Abstract
Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.
Highlights
Collagen, the most abundant structural protein of the vertebrate organism, currently has 27 reported family members [1]
GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F > L > M > A. These results establish GFOGER as a high affinity sequence, which can interact with the ␣2I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content
The cellular context was found to be crucial in determining the binding to these sequences, since, in contrast to adhesion of ␣21-containing HT1080 cells, ␣21-mediated platelet adhesion was poor to GER sequences other than GFOGER unless the peptide contained glycoprotein VI (GPVI) recognition motifs (GPOn) [25, 26], or the platelets were otherwise activated [27] in line with their hemostatic surveillance function
Summary
The cellular context was found to be crucial in determining the binding to these sequences, since, in contrast to adhesion of ␣21-containing HT1080 cells (a constitutively adherent cell line), ␣21-mediated platelet adhesion was poor to GER sequences other than GFOGER unless the peptide contained GPVI recognition motifs (GPOn) [25, 26], or the platelets were otherwise activated [27] in line with their hemostatic surveillance function. This concept was confirmed by non-selective binding of active ␣2I mutants to all sequences
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