Identification of an alpha(3)beta(1) integrin recognition sequence in thrombospondin-1.
A synthetic peptide containing amino acid residues 190-201 of thrombospondin-1 (TSP1) promoted adhesion of MDA-MB-435 breast carcinoma cells when immobilized and inhibited adhesion of the same cells to TSP1 when added in solution. Adhesion to this peptide was enhanced by a beta(1) integrin-activating antibody, Mn(2+), and insulin-like growth factor I and was inhibited by an alpha(3)beta(1) integrin function-blocking antibody. The soluble peptide inhibited adhesion of cells to the immobilized TSP1 peptide or spreading on intact TSP1 but at the same concentrations did not inhibit attachment or spreading on type IV collagen or fibronectin. Substitution of several residues in the TSP1 peptide with Ala residues abolished or diminished the inhibitory activity of the peptide in solution, but only substitution of Arg-198 completely inactivated the adhesive activity of the immobilized peptide. The essential residues for activity of the peptide as a soluble inhibitor are Asn-196, Val-197, and Arg-198, but flanking residues enhance the inhibitory activity of this core sequence, either by altering the conformation of the active sequence or by interacting with the integrin. This functional sequence is conserved in all known mammalian TSP1 sequences and in TSP1 from Xenopus laevis. The TSP1 peptide also inhibited adhesion of MDA-MB-435 cells to the laminin-1 peptide GD6, which contains a potential integrin-recognition sequence Asn-Leu-Arg and is derived from a similar position in a pentraxin module. Adhesion studies using recombinant TSP1 fragments also localized beta1 integrin-dependent adhesion to residues 175-242 of this region, which contain the active sequence.
Krutzsch
H C
HC
Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Choe
B J
BJ
Sipes
J M
JM
Guo
N h
Nh
Roberts
D D
DD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion; D002633:Chemotaxis / Q000187:drug effects; D006801:Humans; D039201:Integrin alpha3beta1; D016023:Integrins / Q000737:chemistry* / Q000378:metabolism; D007797:Laminin / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D039201:Integrin alpha3beta1; D016023:Integrins; D007797:Laminin; D019700:Thrombospondin 1; D008274:Magnesium; D002118:Calcium