monoclonal antibody to the integrin alpha V beta 3, a receptor for vitronectin. A variant peptide, GRG-ESPA, did not alter cell attachment. Based on these observations, we conclude that (1) binding of OPN and the 28 kD peptide to fibroblasts involves binding to alpha V beta 3, (2) a site other than the RGD sequence on OPN is also involved in binding to integrins, and (3) the binding of this second site to alpha V beta 3 is inhibited by RGD-containing peptides.
van Dijk
S
S
Department of Basic Sciences, University of Texas Houston Health Science Center.
D'Errico
J A
JA
Somerman
M J
MJ
Farach-Carson
M C
MC
Butler
W T
WT
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000187:drug effects; D005353:Fibronectins / Q000494:pharmacology; D005881:Gingiva / Q000166:cytology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000494:pharmacology; D053495:Osteopontin; D010446:Peptide Fragments / Q000494:pharmacology; D051381:Rats; D012795:Sialoglycoproteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D005353:Fibronectins; D009842:Oligopeptides; D010446:Peptide Fragments; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; C047981:arginyl-glycyl-aspartic acid