Identification in Collagen Type I of an Integrin α2β1-binding Site Containing an Essential GER Sequence

Abstract

The collagen type I-derived fragment ␣ 1 (I)CB3 is known to recognize the platelet collagen receptor integrin ␣ 2 ␤ 1 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 ␣ 2 ␤ 1 and the recombinant ␣ 2 A-domain, and their ability to support ␣ 2 ␤ 1 -mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an ␣ 2 ␤ 1 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 ␣ 1 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 ␣ 2 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 ␣ 2 ␤ 1 by the peptide CB3(I)-2 containing the putative ␣ 2 ␤ 1 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 ␣ 1 (I)CB3 fragment.Integrins that recognize collagen can modulate cell behavior, including adhesion and spreading, migration, division, metabolism, and the expression of the differentiated phenotype. These important processes are physiologically relevant to growth and development, wound repair, and angiogenesis and in pathological processes such as thrombosis and tumor metastasis.Integrin ␣ 2 ␤ 1 is also an important collagen receptor in hemostasis, where it plays an essential role in the arrest of platelets, under conditions of blood flow, on the collagen fiber surface exposed as a consequence of injury (1-6). Subsequent recognition by the platelet receptor Gp 1 VI of GPP* 2 sequences within the collagen triple helix (7) leads to platelet activation and aggregation with formation of a platelet plug, which serves to stem the loss of blood. Activation of platelets by collagen may also be a cause of thrombosis, especially that associated with rupture of the atherosclerotic plaque, which leads to exposure of underlying collagens (8).Previous fragmentation studies (9) have indicated the presence of a number of integrin ␣ 2 ␤ 1 recognition sites in collagen I, which, with collagen III, represents the main platelet-aggregatory collagen species in the vessel wall and perivascular space (8). In particular, ␣ 2 ␤ 1 -mediated platelet adhesion to fragment ␣ 1 (I)CB3 derived from the ␣ 1 (I) chain of collagen I is as good as to the parent collagen (9, 10). Inhibition studies with short lin...

Mammalian collagen IV

“…Similar to α 1 β 1 , a conformational-dependent binding to collagen I and IV has been shown for α 2 β 1 integrin (Knight et al, 1998). The α 2 β 1 integrin plays an important role in platelet adhesion to collagens and hemostasis in the blood vessel wall (Sixma et al, 1997).…”

Mapping the Ligand-binding Sites and Disease-associated Mutations on the Most Abundant Protein in the Human, Type I Collagen

“…Of the integrin receptors, a number of interactive sites have been mapped. The site at residue positions 435-438 was proposed to mediate platelet ␣ 2 ␤ 1 integrin-collagen binding (24,25); however, this was not subsequently confirmed (18,26). Another site at residues 567-569 was implicated in RGD-dependent cell adhesion (27) but was later shown relevant only for denatured collagen (28).…”

“…Integrin-binding Sites-Type I collagen is considered an important substrate for cell adhesion, and receptors reported to mediate cell-type I collagen interactions include the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins (18,19), cell surface HSPGs (20), and the discoidin domain receptors 1 and 2 (DDR1 and DDR2) (21)(22)(23). Of the integrin receptors, a number of interactive sites have been mapped.…”

Induction of a novel isoform of the lncRNA HOTAIR in Claudin‐low breast cancer cells attached to extracellular matrix

“…2B). To confirm an essential role of integrin α2 in the induction of HOTAIR, we inhibited integrin α2 using its neutralizing antibody (clone JBS2) in lrECM 3D culture of MDA‐MB‐231 and Hs578T cells (Knight et al ., 1998). The integrin α2‐neutralizing antibody (10 μg·mL −1 ) reduced the RNA levels of HOTAIR to 32% and 38% of that in the control IgG‐treated MDA‐MB‐231 and Hs578T cells, respectively (Fig.…”

“…The GAPDH‐ and BRD4‐specific antibodies were purchased from Novus Biologicals (Littleton, CO, USA) and Cell Signaling (Danvers, MA, USA), respectively. An integrin α2β1‐neutralizing antibody (clone JBS2) was purchased from Chemicon (Temecula, CA, USA) (Knight et al ., 1998). A retroviral vector expressing dominant‐negative chicken Src‐K295R mutant (dnSrc) was kindly provided by Joan Brugge at Harvard University (Thomas et al ., 1991).…”