Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence.
The collagen type I-derived fragment alpha1(I)CB3 is known to recognize the platelet collagen receptor integrin alpha2beta1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha2 beta1 and the recombinant alpha2 A-domain, and their ability to support alpha2 beta1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha2 beta1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha2 beta1 by the peptide CB3(I)-2 containing the putative alpha2 beta1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha1(I)CB3 fragment.
Knight
C G
CG
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom.
Morton
L F
LF
Onley
D J
DJ
Peachey
A R
AR
Messent
A J
AJ
Smethurst
P A
PA
Tuckwell
D S
DS
Farndale
R W
RW
Barnes
M J
MJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D003094:Collagen / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010974:Platelet Aggregation; D038981:Receptors, Collagen; D014407:Tumor Cells, Cultured
D016023:Integrins; D010446:Peptide Fragments; D038981:Receptors, Collagen; D003094:Collagen