Prolyl Hydroxylation of Collagen Type I Is Required for Efficient Binding to Integrin α1β1 and Platelet Glycoprotein VI but Not to α2β1

Abstract

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 . Collagen fibrils also bind to the integrin ␣ 2 ␤ 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO) n , respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptorbinding properties of collagen. We show that ␣ 2 ␤ 1 but not ␣ 1 ␤ 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant ␣ 1 ␤ 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use ␣ 2 ␤ 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by ␣ 1 ␤ 1 . These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.The collagens include the most abundant proteins in mammalian tissues providing a scaffold and framework for extracellular matrix assembly. In addition, they can modulate cell behavior through interactions with cellular receptors. All members of the collagen family are built up of three chains and contain at least one triple helix domain formed by repeating GXY sequences (where X is often proline (P) and Y is often hydroxyproline (O)). Such molecules associate to form complex structures, of which the fibril-forming collagens constitute the most abundant matrix component (1). The higher order organization of the collagens, collagen I being the best example, proved crucial to the triggering of specific cell responses; native triple helical structure is essential for cell and platelet adhesion, and the fibrillar structure is required for platelet activation and aggregation (2).The cell surface receptors ␣ 1 ␤ 1 and ␣ 2 ␤ 1 of the integrin family are principally collagen receptors, although they can interact with other extracellular matrix components (e.g. laminins). The specificity of the more recently discovered collagen binding integrins, ␣ 10 ␤ 1 and ␣ 11 ␤ 1 , remains to be determined. Different receptor families have also been identified as containing collagen receptors, such as the membrane-spanning proteoglycans, syndecans (3), and the recently described tyrosine kinase receptors DDR1 and DDR2 (4). In platelets, the recently described glycoprotein VI is a key signaling collagen receptor and is an immunoglobulinn superfamily receptor (5).Several important clues to the understanding of the interaction of ␣ 2 ␤ 1 integrin with collagen arose from the development of triple helical synthetic peptid...

A Streptococcal Collagen-like Protein Interacts with the α2β1 Integrin and Induces Intracellular Signaling

“…The substrate specificity of the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins appears to be similar but not identical. Thus, ␣ 1 ␤ 1 but not ␣ 2 ␤ 1 integrin requires a hydroxyproline residue in the binding site for full activity (51). This requirement could explain our results why ␣ 2 ␤ 1 but not ␣ 1 ␤ 1 mediates cell adhesion to the p176 protein, which lacks hydroxyprolines.…”

Collagen XVI Harbors an Integrin α1β1 Recognition Site in Its C-terminal Domains

“…After its immobilization to multiwell plates, collagen XVI was utilized as attachment substratum for two prototypic cell lines. The rat glioma cell line RuGli was used because these cells mediate their contacts with collagen principally through integrin ␣1␤1, whereas human HT1080 fibrosarcoma cells are known to interact with collagen mainly via integrin ␣2␤1 (27,28). Cells of both lines attached to the immobilized collagen XVI in a dose-dependent manner (Fig.…”

Integrin Activation State Determines Selectivity for Novel Recognition Sites in Fibrillar Collagens

“…2 Thus, other residues (Ser, Pro) may be substituted without wholly compromising binding to ␣ 2 I. However, while a recombinant type I collagen devoid of hydroxyproline bound ␣ 2 ␤ 1 well, it had a poor interaction with both ␣ 1 I and ␣ 1 -expressing cells (43), suggesting selectivity among I domains. GLS was a substantially weaker binder than GLO for all wild-type ␣I domains, but GAS was similar to GAO, the latter presumably being a poor substrate compared with GFO by virtue of the short alanine side chain.…”