We have previously assigned an integrin ␣ 2 ␤ 1 -recognition site in collagen I to the sequence, GFOGERGVEG-POGPA (O ‫؍‬ Hyp), corresponding to residues 502-516 of the ␣ 1 (I) chain and located in the fragment ␣ 1 (I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports ␣ 2 ␤ 1 -dependent cell adhesion and exhibits divalent cation-dependent binding of isolated ␣ 2 ␤ 1 and recombinant ␣ 2 A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin ␣ 1 A-domain and supports integrin ␣ 1 ␤ 1 -mediated cell adhesion. Triple-helical GFOGER completely inhibits ␣ 2 A-domain binding to collagens I and IV and ␣ 2 ␤ 1 -dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits ␣ 1 A-domain binding to collagen I and strongly inhibits ␣ 1 ␤ 1 -mediated adhesion of Rugli cells to this collagen but has little effect on either ␣1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for ␣ 2 ␤ 1 and in collagen I for ␣ 1 ␤ 1 . Other high-affinity sites in collagen IV mediate its recognition of ␣ 1 ␤ 1 .The integrins are important receptors mediating both cellcell contact and cellular recognition of the extracellular matrix. They are heterodimers comprising an ␣ and a ␤ chain and are classified according to the identity of the latter (1). Integrin recognition sequences have been identified in a number of matrix proteins. RGDX 1 (where X is one of several possible amino acids) occurs in a wide variety of adhesive glycoproteins and recognizes several of the integrins. In fibronectin, for example, RGD recognizes a number of integrins, including ␣ 5 ␤ 1 , ␣ V ␤ 3 , and ␣ IIb ␤ 3 (2, 3).Integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 are the major integrin collagen receptors (4 -6). Each recognizes a variety of collagens, including collagen I, the most abundant and widely distributed of all the collagens. Recognition of collagen IV by integrin ␣ 1 ␤ 1 has been reported to involve an aspartyl residue at position 461 in the ␣ 1 (IV) collagen chain and an arginyl residue at the same residue position in the ␣ 2 (IV) chain (7).Integrin ␣ 2 ␤ 1 plays an essential role in platelet adhesion to collagens in the blood vessel wall under flow conditions (8). This adhesion depends on collagen being in the triple-helical conformation (9) and is important in hemostasis, but it may also be a crucial initiator of thrombosis. Fragmentation of collagen I has indicated the presence of several ␣ 2 ␤ 1 -binding sites throughout the molecule recognized by platelets (9). In particular, fragment ␣ 1 (I)CB3 2 is as good as the parent collagen in supporting ...

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...

Fibril-forming (fibrillar) collagens are extracellular matrix proteins conserved in all multicellular animals. Vertebrate members of the fibrillar collagen family are essential for the formation of bone and teeth, tissues that characterise vertebrates. The potential role played by fibrillar collagens in vertebrate evolution has not been considered previously largely because the family has been around since the sponge and it was unclear precisely how and when those particular members now found in vertebrates first arose. We present evidence that the classical vertebrate fibrillar collagens share a single common ancestor that arose at the very dawn of the vertebrate world and prior to the associated genome duplication events. Furthermore, we present a model, 'molecular incest', that not only accounts for the characteristics of the modern day vertebrate fibrillar collagen family but demonstrates the specific effects genome or gene duplications may have on the evolution of multimeric proteins in general.

The integrin ␣1␤1 is a cell surface receptor for collagens and laminin. The ␣1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the ␣1 A-domain in ligand binding and the extent to which it reproduced the ligand binding activity and specificity of the parent molecule, we produced recombinant ␣1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from ␣1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The ␣2 A-domain, from the ␣2␤1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for ␣1. This is the first demonstration of laminin binding by A-domains. Specificity of A-domain-ligand binding was further investigated using the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4. ␣1 A-domain bound to all three fragments, while the ␣2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the first time identify a limited proteolytic fragment of a ligand that contains integrin A-domain binding activity. To gain insight into the contribution that the A-domain makes to ligand binding within the whole integrin heterodimer, we measured binding constants for A-domain-collagen interactions using surface plasmon resonance biosensor technology. The values obtained were similar to those reported for intact integrin binding, suggesting that the A-domain is the major collagen binding site in the ␣1␤1 and ␣2␤1 integrins.