Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III.
A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.
Raynal
Nicolas
N
Department of Biochemistry, University of Cambridge, UK.
Hamaia
Samir W
SW
Siljander
Pia R-M
PR
Maddox
Ben
B
Peachey
Anthony R
AR
Fernandez
Rafael
R
Foley
Loraine J
LJ
Slatter
David A
DA
Jarvis
Gavin E
GE
Farndale
Richard W
RW
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D024061:Collagen Type III / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D038982:Integrin alpha2beta1 / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010973:Platelet Adhesiveness
D024061:Collagen Type III; D038982:Integrin alpha2beta1; D010446:Peptide Fragments